QuaSAR

Quantitative Structure Activity

Relationships of Analgesics,

Narcotic Antagonists, and

Hallucinogens

Editors:

Gene Barnett, Ph.D.

Milan Trsic, Ph.D.

Robert E. Willette, Ph.D.

NIDA Research Monograph 22

DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

Division of Research

5600 Fishers Lane

Rockville, Maryland 20857

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402

Stock Number 017-024-00786-2
The NIDA Research Monograph series is prepared by the Division of Research of

the National Institute on Drug Abuse. Its primary objective is to provide critical reviews of research problem areas and techniques, the content of state-of-theart

conferences, integrative research reviews and significant original research. Its

dual publication emphasis is rapid and targeted dissemination to the scientific

and professional community.

Editorial Advisory Board

Avram Goldstein, M.D.

Addiction Research Foundation

Palo Alto, California

Jerome Jaffe, M.D.

College of Physicians and Surgeons

Columbia University, New York

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric Institute

University of California

San Francisco, California

William McGlothlin, Ph.D.

Department of Psychology, UCLA

Los Angeles, California

Jack Mendelson, M.D.

Alcohol and Drug Abuse Research Center

Harvard Medical School

McLean Hospital

Belmont, Massachusetts

Helen Nowlis, Ph.D.

Office of Drug Education, DHEW

Washington, D.C.

Lee Robins, Ph.D.

Washington University School of Medicine

St. Louis, Missouri

NIDA Research Monograph series

Robert DuPont, M.D.

DIRECTOR, NIDA

William Pollin, M.D.

DIRECTOR, DIVISION OF RESEARCH, NIDA

Robert C. Petersen, Ph.D.

EDITOR-IN-CHIEF

Eleanor W. Waldrop

MANAGING EDITOR

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
QuaSAR

Quantitative Structure Activity

Relationships of Analgesics,

Narcotic Antagonists, and

Hallucinogens
The U.S. Government does not endorse or favor any specific

commercial product or commodity. Trade or proprietary names

appearing in this publication are used only because they

are considered essential in the context of the studies

reported herein.

The National Institute on Drug Abuse has obtained permission

from the copyright holders to reproduce certain previously

published material as noted in the text. Further reproduction of this material is prohibited without specific permission of the copyright holders. With these exceptions, the

contents of this monograph are in the public domain and may

be used and reprinted without special permission. Citation

as to source is appreciated.

Library of Congress catalog card number 78-600104

DHEW publication number (ADM) 78-729

Printed 1978

NIDA Research Monographs are indexed in the Index Medicus.

They are selectively included in the coverage of BioSciences

Information Service, Chemical Abstracts, Psychological

Abstracts, and Psychopharmacology Abstracts.

i v
Foreword

The narcotic analgesics and the hallucinogens, two of the many

classes of drugs used by mankind for many centuries, are still

objects of substantial research, and the source of numerous

questions as to the nature of their biological activity. Features

that these two groups of drugs have in common include a strong

predisposition to abuse, considerable overlap in physicochemical

and pharmacochemical properties, and some possibly similar mechanisms of action. It is therefore no coincidence that we held

a meeting to discuss these compounds together.

Man, in his quest for greater knowledge and understanding, demonstrates extreme resourcefulness as he deliberately turns to every

means and technique available in order to help in the pursuit of

revealing the secrets of nature. This is dramatically illustrated

in this volume, where use is demonstrated of a wide variety of

physicochemical, pharmacochemical, and theoretical methods. The

monograph is one result of an intensive three-day meeting sponsored

by the National Institute on Drug Abuse to bring together research

scientists involved in the investigation of biological properties

of analgesics, narcotic antagonists, and hallucinogens.

It was recognized by the Institute, as well as by others, that

the use of such techniques as quantum mechanics, molecular spectroscopy, tissue and receptor binding studies, chemical modification of molecular structures, and correlation analysis can be of

significant aid in understanding the basic mechanisms of drug

action at the molecular level. For this reason NIDA has judiciously supported a considerable amount of research using these

approaches on drugs of major concern and interest. Much of the

work reported in this volume has been a product of that support.

The recent convergence of the discovery of the endorphins and the

development of significant new and refined research techniques

led us to conclude that it was essential, at this time, to bring

together this interdisciplinary group of both esteemed and newly

budding scientists in order that they could discuss their findings.

v
It will be apparent to the reader that final answers and definitive

conclusions, both about the drugs of interest and the methods of

study, are still not abundant. However, it is clear that much has

been achieved and our continuing interest and support are necessary

to assure that further advances are possible. It is our eventual

goal that, in learning how these useful but abused drug molecules

interact with the human body, we will one day be able to tailor

their structures to provide a more favorable balance of properties,

in order to direct us toward better means of treating drug addiction or abuse, and perhaps eventually to learn how one can prevent

drug abuse altogether.

William Pollin, M.D.

Director

Division of Research

National Institute on Drug Abuse

v i
Contents

FOREWORD

William Pollin . . . . . . . . . . . . . . . . . . . . . . . v

INTRODUCTION

The Editors. . . . . . . . . . . . . . . . . . . . . . . . . . 1

SECTION I. PHARMACOCHEMICAL METHODS . . . . . . . . . . . . . . 6

Absolute Configuration and Psychotomimetic Activity

George M. Anderson III, Gisela Braun, Ulrich Braun,

David E. Nichols, and Alexander Shulgin . . . . . . . . . . . 8

Congeners of DOM: Effect of Distribution. on the Evaluation

of Pharmacologic Data

C.F. Barfknecht, J.F. Caputo, M.B. Tobin, D.C. Dyer,

R.T. Standridge, H.G. Howell, W.R. Goodwin, R.A. Partyka, J.A. Gylys, R.L. Cavanagh . . . . . . . . . . . . . . .16

Mescaline Analogs: Substitutions at the 4-Position

Ulrich Braun, Gisela Braun, Peyton Jacob III,

David E. Nichols, and Alexander T. Shulgin . . . . . . . . . 27

Defining the Histamine H2-Receptor in Brain: The Interaction

with LSD

Jack Peter Green, Harel Weinstein, and Saul Maayani . . . . .38

The Nature of Opioid and LSD Receptors: Structural Activity

Relationship Implications

William R. Martin. . . . . . . . . . . . . . . . . . . . . .60

The Use of Rigid Analogues to Probe Hallucinogen Receptors

David E. Nichols, Herschel J.R. Weintraub, William

R. Pfister, and George K.W. Yim . . . . . . . . . . . . . . .70

SECTION II. HANSCH ANALYSIS AND OTHER EMPIRICAL METHODS . . . 84

QSAR: A Critical Appraisal

Sydney Archer. . . . . . . . . . . . . . . . . . . . . . . .86

QSAR of Agents Involved in Serotonin and LSD Binding Sites

Y.L. Chan, E.J. Lien, and J.C. Shih . . . . . . . . . . . . 103

Structure Activity Studies by Means of the SIMCA Pattern

Recognition Methodology

J. Dunn and Svante Wold . . . . . . . . . . . . . . . . . . 114

vii
Quantitative Relationship Between Antinociceptive and

Opiate Receptor Affinity: The Importance of Lipophilicity

Arthur E. Jacobson. . . . . . . . . . . . . . . . . . . .129

Quantitative Stereo-Structure-Activity Relationships

I. Opiate Receptor Binding

Howard Johnson. . . . . . . . . . . . . . . . . . . . . .146

Progress With Several Models for the Study of the SAR

of Hallucinogenic Agents

Lemont B. Kier and Richard A. Glennon . . . . . . . . . . 159

QSAR of Narcotic Analgetic Agents

E.J. Lien, G.L. Tong, D.B. Srulevitch, and C. Dias . . . .186

SECTION III. MOLECULAR MECHANICS. . . , , . . . . . 197

Quantitative Structure-Activity Relationships in the 2,4,5-

Ring Substituted Phenylisopropylamines

George M. Anderson III, Neal Castagnoli, Jr., and

Peter A. Kollman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199

Assessment of Quantun Mechanical Techniques for Use in Structure

Activity Relationship Development, and Application to Analgesics

and Other Drugs

B. Vernon Cheney, D. J. Duchamp, Ralph E. Christoffersen . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Recent Physicochemical and Quantum Chemical Studies on Drugs

of Abuse and Relevant Biomolecules

Joyce J. Kaufman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Structure-Activity Studies of Narcotic Agonists and Antagonists

from Quantum Chemical Calculations

Gilda H. Loew, Donald S. Berkowitz, and Stanley K. Burt . 278

An Extended Isolated Molecule Method and Its Applications

to the Design of New Drugs: General Aspects

Alfredo M. Simas, Roy E. Bruns, and Richard E. Brown . . .317

Recognition and Activation Mechanisms on the LSD/Serotonin

Receptor: The Molecular Basis of Structure Activity Relationships

Harel Weinstein, Jack Peter Green, Roman Osman, and

W. Daniel Edwards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .333

Conformational Energies and Geometries of Narcotics, Using

a Potential Function Method

Mark Froimowitz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Conformational Study of Lysergic Acid Derivatives in Relation

to their Hallucinogenic and Antiserotonin Activities

Mahadevappa Kumbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374

viii
SECTION IV. SPECTROSCOPIC METHODS . . . . . . . . . .409

Carbon-13 Nuclear Magnetic Resonance Study of the α- and

ß-Isomers of Methadol and Acetylmethadol Hydrochlorides

F. Ivy Carroll, Charles G. Moreland, George A. Brine,

and Karl G. Boldt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .410

Photoelectron Spectroscopic Studies of Hallucinogens:

The Use of Ionization Potentials in QSAR

L.N. Domelsmith and K.N. Houk . . . . . . . . . . . . . . 423

An Assessment of Parameters in QuaSAR Studies of Narcotic

Analgesics and Antagonists

Robert Katz, Steven Osborne, Florin Ionescu, Peter

Andrulis, Jr., Robert Bates, William Beavers, Paul

C.C. Chou, Gilda Loew, and Donald Berkowitz . . . . . . . 441

Conformational Studies on Phenethylamine Hallucinogens

The Role of Alpha Alkyl Substitution

Alexandros Makriyannis and James Knittel. . . . . . . . 464

APPENDIX A. QuaSAR Program . . . . . . . . . . . . . . . . . . . . 479

APPENDIX B. List of Participants. . . . . . . . . . . . . . . . . . . . . 482

Research Monograph List. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

i x

Introduction

This monograph contains the proceedings of a technical

review meeting held by the National Institute on Drug

Abuse during April 20-22, 1978, entitled Quantitative

Structure Activity Relationships (QuaSAR) of Narcotic

Analgesics, Narcotic Antagonists and Hallucinogens. The

meeting agenda is given in Appendix A and a list of

participants is given in Appendix B. The meeting consisted of 6 half-day sessions with the first three

sessions devoted to the opiates while the last half of

the meeting was given to considerations of the hallucinogens. The chairmen were asked to provide an overview

of the discussions for their respective sessions which

were then used in preparing the general commentary at

the beginning of each of the 4 sections of the monograph. The sections are defined according to primary

quantitative method used as follows: I. Pharmacochemical Methods: II. Hansch Analvsis and other Empirical Methods; III. Molecular Mechanics; IV. Spectroscopic Methods. This classification is admittedly

arbitrary due to both the interdisciplinary nature of

the scientists gathered for this meeting and the complexity of the problems under consideration.

The initial quantitative approach to studying a problem

as complex as those being discussed in these proceedings

is to begin with the simplest model which when well defined, in terms of both functionality and parameterization, gives a reasonable zeroth order description of

the system. Thus the kinetics of drug absorption and

tissue penetration are initially described by Fick's

law. Likewise, the time dependence of drug disposition

from site of administration to sites of action may be

considered as a classical kinetic process. The structure of the drug molecule is initially described in

1
simplest quantum mechanical terms with an approximate

Hartree-Fock procedure. The relation between molecular

structure and biological action is roughly correlated on

a one-to-one level. Molecule interaction with the biosystem is initially described by a simple key and lock

model for the concept of drug receptor interaction.

From these elementary or crude approximate models one

hopes to gain sufficient intuitive insight to provide a

rational basis for a more refined quantitative investigation of the problem.

When one begins to refine the approach, the quantitative

investigator will likely turn to a superposition principle. On the basis of preliminary findings a simple

functional description φi, the i th of a set of valid

ones, is weighted in terms of its assumed or actual importance by a factor Xi and the true solution is expressed as

At this point the infinite sum must be truncated to

practical limits by some means, be it experimentation,

trial and error, or intuition.

At this higher level of quantitative sophistication the

simplicity of the Fickian description for molecular

transport must eventually be replaced by procedures

utilizing the Phenomenological Equations of irreversible

thermodynamics, which have been developed and applied

successfully to biosystem transport. This more complex

method of description has identified concomitant and

interacting driving forces and flows existent in the

transport process where one considers a combination (not

necessarily linear) of flows and of driving forces that

necessarily obey the Onsager reciprocal relations. The

process of drug disposition within the biosystem likewise becomes more complex. The current pharmacokinetic

description of drug absorption, distribution, transformation and elimination is described by a superposition of first order kinetic processes where one

often needs to consider nonlinear components in the

process as well when the quantitative level is increased. Likewise the correlation between molecular

properties and biological response is a more complex

procedure involving superposition of parameters. In

molecular structure calculations the quantitative

advances have used the superposition of atomic orbitals

to calculate more accurate molecular orbitals and the

Hartree-Fock form of the quantum state function may be

2
replaced by a configuration interaction function when a

more sophisticated quantitative description is warranted.

Finally, the concept of a drug receptor is becoming

necessarily more complex and several different types of

receptors are considered. Although the concept of multiple modes of interaction of opioids with different

recognition loci on a single type of receptor or a group

of closely related receptors was proposed over ten years

ago to account for this complexity (P.S. Portoghese, J

Med Chem, 8:609 (1965); W.R. Martin, Pharmacol Rev,

19:463 (1967)), until recently this concept had remained

an idea whose time had not arrived. Multiple hallucinogen receptors have now been proposed as well (J.P.

Green et al., this monograph; A.T. Shulgin et al., this

monograph). The complexity of a biomembrane in terms of

molecular architecture and transport properties, its

structure and function, makes the superposition of receptors a reasonable concept. A drug receptor R(r,t)

has an observed response that is both spatial and time

dependent. It is appealing to describe this general

receptor in terms of contributions from localized

receptors, ri(r,t), we have not yet begun (or are only

now beginning) to resolve sufficiently. Then the

observed drug response is expressed in terms of a

general (global) receptor that is a superposition of

localized receptors,

where λi is likely to be time dependent, λi(t).

Conclusions that can be drawn from the three days of

presentations and discussions offer not only guidance

for new directions for further research but also caution

in the use of some of the many procedures and methods

that are a necessary part of interdisciplinary research.

(1) The experimental pharmacologists have urged

extreme caution in utilizing data obtained from some of

the currently applied tests for biological activity,

especially from whole animal or other complex systems.

It was felt that further research and development is

urgently needea both for new methods and to make the old

methods more quantitative.

(2) The Hansch analysis method came under considerable criticism but was also ably defended. It was

concluded that the method does have a domain of validity

in the type of research discussed here. The reason that

this type of method is so controversial, while little

3
criticism is aimed at the quantum chemical methods, is

both apparent and one of the virtues of the Hansch

analysis. This method is straightforward and quite

easy to comprehend although it was urged that greater

care be exercised in the choice of methods and parameters and that greater emphasis be placed on orienting

such studies so as to provide mechanistic insight. The

quantum chemical method, on the other hand, is couched

in the language of the Schroedinger Equation, partial

differential equations, molecular integrals, matrix

elements and jargon such as HOMO, LUMO, SCF-HF-LCAO-HO,

CNDO, etc. Therefore, this difficulty tends to lead the

nontheoretician to accept calculated results with minimal objection.

(3) The theoretical physicists have urged caution

in the application and interpretation of quantum chemical approaches to research. Because of the complexity

of the methods and the ready availability of packaged

computer programs it has been concluded that a critical

technical review of the role of quantum mechanics in

biomedical research may be beneficial in the near

future.

(4) There is a far greater role for spectroscopic

methods in QuaSAR research than indicated by the small

number of presentations included in these proceedings.

The methods presented here study properties of isolated

molecules and solvent-solute interactions. The conclusion is warranted that such studies be extended to

the molecule-receptor complex by utilization of synthetic model membranes as well as in vitro, in vivo and

extracted receptors. This advance is needed to provide

some measurement of the chemicophysical interactions

involved and thus allow more quantitative modeling of

the molecule-receptor complex.

(5) Studies should be directed toward better

opioid receptor characterization. This would include

receptor isolation, studying their chemical and biochemical properties, and ultimately determining their

constitution. Ventures of this magnitude are formidable

tasks and require a long term commitment to basic

research.

(6) In conjunction with the above, a concerted

effort should be made to develop new opioid receptor

probes that would facilitate receptor characterization

and be of aid in other types of pharmacologic studies.

This would include design and synthesis of highly selective affinity labeling agents to tag opioid receptors. One needs only to scan the literature or a

pharmacology text to recognize the broad impact that

agents such as phenoxybenzamine have made in elucidating

mechanisms of drug action. The development of selective

4
receptor probes may have a similar effect in the opioid

field.

(7) The area of endorphins is of fundamental

importance and should continue to be pursued with vigor.

This would include projects directed toward (a) the

isolation and characterization of new endorphins and

antiendorphins, (b) biosynthesis and catabolism of

endorphins, and (c) synthesis of novel endorphins.

(8) There is a lack of basic knowledge concerning

the pharmacokinetics of the transfer of ligands into and

out of the brain. While many reports have dealt with

the relationship between uptake and lipophilicity, no

systematic study has addressed the role of pharmacokinetic parameters in this process. For example, there

are no systematic studies that provide information on

the relationship (if any) between the individual

transfer constants (entry and exit constants) and lipophilicity. Clearly, a knowledge of such rate processes

would be useful in the design of opioids or opioid antagonists having a predictable time-course of action.

This would be particularly relevant to the develoment of

long-acting antagonists.

(9) With respect to the hallucinogen areas discussed, it is apparent that the evolution of new molecules continues to provide further specificity or separation of pharmacological properties. The work on

identifying receptors and further delineating the properties of this little understood group of compounds

should help to provide greater insight into the intimate

processes of the brain and mind.

The proceedings reported in this monograph concentrate

on questions of molecular structure, correlation of

molecular properties with biological activity, and

molecular interactions with the receptor(s). While

these proceedings demonstrate progress in achieving an

understanding of the basic problems, each participant

would readily agree that there is still a long, tortuous, high resistance pathway that must be traversed

before full knowledge is gained.

The Editors

ACKNOWLEDGMENT

We are pleased to acknowledge the special assistance

provided by Dr. Philip Portoghese on scientific matters.

5
Section I

Pharmacochemical Methods

The relationship between structure and biological

activity of ligands that act at opioid receptors is

extremely complex. The fact that opioids possess diverse chemical constitution, divergent absolute stereoselectivities, and a spectrum of pharmacologic profiles

attests to the magnitude of this complexity. The advent

of new testing procedures has opened a new chapter in

this active research area, leading not only to superior

agonists and antagonists, but also to the discovery of

the enaorphins ("The Endorphins," E. Costa and M.

Trabuchi, ed., Raven Press, 1978). Mounting evidence

supports a complex picture consisting of multiple recognition sites with regard to location on the nerve cells

and location in the CNS. This being the case, one

wonders whether the comparison of congeners from distantly related series is valid and whether a unified

analysis of opioids is tenable.

Though a new chapter has been opened, our knowledge of

opioid receptors is still rather rudimentary. After

these recognition sites have been characterized further

and additional basic information concerning the endorphins is obtained, quantum mechanics may be able to make

substantial contributions toward the understanding of

events at the level of the receptor. However, until a

critical mass of basic knowledge is acquired, it would

be unreasonable to expect definitive answers from this

approach, although it should provide guidance along the

way.

To a lesser degree, basic work on the identification and

isolation of hallucinogen receptors and the measurement

of drug-receptor bindings is beginning to provide a more

quantitative understanding of the structural requirements for such activity. Indeed, in his presentation,

Dr. Hartin cautioned that structure-activity studies

6
must carefully take into account the existence of multiple receptors or families of receptors. The work presentea by Dr. Nichols and by Dr. Barfknecht on rigid or

restricted molecules as probes appears helpful. The

work by Green and associates illustrates a good combination of the interrelationship of in vivo and in vitro

data and calculated values. Dr. Shulgin and collaborators have used more traditional approaches in their

attempts to shed light on the subtle distinction in

effects brought about by minor structural changes.

Correlations of these many factors often provide clues

if not answers.

At our present level of knowledge, it appears that

multiple regression analysis on series of compounds

might be useful in assisting in the search for nonaddictive analgesics or therapeutically useful psychotomimetics if the pitfalls and limitations of this technique are recognized by its users. One of the problems

with multiple regression analysis as it is presently

applied to analgesics is that no attempts have been made

to sort out analgesic action from side effects such as

addiction liability. Studies of this type might be done

retrospectively if reliable biological data are available but it would seem that a combined program of synthesis, testing and regression analysis might be more

rewarding. Extreme caution should be exercised in such

studies because of the possibility that different

ligands might bind to multiple receptors. In this

connection, regression analysis should be confined to a

series of closely related compounds in an effort to

avoia such pitfalls. Projects of this type might best

be categorized as development work because the limitations of the approach and the complexity of the

biological system would make very difficult the factoring of receptor-related physical parameters for a

basic understanding of the drug-receptor interaction.

7
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic. and R. Willette, eds.

National lnstitute on Drug Abuse, 1978

Absolute Configuration and

Psychotomimetic Activity

George M. Anderson, III, Gisela Braun,

Ulrich Braun, David E. Nichols, and

Alexander T. Shulgin

Most of the known psychotomimetic agents have at least

one chiral center within their structures but have been

stuaied only as the racemic mixtures. All of those

which have been studied in optically active form are

consistent in that the more potent isomer is the isomer

with the absolute "R" configuration at the chiral center

carrying the nitrogen that corresponds to the amino

group of the phenethylamine moiety. With LSD, the 5-

"R",8-"R" isomer is effective in man within the dosage

range of 0.05-0.1 mg, whereas the diastereo-isomeric 1-

iso-LSD (5-"S", 8-"R") is inactive in man at twenty times

tnis dosage (Hofmann 1959). Four of the ringsubstituted phenylisopropylamine psychotomimetics have

been studied in their optically active forms. With 4-

bromo-2,5-dimethoxyphenylisopropylamine (DOB) the "R"

isomer is effective at 0.5 mg, approximately twice the

potency of the racemate (Shulgin et al. 1971), whereas

the "S" isomer is only about a fifth as potent. The "R"

isomer of 4-methyl-2,5-dimethoxyphenylisopropylamine

(DOM, STP) is reported as being at least four times more

potent as a psychotomimetic than the "S" isomer (Shulgin

1973). These observations with both DOB and DOM are

parallel to those observed in biochemical studies (Dyer

et al. 1973) and in animal models (Benington et al.

1973), although these latter studies were conducted at

nearly lethal dosages. The ethyl homolog of DOM, 4-

ethyl-2,5-dimethoxyphenylisopropylamine (DOET), has been

studiea as its optical isomers (Snyder et al. 1974) and

here again the "R" isomer is approximately four times

the activity of the "S" counterpart. Finally, the "R"

isomer of 3,4-methylenedioxyphenylisopropylamine (MDA)

is reported to be three-fold more potent than its

optical enantiomer (Marquardt 1978).

Amphetamine itself, on the other hand, is at nominal

dosages a stimulant rather than a psychotomimetic drug.

In direct comparisons of its optical isomers in man, it

8
is the "S" or dextrorotatory form that is the more

active. In most titration measurements in clinical

stuaies it is accepted as being about twice as potent as

the "R" isomer (Smith and Davis 1977), although the

levo, or "R", isomer has been reported to be as effective as tne "S" isomer in the development of a psychotic

syndrome (Angrist et al. 1971). These comparisons are

presented in table I.

TABLE I

RELATIVE POTENCIES OF THE OPTICAL

ISOMERS OF SEVERAL CNS AGENTS

Order of

Drug CNS Action

Chiral

carbon potency Factor

LSD Psychotomimetic

DOB

DOM

DOET

MDA

Amphetamine Stimulant

Several other clinically used CNS stimulants contain the

same chiral center as does amphetamine but they have

been studied in man only as the "S" form (as with phenaimetrazine) or in the racemic form (as with fenfluramine, mephentermine, methylphenidate and fenethylline).

There are no reports with these drugs of the "R" isomer

having been evaluated in man.

In addition to optical properties, there is another

structural feature that can be employed to separate the

various phenethylamine-type psychotomimetics into two

groups, i.e., the effect on the potency of the drug with

methylation of the primary amino group. Six pairs of

homologs have been studied and can be compared as to

tneir psychotogenic potency. Four of these compound

pairs (all trisubstituted on the aromatic ring) show a

arop of from a half to a whole order of magnitude upon

N-methylation. Two of these, DOB and DOM, upon Nmethylation are less effective as psychotomimetic agents

by a factor of ten. The other two, 3,4,5-trimethoxypnenylisopropylamine and the 2,4,5-counterpart (TMA and

TMA-2, respectively), are decreased in potency by a

factor of three. The remaining psychotomimetic, MDA, is

unique in that upon N-methylation, it maintains substantially an unchanged potency (Shulgin and Nichols 1978)

although undergoing subtle changes in the qualitative

nature of the induced intoxication. The drug amphetamine, although a stimulant at normal clinical dosages,

can exhibit dramatic psychotomimetic properties when

used chronically. The N-methyl homolog methamphetamine

is similarly psychotoxic and thus these represent a pair

of compounds resembling in this respect MDA but being

9
quite dissimilar from the other phenethylamine psychotomimetics. These relationships are summarized in table

II.

TABLE II

RELATIVE POTENCIES OF SEVERAL PRIMARY AMINES AND

THEIR N-METHYL HOMOLOGS AS PSYCHOTOMIMETIC AGENTS

Parent NH2 drug Relative potencies

NH2 vs. NHCH3

dl-DOB ×10

dl-DOM ×10

dl-TMA >×3

dl-TMA-2 >×3

dl-MDA =

d-Amphetamine =

It is apparent that the N-methyl homolog of MDA, Nmethyl 3,4-methylenedioxyphenylisopropylamine or MDMA,

might be a drug with unusual properties. Its parent,

MDA, is a drug with the expected absolute configuration

for psychotomimetic activity, but it is unlike the other

psychotomimetics in that it does not suffer a diminution

of potency upon N-methylation.

This report discusses the comparative pharmacology and

psychopharmacology of MDMA which has been prepared in

the form of its optical isomers. The two bases "R" (-)

MDMA and "S" (+) MDMA were prepared synthetically as

snown (for the "R" isomer) in the scheme:

"R"-MDA "R"-MDMA

3,4ace-Methylenedioxybenzyl methyl ketone (piperonylacetone) was coupled with (-) "R" benzylmethylamine, and

the resulting Schiff base reduced with Raney nickel.

Catalytic reduction of this "R,R" secondary base proviaea "R"-MDA, (a)D= -24.7, agreeing in physical and

chromatographic properties with a sample obtained from

NIDA, Rockville, Maryland. This base was formylated in

methyl formate in a sealed tube and tne resulting forma10
mide had a reversed rotation (α)D= +12.4 (in ethanol)

and m.p.= 99-101. Reduction of this amide in THF with

LAH provided the desired "R"-MDMA, (α)D=-18.2, m.p.=

181-183 as the HCl salt. The "S" isomer was prepared in

an exactly parallel manner, with the primary amine

showing (α)D= +25.3, the amide (α)D= -12.6, m.p.= 101-

102, and the final "S"-MDMA with (α)D= +17.2 and a m.p.=

184-185. Racemic MDMA m.p.= 150-151.

An evaluation of the two optically active isomers, in

comparison with the racemic form of MDMA, was made in

rabbits, using the evoked rectal hyperthermia as a

measure of central activity. The literature procedure

(Aldous et al. 1974) was followed, and the two methods

suggested for the evaluation of potency were employed:

Methoa A utilized the mean maximum temperature rise

(determined by a plot of ΔT vs. log dose), Method B

employed an integration of the temperature-time curve as

a measure of effectiveness. All compounds were assayed

in at least three doses, in duplicate animals, and all

values were normalized against racemic DOM, which was

given the arbitrary value of 100. The results for MDMA

in its optically active form and as the racemate are

given in table III. The values obtained for MDA are

presented for purposes of comparison.

TABLE III

RABBIT HYPERTHERMIA, RELATIVE POTENCY OF TEST COMPOUNDS,

WITH dl-DOM = 100

Compound Method A Method B

(Mean Maximum) (Integral)

dl-MDMA 1.96 1.15

"R"-MDMA 0.71

4.81

0.5*

"S"-MDMA 1.30

dl-MDA 2.46 1.51

"R"-MDA 3.48 2.29

"S"-MDA 2.93 0.91

*Assays were conduced near the LD50, and several

animals were lost during the experiment.

Two features are immediately apparent from table III,

both of which are in exact accord with the human

intoxication aspects which will be discussed below.

First, it is apparent, employing either method of

evaluation, that MDMA is about two orders of magnitude

less potent than the reference compound DOM and that it

is slightly less potent than the N-desmethyl homolog

MDA. Second, although it is clearly apparent that the

"R" isomer of MDA is more effective than the "S" isomer

with either method of calculation, with MDMA this poten11
cy assignment is reversed. "S"-MDMA is more effective

as a CNS agent than is "R"-MDMA. Less apparent from the

table, but a point to be brought out during the discussion below concerning human clinical evaluation, is the

disconcerting absense of additivity between the component isomers in comparison with the activity of the

racemate.

The three chemical species racemic MDMA, "R"-MDMA and

"S"-MDMA, were evaluated in normal human subjects as

their hydrochloride salts, orally, by established procedures (Shulgin et al. 1969). A plot of the quantitative response factor (judged as any of five points,

-,±,+,++, and +++, depending upon the degree and the

disruptiveness of the induced intoxication) against

aosage, for each of the three chemical species, is shown

in figure I. All responses at a given dose for a given

compound are averaged and plotted as a single data

point.

FIGURE I

HUMAN RESPONSE TO "R", "S" AND dl-MDMA

The data in figure I represent 35 clinical trials. The

effective dosage for racemic MDMA has been presented as

being in the range 75-150 mg (Shulgin and Nichols 1978)

and 100-160 mg (Shulgin et a1.1978). Both values range

in complete accord with the response of ++ being representative of a nominal intoxication as seen in the

figure. The "S" isomer is more active than its optical

enantiomorph, being effective within the dosage range of

80-120 mg. This range, however, is a larger dosage than

is calculated (50-80 mg) by halving the racemate. As

the contribution of the "R" isomer is little if any at

12
these levels, it must be concluded that the racemate is

more effective as a CNS agent than would be expected or

calculated from the separate activities of the component

optical isomers. No acceptable value for the effective

dose of the "R" isomer is clear from these data, but it

would-appear that an effective dose might lie in the

vicinity of 300 mg. Qualitatively, most of the sensory

and interpretative properties reported for the racemate

are seen in the "S" isomer, including the frequent

physical toxicity manifestations of mydriasis and jawclenching. The "R" isomer is free of both side effects,

even at the highest doses assayed. However, two subjects who experienced color enhancement on the racemate

observed this peculiarity only with the (otherwise

ineffective) "R" isomer. It must be concluded, within

the data presently in hand, that with this one psychotomimetic compound, MDMA, the active optical isomer is the

absolute "S" isomer with the configuration of dextroamphetamine, in contrast with the previous generality

that the activity of racemic psychotomimetic compounds

could be largely accounted for by the "R" isomer.

The inability of the component isomers to adequately

account for the action of the racemate is unusual but

not without precedent with similar compounds in the

literature. There are numerous reports where there is a

clear interaction between the component isomers of

racemic compounds in in vitro studies; this is well

established in the metabolism of both amphetamine (Gal

et al. 1976) and DOM (McGraw et al. 1977). In vivo

studies in animals with 3,4-dimethoxyphenylisopropylamine (3,4-DMA) (Barfknecht and Nichols 1972) showed

that the "R" isomer was only a third as active as the

"S" isomer, and that neither could duplicate the

characteristic effects of the racemic mixture.

The purpose of this report is not to explain or theorize

on the persistence of activity following the N-methylation of MDA or on the unexpected reversal of activity

assignment to the optical isomers. It is, rather, to

present these findings and to let them provoke the

necessary changes in receptor-site theory that must be

made. Since the effects of MDA to some extent, and of

MDMA to a very large extent, are far removed from that

constellation of symptoms usually associated with

psychotomimetic drugs such as TMA, DOM, and LSD, it is

appealing to explain the difference of assymetric requirements in accord with these differences in qualitative responses, and to conclude that there is some

different receptor site being acted upon in this case.

There is no insistence that all psychotomimetic agents

act by a single mechanism. It has been proposed (Cheng

et al. 1974) that hallucinogenic drugs might be usefully

classified either as direct acting or as indirect acting

releasing agents. Most psychotomimetics appear to act

13
as direct serotonin agonists, but materials such as

para-methoxyphenylisopropylamine (PMA) and MDA do not

fit this model quantitatively (Nichols et al. 1977).

Although no human studies have been conducted with the

isomers of PMA, both PMA and MDA have been demonstrated

to be indirect acting sympathomimetics (Nichols et al.

1974; Paton et al. 1975).

MDMA probably does not act by demethylation, as the

reversal of optical requirements speaks against such a

mechanism. This is further supported by the absense of

cross-tolerance between these two drugs in man. Perhaps

there is a stimulant action from one of the isomers

which enhances or potentiates the (otherwise) dormant

potential of the other. Such questions can be answered

only by further experiments.

REFERENCES

Aldous, F.A.B., Barrass, B.C., Brewster, K., Buxton,

D.A., Green, D.M., Pinder, R.M., Rich, P., Skeels, M.,

and Tutt, H.J. J Med Chem, 17:1100, 1974.

Angrist, B.M., Shopsin, B., and Gershon, S. Nature,

234:152, 1971.

Barfknecht, C.F., and Nichols, D.E. J Med Chem, 15:109,

1972.

Benington, F., Morin, R.D., Beaton, J., Smythies, J.R.,

and Bradley, R.J. Nature, 242:185, 1973.

Cheng, H.C., Long, J.P., Nichols, D.E., and Barfknecht,

C.F. J Pharm Exptl Therap, 188:114, 1974.

Dyer, D.C., Nichols, D.E., Rusterholz, D.B., and

Barfknecht, C.F. Life Sci, 13:885, 1973.

Gal, J., Wright, J., and Cho, A.K. Res Commun Chem Path

Pharmacol, 15:525, 1976.

Hofmann, A. Acta Physiol Pharmacol Neer, 8:240, 1959.

Marquardt, G., see Shulgin, A.T. In: Iversen, Iversen,

and Snyder, eds. Handbook of Psychopharmacology Vol.

II. New York: Plenum Press, 1978. pp. 243 et sec.

McGraw, N.P., Callery, P.S., and Castagnoli, N. J Med

Chem, 20:185, 1977.

Nichols, D.E., Ilhan, M., and Long, J.P. Arch Int

Pharmacodyn, 212:83, 1974.

14
Nichols, D.E., Shulgin, A.T., and Dyer, D.C. Life Sci,

21:569, 1977.

Paton, D.M., Bell, J.I., Yee, R., and Cook, D.A. Proc

West Pharmacol Soc, 18:229, 1975.

Shulgin, A.T., J Pharm Pharmacol, 25:271, 1973.

Shulgin, A.T., and Nichols, D.E. Psychopharmacology of

the Hallucinogens In: Stillman, A., and Willette, A.,

eds. New York: Pergamon Press, 1978.

Shulgin, A.T., Sargent, T., and Naranjo, C. Nature,

221:537, 1969.

Shulgin, A.T., Sargent, T., and Naranjo, C. Pharmacology, 5:103, 1971.

Shulgin, A.T., Braun, U., and Braun, G. N-Substituted

Analogs of 3,4-Methylenedioxyphenylisopropylamine.

Presented at the ACS Conference, March 1978, Anaheim,

California.

Smith, R.C., and Davis, J.M. Psychopharmacology, 53:1,

1977.

Snyder, S.H., Unger, S., Blatchley, R., and Barfknecht,

C.F. Arch Gen Psychiat, 31:103, 1974.

AUTHORS

George M. Anderson, III, Department of Pharmaceutical

Chemistry, School of Pharmacy, University of California,

San Francisco, California 94143.

Gisela Braun, Ph.D., Pharmakologisches Institut der

Universitat, Reuterstrasse 2b, 5300 Bonn, West Germany.

Ulrich Braun, Ph.D., Pharmakologisches Institut der

Universitat, Reuterstrasse 2b, 5300 Bonn, West Germany.

David E. Nichols, Ph.D., Department of Medicinal

Chemistry and Pharmacognosy, School of Pharmacy and

Pharmacal Sciences, Purdue University, West Lafayette,

Indiana 47907.

Alexander T. Shulgin, Ph.D., 1483 Shulgin Road,

Lafayette, California 94549.

15
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Congeners of DOM: Effect of

Distribution on the Evaluation of

Pharmacologic Data

C. F. Barfknecht, J.F. Caputo, M.B. Tobin,

D. C. Dyer, R.T. Standridge, H.G. Howell,

W. R. Goodwin, R.A. Partyka, J. A. Gylys, and

R. L. Cavanagh

INTRODUCTION

The study of structure-activity relationships (SAR) has

intrigued medicinal chemists for decades. In the class

of compounds termed psychotomimetics, the group of

mescaline-type agents (1) has been studied extensively.

The SAR of the group may be summarized as (Shulgin 1976;

Barfknecht, Nichols and Dunn 1975):

1. The presence of an α-methyl (R3) enhances the potency, while R

3

 = H or R

3

 = ethyl or larger alkyl group

yield compounds which may be psychoactive, but not

psychotomimetic.

2. Three aromatic ring substitutents enhance the potency over mono-or disubstituted ring systems. The

location of the three substitutents in the 2,4,5

position is optimal.

3. In the compounds with an a-methyl, the para position is the primary site of metabolism (O-demethylation

for p-methoxy and p-hydroxylation for p-hydrogen).

16
p-Methyl or p-halo substitutents retard the metabolism

at this site. In addition, compounds with these parasubstitutents are significantly more potent than the pmethoxy or p-hydrogen analogs.

4. Substitution on the nitrogen abolishes psychotomimetic

activity. The N-methyl analog of DOM is an antagonist.

5. When the α−methyl group is present, two enantiomers

exist. The R(-) is more potent than the S(+) enantiomer.

The absolute stereochemistry of the more potent enantiomer

correlates with the stereochemistry of the corresponding

position in LSD.

6. The ability of a compound to reach its site of action

is dependent upon the drug distribution between lipid

and aqueous phases. Changes in the ring substitution

have a major influence on distribution.

In the group, the prototype synthetic agent is called

DOM (2, STP), 1-(2,5-dimethoxy-4-methylphenyl)-2-

aminopropane. Although many studies have been reported

on the pharmacologic and biochemical effects of

methoxylated phenylisopropylamines, the mechanisms by

which these molecules exert the psychotomimetic effect

remain unclear.

A breakthrough in the SAR was the discovery that l-

(2,5-dimethoxy-4-methylphenyl)-2-aminobutane (3) does

not exhibit psychotomimetic effects (Standridge et al.

1976). Preliminary evaluation indicated that its actions

could be exploited for therapeutic purposes.

A research effort was launched to explore the SAR of 3

and to develop animal models for differentiating between

the effects of 2 and 3. This paper will present the

synthesis of two congeners, the evaluation of the

compounds in the whole animal model and by in vitro

assays, and finally the interpretation and correlation

of these results in light of the distribution studies.

3 differs from 2 only by a single additional methylene

in the α-side chain. This rather minor structural

modification has a pronounced effect on pharmacologic

17
effects. 2-Amino-1-(2,5-dimethoxy-4-methylphenyl)-2-

methyl propane (4, BL-4041A) and 1-amino-1-(2,5-

dimethoxy-4-methylbenzyl)-cyclopropane (5, BL-4358A)

were designed to incorporate aspects of both 2 and 3.

From an empirical formula standpoint, 4 has one additional

hydrogen and 5 has one less hydrogen than 3. From steric

and electronic viewpoints, these molecules-should differ

from both 2 and 3 and from each other.

CHEMISTRY

Preparation of 4 was accomplished by a route previously

used for a closely related compound (Standridge et al.

1976). 2,5-Dimethoxy-4-methylbenzyl chloride was

condensed smoothly with isobutyric acid to yield 2-(2,

5-dimethoxy-4-methylbenzyl)-2-methylpropanoic acid.

This acid was then converted to the amine (4) through

the carbobenzyloxy derivative.

1 8
Application of the previous synthetic scheme to the preparation of 5 failed. Another route involved condensation

of 2,5-dimethoxytoluene with 1-carbethoxycyclopropanecarbonyl chloride to give the substituted cyclopropyl

phenyl ketone. The ketone could be removed and the ester

converted to the amine (5) via the acid. The actual

execution of this proposal proved to be considerably

more complicated than anticipated.

While the scheme provided the desired intermediate, the

side reactions and purification difficulties which were

encountered necessitated the search for a more satisfactory route.

The successful route involved modification of a synthetic

procedure developed by Kaiser and Weinstock (Kaiser and

Weinstock 1972). The substituted benzaldehyde was

condensed with diethyl malonate. After catalytic

hydrogenation, the substituted malonate was alkylated

with dimethylaminoethyl chloride and quaternized. The

quaternary hydroxide underwent a Hoffman elimination -

condensation, yielding the desired cyclopropanecarboxylic

ester. After saponification and purification, the acid

was converted to 5 in a manner similar to that used for

4.

19
PHARMACOLOGY

The goal of the biological evaluation in this group of

compounds is to differentiate between those compounds

which may have useful psychoactivities and those which

are psychotomimetic and thus are eliminated from further

consideration. The pharmacology of substituted

phenylalkylamines is highly complex and will not be

explored here.

Earlier, the spasmogenic effect of 2 and 3 on smooth

muscle of the rat fundus was reported (Standridge et al.

1976). 2 was substantially more potent than 3 and was

approximately one quarter the potency of serotonin. It

20
has been suggested that stimulation of serotonin

receptors may be related to psychotomimetic activity

(Barfknecht et al. 1973; Dyer et al. 1973). The relative potencies of 2,3,4 and 5 on the rat fundus system

are summarized in Table I. Compounds 2 and 5 exhibited

greater potencies than 3 and 4.

Table I. Comparative Pharmacological Effects on the

Cat Behavior and Rat Gastric Fundus Tests

Cat Behavior Rat Gastric Fundus

Compound N Dose Mean ± SE ED50 Relative

Mg/kg sc Molar Potency

2 4 1 13.3 ± 1.8 1.28 × 10-7 26.8

(0.81 - 2.02)

4 10 7.14 × 10-5 0.048l 3 1.3 ± 0.50

(5.06 - 10.1)

4 4 10 0.75 ± 0.48 4.69 × 10-6 0.73

(2.81 - 8.20)

5 2 5 15 (14,16)2

5.21 × 10-7 6.58

(1.52 - 10.2)

1

Relative spasmogenic potency in terms of serotonin

(100); ED50 = 3.43 × 10-8m, (2.49 - 4.91), 95 percent

confidence limits in parenthesis.

2

Mean and individual values in parenthesis.

The cat behavior test, a modification of a published

procedure (Wallach, Friedman, and Gershon 1972), has

been used to determine psychotomimetic activity. The

animal reactions to drugs are observed and categorized

in several classes: body posture (arching of body and

tail), limb extension (legs and claws), body muscle

rigidity, abnormal limb position (cataleptic-like

reactions), motor coordination, mouth opening or tongue

protrusion, hostility reactions (clawing, biting, hissing) , loss of contact to environment, autonomic reactions

(piloerection, myosis, salivation, emesis). The drug

effects are quantitated by assigning a point value for

the occurrence. The effects of the compounds in this

test are summarized in Table I. Both 3 and 4 were weak

and exhibited a similar potency in contrast to 2 and 5

which induced markedly stronger effects. The conclusion

drawn from these studies was that 5 had undesirable

psychotomimetic properties, while its chemical relative

(4) did not. Since the structural similarities of 4 and

5 are so great, further demonstration of the different

pharmacologic effects was needed.

21
The sheep umbilical artery strip preparation has been

shown to be serotonergic and to be stimulated by 2 and

other psychotomimetic compounds. By way of contrast, 3

was found to be ineffective as an agonist (Dyer 1976).

The dose-response relationships of serotonin, 4,5 on the

sheep umbilical arteries are presented in Figure I.

Consistent with the data in Table 1, 5 was shown to be an

agonist, while 4 was much less effective.

Figure I. Comparative Pharamacological Effects of 4 and

5 on the Sheep Umbilical Artery Preparation

22
DISCUSSION

With the results of three pharmacologic studies in agreement that 5 was psychotomimetic and 4 was not, it was

tempting to ascribe these differences to interactions at

the receptor site. However, from inspection of molecular

models there were no obvious dissimilarities in steric

requirements or preferred conformations. If we were to

propose conformational differences as the source of the

pharmacologic effects, detailed studies of the crystal

structures, solution conformations, and molecular orbital

calculations of preferred conformations would have to be

undertaken. Even then, it would be necessary to show

that these data were applicable to the pharmacologic

assay situations.

For the past decade, the importance of the physical

distribution of molecules on their apparent pharmacological

actions and their relative potencies has received greater

attention. Lead by the work of Hansch and his colleagues

(Hansch 1969), we have become aware of partition coefficients and their influence on the interpretation of

pharmacologic data. Recently, the use of the distribution

coefficient has been shown to offer advantages over the

partition coefficient in assessing the role of distribution (Scherrer and Howard 1977). The distribution

coefficient, D, is defined as the ratio of the concentration of drug in the lipid phase to the concentration

of total ionized and unionized drug in the aqueous phase

at a given pH. It is assumed that only the unionized

drug partitions into the lipid phase.

The relationship between log D and log P is shown in the

following equation:

The significance of this relationship is embodied in the

second term on the right-hand side of the equation. This

term is the correction factor for ionization of the involved species. When the pKa of the molecule is equal

to the pH of the aqueous phase, this factor approaches

zero. When the pKa becomes greater than the pH, the

correction term becomes a larger negative value. Thus,

the log P of an amine will always be equal to or greater

than the log D when the pH is 7.4 (physiological pH).

The distribution coefficients were determined in 1-

octanol/water according to published procedure at

ambient temperature (Fujita, Iwasa, Hansch 1964). The

aqueous phase was buffered to pH 7.4 with phosphate

buffer. The distribution data are shown in Table II.

23
Table II. Distribution Data on 4 and 5

Compound D LogD S

1

4

2

0.542 -0.266 0.038

5

3

14.55 1.162 1.028

1

S is the standard deviation

2

D was calculated as the mean of six determinations

3

D was calculated as the mean of four determinations

If one were to explain the pharmacologic data without

considering the distribution data, one might say that

the evidence clearly suggests that 5 is hallucinogenic

and 4 is not. Then one would be tempted to invoke some

receptor interaction difference to explain the situation.

However, with the distribution data available, one has

such a clear difference in distribution that any thought

of ascribing the pharmacologic effects solely to receptor

site interactions must await further studies of binding

or molecular efficacy. Thus both molecules (4 and 5)

may well exhibit the hallucinogenic profile, If they

both were able to attain the necessary concentration at

the receptor site.

Pharmacological screening is designed to find useful

effects without regard to mechanism of action or the

factors that may be contributing to the potency. It is

only necessary to investigate distribution, metabolism,

and molecular factors when the events on the receptor

level are to be analyzed. These two strategies

(pharmacological screening and molecular investigation)

are designed to answer different questions and provide

complementary information.

In applying the information gained in this study to

other situations, one must not expect the cyclopropyl

analog of every compound to have a significantly

different distribution from that of its gem-dimethyl

analog. Rather, the data presented here shows that one

must consider the effect of the pKa on the distribution

and the effect of structure on the pKa. The cyclopropyl

group of 5 affects the pKa of the amino function by

means of its pseudo-π character. The result is that

less of the compound is protonated at pH 7.4 and less is

present in the aqueous phase. The gem-dimethyl grouping

does not affect the pKa of the amino appreciably. Thus

more of the compound is protonated at pH 7.4 and more

is present in the aqueous phase, resulting in a distribution of 4 similar to that anticipated for 2 or 3.

24
This study has provided an insight into the complex

process of evaluating the results of various pharmacologic

tests and interpreting the data with and without considering the physical distribution of molecules in biological

fluids. It is evident that distribution may play a

significant role in all pharmacologic test systems,

including the isolated in vitro preparations where

traditionally it has not been considered. Further work

to study the influence of physical factors in isolated

systems is under investigation.

REFERENCES

Barfknecht, C.D., Nichols, D.E., and Dunn III, W.J.

Correlation of psychotomimetic activity of phenethylamines and amphetamines with 1-octanol-water partition

coefficients. J Med Chem, 18:208-210, 1975.

Barfknecht, C.F., Nichols, D.E., Rusterholz, D.B.,

Long, J.P., Engelbrecht, J.A., Beaton, J.M., Bradley,

R.J., Dyer, D.C. Potential psychotomimetics 2-amino1,2,3,4-tetrahydronaphtahalene analogs. J Med Chem, 16:

804-808, 1973.

Dyer, D.C. Comparison of the effects of R-(-)-2-amino1-(2,5-dimethoxy-4-methylphenyl) propane (DOM), R-(-)-

2-amino-1-(2,5-dimethoxy-4-methylphenyl) butane (BL-3912A)

and 5-hydroxytryptamine on non-inneruated vascular smooth

muscle. Res Comm Chem Path Pharmacol, 14:449-454, 1976.

Dyer, D.C., Nichols, D.E., Rusterholz, D.B., and

Barfknecht, C.F. Comparative effects of stereoisomers

of psychotomimetic phenylisopropylamines. Life Sci,

13:885-896, 1973.

Fujita, T., Iwasa, J., and Hansch, C. A new substituent

constant, π, derived from partition coefficients. J

Amer Chem Soc, 86:5175-5180, 1964.

Hansch, C. A quantitative approach to biochemical

structure-activity relationships. Acc Chem Res, 2:232-239,

1969.

Kaiser, C. and Weinstock, J. Cyclopropanes via abnormal

Hofmann elimination: ethyl 1-benzylcyclopropane

carboxylate. Org Syn, 52:1777, 1972.

Scherrer, R.A. and Howard, S.M. Use of Distribution

coefficients in quantitative structure-activity relationships. J Med Chem, 20:53-58, 1977.

Shulgin, A.T. Psychotomimetic agents. In: Gordon,

Maxwell, ed. Psychopharmacological Agents, Vol. IV. New

York: Academic Press, 1976. pp. 59-146.

25
Standridge, R.T., Howell, H.G., Gylys, J.A., Partyka,

R.A., and Shulgin, A.T. Phenylalkylamines with potential

psychotherapeutic utility 1. 2-amino-14-(2,5-dimethoxy4-methylphenyl) butane. J Med Chem, 19:1400-1404, 1976.

Wallach, M.B., Friedman, E., and Gershon, S. 2,5-

Dimethoxy-4-methylamphetamine (DOM), a neuropharmacological examination. J Pharmacol Exp Ther, 182:145-154,

1972.

AUTHORS

C.F. Barfknecht, Ph.D., J.F. Caputo, Ph.D., and M.B. Tobin,

B.S., Division of Medicinal Chemistry and Natural Products,

College of Pharmacy, University of Iowa, Iowa City, Iowa

D.C. Dyer, Ph.D. Department of Pharmacology, College of

veterinary Medicine, Iowa State University, Ames, Iowa

J.A. Gylys, Ph.D., and R.L. Cavanagh, Ph.D., Department of

Pharmacology, Bristol Laboratories, Syracuse, New York

R.T. Standridge, Ph.D., H.G. Howell, Ph.D., W.R. Goodwin,

Ph.D., and R.A. Partyka, Ph.D., Department of Chemical

Research, Bristol Laboratories, Syracuse, New York

26
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Mescaline Analogs: Substitutions at

the 4-Position

Ulrich Braun, Gisela Braun, Peyton Jacob III,

David E. Nichols, and Alexander T. Shulgin

Mescaline, 3,4,5-trimethoxyphenethylamine, is one of the

longest known and best studied of the psychotomimetic

drugs. It has served as the structural paradigm for the

synthesis and study of a large number of analogs. Many

of the molecular changes have resulted in increased

potency, and have established structural parameters felt

to be necessary for maximum neurotoxic activity. One of

these is the extension of the carbon chain from two

carbons to three, by the addition of an alpha-methyl

group adjacent to the basic nitrogen. This simple homologation appears to protect the nitrogen atom from metabolic removal, and to effectively increase the potency

of the drug. In the examples in the earlier literature

where direct comparisons between the two-carbon and the

three-carbon counterparts were made, there was certainly

an increase in potency. However, the metabolic argument

is clouded by the fact that in those examples where the

chain was extended to four carbons (providing as complete a structural hindrance to metabolic attack as a

three-carbon chain) there was also a consistent decrease

in biological activity.

A second parameter is the positioning of the groups of

the aromatic ring. The relocation of substituents from

the 3,4,5-orientation of mescaline to the 2,4,5-pattern

has, again in the earliest reports (Shulgin 1964), resulted in a substantial increase in psychotomimetic potency. As a result of these generalizations, both the

two-carbon phenethylamines and the 3,4,5-"mescalinelike" substitution pattern have been largely ignored in

the synthesis and evaluation of psychotomimetic drugs

and have played only a small role in structure activity

relationship studies. In recent years, several discoveries have renewed interest in compounds more closely

allied to mescaline in structure. First, a number of

potent ring-substituted phenethylamines have been

reported, chemicals that are the two-carbon analogs of

27
known psychotomimetic phenylisopropylamines. Second,

there has been an increasing awareness of the importance

of the 4-position in the substitution pattern of the

2,4,5-orientation. Third, there have been recent correlations between psychotomimetic potency and physical

properties such as lipophilicity, which have suggested

that modest chain-lengthening within a set system of

aromatic substituents might affect biological potency

(Barfknecht et al. 1975). The purpose of this paper is

to review these parameters, to present experimental data

concerning several new compounds, and to discuss the

possible reasons for their activity.

Initially, mescaline was the only two-carbon psychotomimetic known. Although many variations on its structure had been made leading to a variety of compounds

with greatly increased potency, it was only recently

that the logical step was taken of investigating the

two-carbon counterparts of the more potent phenylisopropylamines. The phenethylamines which have been

studied, and their reported potencies in human subjects,

are presented in table I in direct comparison to the

corresponding substituted amphetamine homologs. It is

immediately apparent that, in all cases, the threecarbon homolog is more potent than the corresponding

phenethylamine, sometimes by as much as an order of

magnitude. In some entries, the absence of defined

action in man makes the comparison between the two

groups uncertain.

Structural changes at the 4-position of the psychotomimetic phenylisopropylamines can modify both the

quantitative and the qualitative effects that are

produced. The potency increases as the nature of the

group in the 4-position varies from H< OR < SR< R < X

wherein R is an alkyl group and X is a halogen. Within

each of these families, small groups of close homologs

have been studied and the comparative quantitative relationships of these are shown in table II. In general,

those series that represent progressive homologous sets

of compounds have their maximum potency with the methyl

or the ethyl substituent. However, of particular interest to this study is the unusual enhancement of activity

seen in the ethoxy compound 3,5-dimethoxy-4-ethoxyphenylisopropylamine. The relatively minor change of

potency seen upon replacing a methoxy group with an

ethoxy group, or a methyl group with an ethyl group, at

the 4-position (as seen in the comparison of TMA-2 to

MEM, of para-DOT to aleph-2, and of DOM to DOET) is

exaggerated when this alkoxy group is flanked with

methoxy substituents (see table II). The unusual five

fold increase in activity of 3,5-dimethoxy-4-ethoxyphenylisopropylamine over the 4-methoxy counterpart TMA

may emphasize the importance of steric considerations in

the action of these drugs. In the case of the 3,4,5-

28
TABLE I

HUMAN ACTIVITY OF 2-CARBON AND ANALOGOUS 3-CARBON-CHAIN

PSYCHOTOMIMETICS (a)
a.

b.

c.

d.

e.

f.

g.

h .

i .

j.

k.

l.

m.

n.

o.

p.

q.

r.

s.

REFERENCES FOR TABLE I

Compounds listed with > are inactive in man at the

stated values, which are total doses.

Brown, W.T., McGeer, P.L., and Moser, I. Can Psychiat

J, 31:91, 1968.

Shulgin, A.T., Sargent, T., and Naranjo, C. Nature,

221:537, 1969.

Vojtechovsky, M., and Krus, D. Acta Nerv Sup, 381,

1967.

Shulgin, A.T. In: Iversen, Iversen, and Snyder, eds.

Handbook of Psychopharmacology Vol. II. New York:

Plenum Press, 1978.

Alles, G.A. In: Abramson, ed. Neuropharmacology

(Trans 4th Conf) J. Macy Jr. Foundation: 1959.

Shulgin, A.T. Lloydia, 36:40, 1973.

Peretz, D.I., Smythies, J.R., and Gibson, W.C. J

Ment Sci, 101:317, 1955.

Shulgin, A.T., Bunnell, S., and Sargent, T. Nature,

189:1011, 1961.

Slotta, K.H., and Huller, J. Z Physiol Chem, 238:14,

1936.

Jansen, H.P.J.M. Rec Trav Chim, 50:291, 1931.

Dittrich, A. Psychopharmacologia, 21:229, 1971.

Shulgin, A.T.

Shulgin, A.T.,

1:93, 1975.

Shulgin, A.T.,

cology, 5:103,

Shulgin, A.T.

Shulgin, A.T.

J Psyched Drugs, 8:169, 1976.

and Carter, M.F. Psychopharm Commun,

Sargent, T., and Narango, C. Pharma1971.

logical Agents

1976.

Shulgin, A.T.,

cology, 10:12,

Present paper.

Unpublished data.

Vol. 4. New York: Academic Press,

In: Gordon, ed. PsychopharmacoSargent, T., and Naranjo, C. Pharma1973.

30
TABLE II

RELATIVE POSTENCIES IN MAN OF DIMETHOXYPHENYLISOPROPYLAMINE

PSYCHOTOMIMETICS WITH VARIOUS SUBSTITUENTS ON THE 4-POSITION
REFERENCES FOR TABLE II

Shulgin, A.T. J Psyched Drugs, 8:169, 1976.

Shulgin, A.T. In: Inverson, Iversen, and Snyder,

eds. Handbook of Psychopharmacology Vol. II., New

York: Plenum Press, 1978.

Shulgin, A.T. Unpublished data.

Shulgin, A.T., and Nichols, D.E. In: Stillman, R.,

and Willette, R. eds. Psychopharmacology of Hallucinogens. New York: Pergamon Press, 1978.

Shulgin, A.T., and Dyer, D.C. J Med Chem, 18:1201,

1975.

A > symbol indicates the absence of any activity at

the stated dosage.

Shulgin, A.T., Sargent, T., and Naranjo, C. Pharmacolony. 5:103, 1971.

Peretz: D.I., Smythies, J.R., and Gibson, W.C. J

Ment Sci, 101:317, 1955.

Shulgin, A.T., Bunnell, S., and Sargent, T. Nature,

189:1011, 1961.

Nichols, D.E., Shulgin, A.T., and Dyer, D.C. Life

Sci, 21:569, 1977.

a.

b.

c.

d.

e.

f .

g.

h.

i.

j.

32
substitution pattern, an ethoxy group in the 4-position

is of necessity directed away from the plane of the

aromatic ring.

These possible steric effects have been evaluated by an

approach involving partition coefficients. In this way,

an estimate of comparative lipophilicity can be made

since this property is felt to influence the ease of

membrane transport and thus eventual availability to the

site of action. A number of psychotomimetic phenylisopropylamines have been studied in an octanol-water

partition system, and the correlation of the resulting

values with central activity has provided a relationship

that suggests an optimum lipophilicity for maximum biological activity (Barfknecht et al. 1975). These partition values have been correlated to serotonin receptor

stimulation capability (Nichols and Dyer 1977) and have

recently been extended to a number of phenethylamine

compounds (Nichols et al. 1977).

We have undertaken a project directed towards an investigation of a number of compounds that represent a

return to the 3,4,5-orientation and the two-carbon chain

features of mescaline, but that are modified in some way

by the substituent that is found at the 4-position.

These are compounds of the general structure:

wherein X = R, OR, SK and halogen. In this report we

will discuss the chemistry and the psychopharmacology of

the first three compounds studied in this direction.

Two of the compounds described here are 4-alkoxy homologs of mescaline. These have been prepared by the

appropriate alkylation of syringaldehyde with either

ethyl or propyl iodide followed by the formation of a

nitrostyrene with nitromethane. These intermediates

were then reduced with LAH to form the product amines 4-

ethoxy-3,5-dimethoxyphenethylamine (1) and 4-propoxy3,5-dimethoxyphenethylamine (2), respectively. These

products have been called escaline and proscaline in

keeping with the well-established trivial name mescaline

for the 4-methoxy counterpart. These reactions are

shown in scheme I.

33
SCHEME I

The third compound studied is the 4-thio analog of

mescaline, 4-methylthio-3,5-dimethoxyphenethylamine.

This was prepared (see scheme II) by the reaction of

lithiated m-dimethoxybenzene with dimethyldisulfide to

form 2,6-dimethoxythioanisole. This underwent bromination uniquely adjacent to the methoxy group, and the

resulting bromodimethoxythioanisole underwent a smooth

benzyne reaction with acetonitrile to form the benzyl

cyanide shown, which was reduced to the desired end

product (3) with aluminum hydride in THF.

SCHEME II

These three mescaline analogs (table I) have been

assayed in normal subjects by procedures previously

outlined (Shulgin et al. 1969). Both of the alkoxy

homologs are effective as psychotomimetics at dosages of

60 mg orally, with clear threshold effects being noted

34
in some subjects at levels as low as 10 mg. These bases

differ from mescaline in that the onset of action occurs

sooner (within the first hour) and there is no nausea

noted, but otherwise the time course and much of the

qualitative content of the intoxication are similar to

those of mescaline. The sulfur compound thio-mescaline

(3) is also of unexpectedly high potency and is an

effective psychotomimetic in man at oral doses of 30 mg.

The initial indicators of intoxication are apparent

during the second half of the first hour following

administration, and a plateau of intoxication is maintained for approximately three hours. The qualitative

content of the experience resembles LSD more closely

than it does mescaline in that there are few reports of

color enhancement but rather considerable involvement

with intellectualization. The overall content of the

intoxication is closely related to the "aleph" effects

which are characterteristic of the several 4-thioalkyl2,5-dimethoxyphenylisopropylamine compounds listed in

table II.

There are several appealing explanations for the unexpectedly high potency of these three compounds. The

most direct explanation is the one suggested above, that

the presence of two methoxy groups adjacent to the 4-

position (the 3,4,5-orientation) forces the group in

that position completely out of the plane of the

aromatic ring. With the 4-methoxy group as found in

mescaline, there is only a minor protuberance from the

plane of the ring and there is a relatively low order of

potency. However, with the ethoxy and the propoxy

groups the alkyl "tail" of the alkoxy group is conspicuously inserted into the surrounding area. This steric

necessity may inordinately affect both the pharmacokinetics and the pharmacodynamics of the molecule.

The aliphatic nature of this "tail" could modify the

local lipophilicity of the molecule, which will influence its bioavailability. These effects might be

related to partition properties, as mentioned earlier.

There could also be a major dissymetry introduced into

the molecule by this out-of-plane forcing which would

potentially change the closeness of fit of the drug at

some receptor site. Further, the change of the orbital

hybridization of the heteroatom at the 4-position,

especially in the case of the 4-methylthio example,

could allow a change of availability of the molecule to

metabolism and thus to eventual distribution in the

body. An immediate challenge to these possibilities may

be found in the 4-thioethyl compound, which has not yet

been evaluated pharmacologically. If the enhancement

seen from the methoxy to the higher alkoxy (6-fold increase over mescaline) and the enhancement seen from the

metnoxy to the methylthio (12-fold increase over mescaline) may be expected to be general in applicability,

35
the ethylthio compound may be expected to be an exceptionally potent psychotomimetic.

Yet another group at the 4-position deserves more

attention than it has received. The 4-alkyl analog of

mescaline, 4-methyl-3,5-dimethoxyphenethylamine, has

been prepared and studied in cats (Benington, Morin, and

Clark 1960). Although the geometric change induced by

the replacement of the methoxy group with the methyl

group is minor, there was nonetheless a dramatic change

in animal response observed. A rage reaction was

elicited, a property not observed with mescaline itself.

With longer-chain homologs, the "tail" of the molecule

would again be forced out of the plane of the aromatic

ring, but there would be less metabolic susceptability.

Studies of alkyl and thioalkyl analogs of mescaline are

currently underway.

36
REFERENCES

Barfknecht, C.F., Nichols, D.E., and Dunn, W.J. III.

Correlation of psychotomimetic activity of phenethylamines and amphetamines with 1-octanol-water partition

coefficients. J Med Chem 18:208-210, 1975.

Benington, F., Morin, R.D., and Clark, L.C. Mescaline

analogs. X. 3,4-Dimethyl-3,4-Dichloro-, and 3,5-Dimethoxy4-methyl- -phenethylamines. J Org Chem, 25:2066-2067,

1960.

Nichols, D.E., and Dyer, D.C. Lipophilicity and serotonin

agonism activity in a series of 4-substituted mescaline

analogues. J Med Chem, 20:299-301, 1977.

Nichols, D.E., Shulgin, A.T., and Dyer, D.C. Directional

lipophilic character in a series of psychotomimetic phenethylamine derivatives. Life Sci, 21:569-576, 1977.

Shulgin, A.T. Psychotomimetic amphetamines: Methoxy

3,4-Dialkoxyamphetamines. Experientia, 20:366-369,

1964.

Shulgin, A.T., Sargent, T., and Naranjo, C. Structureactivity relationships of one-ring psychotomimetics.

Nature, 221:537-541, 1969.

AUTHORS

Gisela Braun, Ph.D., Pharmakologisches Institut der

Universitat, Reuterstrasse 2b, 5300 Bonn, West Germany.

Ulrich Braun, Ph.D., Pharmakologisches Institut der

Universitat, Reuterstrasse 2b, 5300 Bonn, West Germany.

Peyton Jacob III, Ph.D., 2 Vianne Court, Orinda,

California 94563.

David E. Nichols, Ph.D., Department of Medicinal

Chemistry and Pharmacognosy, School of Pharmacy and

Pharmacal Sciences, Purdue University, West Lafayette,

Indiana 47907.

Alexander T. Shulgin, Ph.D., 1483 Shulgin Road,

Lafayette, California 94549.

37
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Defining the Histamine H2-

Receptor in Brain: The Interaction

With LSD

Jack Peter Green, Harel Weinstein, and Saul

Maayani

Numerous studies have shown that LSD (D-lysergic acid diethylamide)

affects serotonergic, dopaminergic, adrenergic, and tryptaminergic

systems in the central nervous system (see Mandell and Geyer, 1976;

Martin and Sloan 1977; Freedman and Halaris 1978). To this list

is now added a histaminergic system. For LSD is a competitive

antagonist of the histamine H2-receptor in brain (Green et al. 1977).

In describing this action of LSD, we giva an account of the established criteria that we followed to define the receptor with which

LSD reacts. This definition fostered an analysis of the relationship between the molecular structure of known H2-antagonists and

LSD. This results in the correct prediction of a new H2-antagonist.

The action of LSD on a histamine receptor in brain may be relevant

to the neural effects of LSD. For histamine may be a transmitter

in brain. Evidence for this function of histamine remains circumstantial but perhaps no more so than that offered for most other

biogenic amines that are classified as putative transmitters.

In fact, as recently reviewed (Green, Johnson and Weinstein 1978),

studies of mammalian brain show that histamine meets most criteria

for having transmitter function. It has non-uniform distribution

with highest concentration in the hypothalamus. Subcellular fractions of brain containing nerve-endings are rich in histamine.

Brain contains the specific enzyme that decarboxylates histidine to form histamine as well as the specific enzyme that metabolizes histamine. After destroying fibers in the median forebrain

bundle or the afferent fibers to the hippocampus the activity of

the specific histidine decarboxylase falls in brain regions distal

to the lesion. Histamine is released from brain slices by potassium ions in a process that is dependent on ionic calcium. Histamine turns over rapidly: unlike other aromatic biogenic amines,

it is not taken up by presynaptic terminals but is instead metabolized to 3-methylhistamine which is then oxidatively deaminated by

monoamine oxidase B. Neurons respond to histamine, e.g., it decreases the firing rate of cerebral cortical and brainstem neurons

and increases the firing rate of hypothalamic neurons. Especially

provocative is the observation that electrical stimulation of

3 8
afferent fibers to the cerebral cortex or hippocampus reduces their

firing rate, and part of this effect of electrical stimulation can

be blocked by histamine H2-antagonists. As described below, histamine increases adenylate cyclase activity in brain, and this effect is blocked by specific antagonists.

Evidence that LSD and D-2-bromo-LSD block the histamine H2-receptor

is presented here, and the implications are discussed.

THE CLASSIFICATION OF A RECEPTOR IN A TISSUE

Studies aimed at revealing the mechanisms of action of a drug by

observing effects in intact animals are especially difficult when

applied to the brain because of the awesomely complex multineuronal

interactions. For example, treating rats with either LSD or mescaline decreased the firing rate of the raphe neurons but only LSD

did this when applied directly to the neurons (Haigler and

Aghajanian 1973), mescaline affecting the firing rate by acting

indirectly (Gallagher and Aghajanian 1976). The receptors that a

drug acts upon can be revealed less ambiguously by studies on

isolated preparations in vitro. Towards this end, two systems have

been widely used in recent years for studies of receptors in brain

and other tissues. One is the study of high affinity binding of

ligands to membranes. The other is the measurement of adenylate

or guanylate cyclase activity which can reflect interactions of

ligands with the receptor if the receptor is coupled to the enzyme.

Direct actions of drugs on the cyclase can be ruled out. High

affinity binding requires a ligand of high specific activity and

of high affinity for the receptor. Both these criteria mere met

with tritlated pyrilamine, an H1-antagonist, which was used to

label the Hi-receptor in guinea pig (Hill, Young, and Marrian 1977).

The low affinity of the known H2-antagonists for the H2-receptor

requires a very highly labeled ligand to study the H2-receptor,

and this is not available. Adenylate cyclase activity was therefore used.

Whatever system is used, it is imperative that, whenever possible,

the receptor regulating a physiological or biochemical function be

defined, and that the kinetics of the interaction with the agonist

and antagonist be described. Agonists with high selectivity for a

specific receptor, e.g., isoproterenol for the ß-adrenergic receptor

or dimaprit for the histamine H2-receptor, are useful but uncommon

tools. The receptor may be defined by measuring the relative potencies of a series of agonists. For example, histamine stimulates

rat gastric acid secretion, relaxes the rat uterus, and increases

guinea pig atria1 rate. The agonist activities relative to histamine of a series of agonists on these effects were indistinguishable, which suggested that the receptors were the same or at least

very similar (Ash and Schild 1966; Black et al. 1972): they were

classified as histamine H2-receptors. Earlier, the relative potencies of agonists in contracting the guinea pig ileum and the rat

stomach were in close agreement, implying that the receptors were

very similar or the same: they were classified as histamine Hlreceptors. Such classifications demand that the slope of the doseresponse curves for each agonist be the same; that all agonists

39
produce the same level of maximum response, differing only in affinity; and that the time of onset and offset of each agonist be

similar. These criteria were met in a study on the relative potencies of a series of tryptamines in contracting the isolated stomach

strip of the rat, an effect blocked by LSD and its 2-bromo analogue;

the relative potencies paralleled their potencies in blocking LSD

binding to brain membranes (Green et al. 1978).

While agonists have proved extremely useful in helping to classify

receptors, they may yield discordant results. For example, the

potency of dimaprit, an H2-agonist, varies as much as fiftyfold

relative to histamine on different H2-receptors (Durant, Ganellin,

and Parson 1975; Parsons et al. 1977). The use of competitive

antagouists has provided less ambiguity in the classification of

receptors. The effect of a substance directly on the tissue respouse, e.g., a direct action on muscle contraction is not annulled

by a competitive antagonist, while the effect mediated by activation

of the receptor is. Furthermore, the displacement of the doseresponse curves by the antagonist is not dependent on the affinity

of the agonist. Parallel displacement of the log dose-response

curve by the antagonist suggests that the agonist and antagonist

are acting on the same receptor. From the ED50 values in the

presence and absence of antagonist, one can estimate the apparent

dissociation constant, KB, by estimating the concentration of antagonist required to give a dose ratio of two, i.e., the concentration of antagonist that requires doubling the concentration of

agonist to obtain the same effect as obtained in the absence of

antagonist. A more rigorous way to classify a receptor is to measure the displacement of the dose-response curves of an agonist by

different concentrations of the antagonists. Antagonism is expressed by the dose-ratios (DR) of agonist needed to produce equal

responses, e.g., the ED50, in the presence and absence of different

concentrations of antagonists (B). Simple competitive antagonism

results in a straight line of 1 when log (DR-1) is plotted against

log (B); the intercept with the abscissa is -log KB, which is

referred to as pA2 (Schild 1947; Arunlakshana and Schild 1959). Yet

more reassuring is to estimate the pA2 value of an antagonist with

different agonists. The Hl-receptor (Ash and Schild 1966) and the

H2-receptor (Black et al. 1972) were defined in this way. Agonists

with very different relative potencies yielded indistinguishable

pA2 values. The use of more than one agonist - and of more than

one antagonist as well- can help to avoid false inferences due to

adventitious effects, e.g., effects on release, uptake, metabolizing enzymes - any of which can vitiate the estimate of pA2.

A judicious and mindful use of these methods, this "taxonomy of

receptors" (Black 1976), has led to order and understanding and

fecundity. It yields new drugs such as the ß-adrenergic blockers

and, more recently, cimetidine, the histamine H2-antagonist which

is proving so effective in the treatmeut of duodenal ulcer. This

and other H2-antagonists were then used as tools to reveal the H2-

receptor and its associated functions in many tissues (Chand and

Eyre 1975) and the action of known drugs on this receptor - one

consequence of which was the discovery of new H2-antagonists as

described below. Knowing that a receptor for a mediator is

40
Table 1. Apparent dissociation constants (KB) & pA2 values of

histamine H2-antagonists on adenylate cyclase in the guinea pig

hippocampus homogenates compared with pA2 values on pharmacological preparations

Adenylate cyclase activity H2-receptor in

pharmacological

Antagonists Hippocampus (guinea pig) preparations*

ImidazolylpropylK B

4.68 × 10-4

pA2 pA2

3.33 3.50

methylthiourea

N

α

-guanylhistamine 7.94 × 10-5 4.14 3.90

Imidazolylpropyl- 3.16 × 10-6 5.50 4.65

guanidine

Thiaburimamide 2.39 × 10-6

8.71 × 10-7

5.62 5.50

Metiamide

6.03 × 10-7

6.06

Cimetidine 6.22

*Guinea pig atrium and rat uterus (Ganellin 1978)

6.03

6.10

homogeneous in different tissues provides prediction of side-effects

and contraindications, e.g., that ß-adrenergic blockers would be

contraindicated in patients with asthma. Showing that the receptor

for a mediator in different tissues is homogeneous provides convenience: the search for antagonists of histamine at the H2-receptor

was facilitated after it was established that the receptor associated with gastric secretion was indistinguishable from that associated

with guinea pig atrial rate, for effects on the latter are more accurately and rapidly measured. Of more pervasive importance, the

demonstration of a homogeneity of a receptor in different tissues

offers the opportunity to study the receptor in a more tractable

milieu. This is salient to the subject of this discussion: the

histamine H2-receptor linked to adenylate cyclase in brain is not

distinguishable from that in the guinea pig heart, in the rat uterus

or in the parietal cells of the stomach. The cyclase system and the

peripheral systems can now be studied as a target of psychotropic

drugs. And the relationship between chemical structure and biological activity becomes amenable to study on a well defined system.

Although these procedures, which rest on receptor theory, have been

used for over fifty years, there appears to be a surprising innocence

of them. For example, it has been repeatedly asserted that the histamine stimulated adenylate cyclase in brain slices is linked to the

H1-receptor. The evidence offered to support this is that the effect

of histamine on cyclase is blocked by Hl-antagonists. In all these

studies, the Hl-antagonists were used at concentrations of 10-6 M to

10-4M. The pA2 values for the commonly used Al-antagonists, pyrilamine (also called mepyramine) and tripellenamine, are 9.4 and 8.5

respectively or, in KB values, 4 × 10-10 M and 3.2 × 10-9 M. Testing

an antagonist at a concentration greater than 100 times the KB is

likely to block more than the specific receptor. Non-specificity of

many antagonists at high doses is well known; the H1-antagonists in

suitable concentrations are anticholinergic (see Nauta and Rekker

1978; van den Brink and Lien 1978). More to the point, at concentrations of 10-6 M and greater, H1-antagonists block the H2-receptor

41
Table 2. Effect of histamine on adenylate cyclase activity in

fresh membranes from different regions of the guinea pig brain*

linked to adenylate cyclase in homogenates of both brain and heart

ventricle of the guinea pig (see below).

The H2-antagonists have lower affinity for their receptor than have

the Hl-antagonists for the H1-receptor. Cimetidine has a pA2 value

of 6.10 (KB = 8 x l0-5 M). Clearly, a higher concentration of H2-

antagonist, e.g., cimetidine, is needed to block the H2-receptor,

than the concentration of Hl-antagonist needed to block the Hlreceptor. Yet in some work purporting to define a receptor, equal

concentrations of each type of antagonist is used, commonly 10-6 M,

which is near the KB of the H2-antagonist but a thousand times the

KB of the H1-antagonist. Not surprisingly, at these concentrations

the H2-antagonist has no or slight effect while the H1-antagonist

blocks histamine, prompting the (false) inference that the activity

is linked to the H1-receptor. Confusion can also result from the

use of only one concentration of agonist, e.g., histamine. As the

antagonism is competitive and surmountable by increasing doses of

agonist, one dose of agonist is unrevealing, for if the concentration is too high, it will surmount the effect of the antagonist,

as shown by a glance at the dose-response curves below.

THE HISTAMINE RECEPTOR IN BRAIN

Parallel displacement of the dose-response curves to histamine by

metiamide, an H2-antagonist, indicated that histamine-stimulated

adenylate cyclase in guinea pig brain homogenates is linked to an

H2-receptor. The pA2 of metiamide on this system was the same as

that on known H2-systems. The relative potencies of two other

agonists, 4-methylhistamine and 2-methylhistamiae, were also consonant with stimulation of the H2-receptor (Hegstrand, Kanof, and

Greengard 1976). This conclusion was supported by observations on

the same preparation with additional agonists and six H2-antagonists. The pA2 values of the six antagonists (Green et al. 1977)

were very similar to those on the H2-receptors in pharmacological

42
preparations (see Ganellin 1978) and on the H2-receptor linked to

adenylate cyclaae in guinea pig cardiac ventricle (C.L. Johnson,

unpublished work). Table 1 shows the results on the guinea pig

hippocampus. Simple competitive antagonism was further shown by

a Schild plot with cimetidine as antagonist which resulted in a

straight line of slope 1. When dimaprit was used as agonist instead of histamine, the points fell on the same line (Green et al.

1977). Dimaprit is virtually an exclusive H2-agonist, having less

than 0.0001 per cent of the activity of histamine on H1-receptors

(Parsons et al. 1977). Homogenates of the cerebral cortex and

hippocampus were not distinguishable in their response to agonists

and antagonists. It is thus clear that histamine stimulated adenylate cyclase in homogenates of guinea pig hippocampus and cortex is

linked to an H2-receptor. (No extrapolations can be made to brain

slices.)

Table 2 shows that the histamine stimulated adenylate cyclase activity is more sensitive in homogenates of the cerebral cortex and

hippocampus than in other parts of the brain. Neither of these

regions is especially rich in histamine. This distribution confirms previous findings (Hegstrand, Kanof, and Greengard 1976),

except that we showed activity in the hypothalamus and central gray.

In the cortex of guinea pigs, activity was highest in subcellular

fractions containing nerve endings (Kanof, Hegstrand, and Greengard

1977). The activity was present in homogenates of the rat hippocampus (Green et al. 1977) and the rat cortex (Hegstrand, Kanof,

and Greengard 1976), but the corresponding portions of guinea pig

brain were more sensitive.

It may be worth noting that adenylate cyclase in the hippocampus

homogenates is more sensitive to histamine than to norepinephrine

and dopamine. At a concentration of 10-4 M, the respective percentage increases were 107.5, 37.0, and 24.7. Cimetidine, 10-4 M,

reduced histamine activation by 65.5 per cent without reducing

activation by the catecholamines (Green et al. 1977).

The H1-antagonists also antagonized histamine stimulated adenylate

cyclase, as shown for cyproheptadine (Fig. 1). But to do so required

concentrations of pyrilamine and tripellennamine about a thousand

times those required to block the H1-receptor. For cyproheptadine,

the corresponding ratio was ten times (Table 3). These differences

in pA2 values show that the H1-antagonists were not blocking the

H1-receptor linked to cyclase. Further, the H1-antagonists blocked

stimulation of the cyclase by dimaprit which is selective for the

H2-receptor and is virtually devoid of activity on the H1-receptor.

Hence, the H1-antagonists at high concentration block the H2-receptor.

Cyproheptadine has greater affinity for the H2-receptor (KB = 3.7 ×

10-8 M) than any substance yet described, including the H2-antagonists

that have been used clinically, metiamide and cimetidine.

CYPROHEPTADINE AS ANTAGONIST OF MANY RECEPTORS

Although often called a "serotonin antagonist", cyproheptadine has

no special penchant for serotonin receptors. Table 4 shows the KB

43
Fig. 1. Adenylate cyclase activity of the guinea pig hippocampus

in response to varying concentrations of histamine in the absence

(D) and presence of 10-7 M (•)or 10-6 M (o) cyproheptadine. Each

point represents the increase in adenylate cyclase activity above

basal level (no histamine) and is the mean of triplicate determinations on a single enzyme preparation.

(and pA2) values of cyproheptadine on different receptors, listed

in order of affinity. It has highest affinity for the H1- and H2-

receptors and least for the serotonin receptor. The values for

the serotonin and LSD receptors were estimated from published IC50

values as noted in the table. Cyproheptadine also antagonizes

acetylcholine (Rocha e Silva and Leme 1972) and epinephrine (Stone

et al. 1961).

COMPETITIVE BLOCKADE OF THE H2-RECEPTOR BY LSD AND D-2-BROMOLYSERGIC

ACID DIETHYLAMIDE (BrLSD)

Because cyproheptadine has high affinity for the H2-receptor and

it blocks 5-HT, it seemed appropriate to test LSD which also has

affinity for 5-HT receptors. LSD alone did not increase adenylate

cyclase activity in homogenates of the guinea pig hippocampus and

cortex [as it does in rat striatum (Von Hungen, Roberts, and Hill

1975)]. But it blocked histamine or dimaprit stimulated cyclase

in a competitive number as shown by a Schild plot. The pA2 value

of LSD on hippocampal homogenates was 5.95, on cortical homogenates

6.07, not significantly different. This value is very nearly that

of cimetidine, 6.22, and metiamide, 6.06, the two potent and

selective H2-antagonists. Inactive in this system were L-LSD,

44
Table 3. The pA2 values of histamine H1 antagonists on the

histamine H2 receptor linked to adenylate cyclase in guinea

pig hippocampus homogenates compared with the pA2 values on

the H1 receptor in guinea pig ileum

pA2 on H2 linked

adenylate cyclase

activity:

hippocampus,

pA2 on H1 receptor:

ileum,

Antagonist guinea pig

Pyrilamine 5.18

Tripellenamine 5.4

guinea pig

9.4*

8.5†

Cyproheptadine 7.43 8.3‡

*Arunlakshana and Schild 1959

†Marshall 1955

‡Rocha e Silva and Garcia Leme 1972

psilocin and mescaline, all at 10-4 M. But Br-LSD was a competitive antagonist, as shown by the Schild plot. Its pA2 value was

7.16, thus showing an affinity about tenfold that of either LSD,

cimetidine, or metiamide.

PREDICTION OF A NEW H2-ANTAGONIST

There is no ostensible similarity in chemical structure between

LSD (or BrLSD) and the selective H2-antagonists when they are

drawn in the conventional way (Fig. 2). But one of the likely

conformers of metiamide and cimetidine has a topological congruency with LSD, as shown for cimetidine in Fig. 3. The molecules neatly superimpose, the oxygen of the amide group of LSD

being congruent with the nitrogen of the cyano group of cimetidine or the sulfur atom of the throurea group in metiamide. After

making this observation, we wrote:

The residue in D-LSD that is sterically congruent with

metiamide is hexanoic acid-4-aza-4-methyl-6-(3-pyrrolyl)-

N,N-diethylamide. This and the indolyl and imidazol

analogs and their derivatives may have H2-antagonists

activity (Green et al. 1977).

We tested hexanoic acid-4-aza-4,5-dimethyl-6-(3-indolyl)-N,Ndiethylamide (SK&F 10856), obtained from Dr. Carl Kaiser. The

structure is shown (Fig. 4). A Schild plot proved it to be a competitive antagonist of histamine and dimaprit at the H2-receptor

linked to adenylate cyclase, with pA2 = 6.10 (KB = 7.9 x 10-7 M),

not different from that of LSD. We plan to test the pyrrolyl and

imidazoyl analogs.

THE H2-RECEPTOR AND THE PHARMACOLOGY OF LSD AND BrLSD

Both LSD and BrLSD are competitive antagonists of histamine at the

H2-receptor linked to adenylate cyclaee in guinea pig hippocampus

and cortex. The pA2 value of D-LSD was 6.0, very nearly that of

cimetidine, 6.22, and of metiamide, 6.06, the two potent H2-antagonists. The L-isomer, which has no measureable central effects, did

45
Fig. 2. The structural formulas of LSD, metiamide, and cimetidine

46
Fig. 3. Comparison ofthe molecular structures of LSD and

cimetidine

47
Table 4. Estimated KB and pA2 values of cyproheptadine for

different receptors

Receptor

Histamine H1*

Histamine H2†

LSD ‡

Bradykinin*

Haloperidol§

KB(M)

5 × 10-9

8.3

3.7 × 10-8

p A 2

7.4

5.0 × 10-8 7.3

6.3 × 10-8 7.2

Angiotensin*

6.5 × 10-8

2.5 × 10-7

Dopamine§ 1.0 × 10-6

Serotonin‡ 1.6 × 10-6

*Rocha e Silva and Leme 1972

†Green et al. 1977

7.2

6.6

6.0

5.8

‡Estimated from IC50 values of high affinity binding by rat

cortex, from Bennett and Snyder 1976; according to:

KB = IC50/1 + [A]/KA where [A] is the agonist concentration

and KA is the dissociation conetant of the agonist

§Creese, Burt and Snyder 1976

not antagonize the histamine-activated adenylate cyclase. BrLSD

(pA2=7.2) was about ten times more potent than D-LSD (or cimetidine)

in inhibiting histamine-stimulated adenylate cyclase activity

(Green et al. 1977).

The affinity of LSD for the H2-receptor linked to adenylate cyclase

cannot be directly compared with its affinities for other receptors

linked to adenylate cyclase in brain because the pA2 values are not

available. From Table 5 it would appear that LSD and BrLSD may be

more effective in blocking histamine stimulated cyclase than in

blocking norepinephrine or dopamine stimulated cyclase.

However, the pA2 values (Table 6) show that LSD has a greater affinity for the dopamine receptor then for the H2-receptor. LSD has

an even greater affinity for the serotonin receptor. BrLSD has the

came pA2 values for the serotonin, dopamine and H2-receptors. And

it has a greater affinity than LSD for both the haloperidol binding

sites and for the H2-receptor. Other work has shown that LSD and

BrLSD have the same affinity for LSD binding sites in cortex

(Bennett and Snyder, 1976).

Although adenylate cyclase measurements suggest that BrLSD has a

greater affinity for the dopamine receptor (Table 5), high

affinity binding studies of dopamine show that BrLSD has slightly

less affinity than LSD for the dopamine binding sites (Creese, Burt,

and Snyder, 1976; Table 6). This paradox suggests the presence of

dopamine receptors that are not linked to cyclase, a receptor that

has a greater affinity for LSD than for BrLSD. LSD, is contrast

with BrLSD, is an agonist as well as an antagonist at striatal

receptors linked to cyclase (Von Hungen, Roberts, and Hill 1975;

Bockaert et al. 1976). Perhaps BrLSD has leas affinity for another

(presynaptic?) dopamine receptor. BrLSD has about five times more

affinity for haloperidol binding sites than has LSD (Creese, Burt,

and Snyder 1976). As haloperidol binds to the postsynaptic dopaminergic receptors, it was inferred that BrLSD is a better dopamine

48
Fig. 4. The structure of hexanoic acid-4-aza-4,5-dimethyl-6-

(3-indolyl-N,N-diethylamide. It is shown in a conformation

congruent with LSD and cimetidine in fig. 3.

antagonist than is LSD. Supporting this inference are experiments

in rat striatum showing that BrLSD is more potent than LSD in increasing the lwels of DOPA (Persson 1977a) and of a dopamine metabolite, 3,4-dihydroxyphenylacetic acid (Persson 1977b). All

these biochemical studies are consistent with observations on unit

cell activity of the subatantia nigra, where LSD was more effective

than BrLSD in decreasing the firing rate of dopamine-containing

neurons (Trulson, Stark, and Jacobs 1977; Christoph, Kuhn, and

Jacobs, 1977). Perhaps correlating with this difference in affinity for postsynaptic dopamine receptors is that head twitches induced in mice by LSD (Corne and Pickering 1967) and LSD stimulated

striatal cyclase (Von Hungen, Roberts, and Hill 1975) were blocked

by BrLSD.

Agonist activity of LSD is also greater than that of BrLSD on the

flexor reflex of the dog. LSD, like tryptamine, enhanced the

flexor reflex (Martin and Sloan 1974), but a dose of BrLSD 100

49
Table 5. Comparative effects of LSD, L-LSD, and BrLSD in blocking

the adenylate cyclase activity in broken cell preparations,

stimulated by histamine, norepinephrine and dopamine, all at

10-4 M. Values are percentage inhibition.

Drug Histamine* Norepinephrine† Dopamine

LSD, 10-4 M - 75 81†

LSD, 10-5 M 56 - -

L-LSD, 10-4 M 0 - 0‡

BrLSD, 10-4 M - 90 98†

BrLSD, 10-5 M 97 - -

*Guinea pig cortex and hippocampus (Green et al. 1977)

†Rat cortex (Von Hungen, Roberts, and Hill 1975)

‡Rat striatum (Von Hungen, Roberts, and Hill 1975)

times higher did not (W.R. Martin, personal communication).

The differences between the pA2 values for LSD and BrLSD on the

serotonin receptors could also reflect a difference between

affinities for presynaptic (agonist) and postsynaptic (antagonist)

serotonin sites. LSD has greater affinity for the serotonin receptor than does BrLSD. LSD, like serotonin, completely inhibits the

firing of neurons in the midbrain raphe at low doses (10 μg/kg)

whereas BrLSD only partially inhibits these neurons even at a dose

200 times greater (Aghajanian, Foote, and Sheard 1970). Microiontophoretically applied BrLSD also failed to totally inhibit the

firing (Aghajanian 1976). Both BrLSD and LSD inhibited postsynaptic

sites on the ventral lateral geniculate and amygdala (Aghajanian

1976). Some of this work recalls studies of peripheral systems.

LSD and BrLSD are about equally potent antagonists at the postsynaptic serotonin receptors in the rat uterus (Cerletti and Doepfner

1958) and stomach (Vane 1959). On one peripheral tissue, the

effects of LSD and BrLSD are very neatly defined. LSD is a serotonin agonist in human and sheep umbilical vasculature, and BrLSD

is an antagonist of both LSD and serotonin (Dyer and Gant 1973).

It thus seems clear from studies on neural tissue using biochemical

and eleetrophysiological techniques that LSD and BrLSD have affinities for the same receptors and that LSD has greater activity at

(and perhaps a greater affinity for) functionally "presynaptic"

sites, and BrLSD has greater affinity for some "postsynaptic" sites.

The sites that LSD and BrLSD share are very likely to be implicated

in the hallucinogenic action of LSD. For BrLSD blocks the hallucinogenic effect of LSD in man (Ginzel and Mayer-Gross 1956; Bertino,

Klee, and Weintraub 1959) at a dosage interval before cross-tolerance

is seen (Isbell, Miner, and Logan 1959). The effect of a dose of LSD

of 1 μg/kg was blocked by a dose of BrLSD of 32 to 64 μg/kg (Bertino,

Klee, and Weintraub 1959).

Perhaps of greater interest is that BrLSD causes behavioral changes

in laboratory animals and in man. Larger doses of BrLSD than LSD

are needed to show changes in animal behavioral paradigms but they

occur (e.g., Uyeno 1968; Uyeno and Mitoma 1969). In man there is

one report of hallucinations occurring after ingestion of 0.5 mg of

5 0
Table 6. pA2 values of LSD and BrLSD for different receptors

Receptor LSD BrLSD

Serotonin* 8.3 7.2

Haloperidol†

7.7 8.4

Dopamine†

Histamine-H2

‡

7.5 7.2

6.0 7.2

*Estimated from IC50 value of high affinity binding by rat cortex,

from Bennett and Snyder 1976; according to: KB = IC50/1 + [A]/KA

where [A] is the agonist concentration and KA is the dissociation

constant of the agonist

†Creese, Burt, and Snyder 1977

‡Green et al. 1977

BrLSD (Richards et al. 1958). Others have observed severe psychic

effects without hallucinations. Bertino, Klee. and Weintraub (1959)

wrote:

Six of the 10 subjects receiving 128 or 256 μg./Kg. exhibited mental effects. These consisted of a "drunk

feeling," "things seem bright," "devil-may-care attitude," "a feeling of tiredness, euphoria, anxiety, and

impaired concentration."

Psychophysiological effects of 2-brom-LSD were reported

by subjects at dosages as low as 32 μg./Kg. and increased

in severity with increasing dosage. The psychic effects,

though milder, were qualitatively the same as those with

lysergic acid diethylamide (LSD-25).

Schneckloth et al. (1957) wrote:

Thus, when constant intravenous infusions of bromo-LSD

were given to 2 normal subjects (cases 5 and 6) both

experienced psychic changes, which became more severe

as the infusion continued and persisted for 3 to 4

hours after the infusion was stopped. No hallucinations

were noted but there were feelings initially of drowsiness, depression, anxiety and apprehension, followed by

feelings of irritation, restlessness, and tenseness, and

later, intensely disagreeable sensations of unreality

and depersonalization, inexplicable feelings of strangeness and mild confusion.

What contribution blockade of the histamine H2-receptor makes to the

behavioral effects of BrLSD and LSD is not known. The availability

of an H2-antagonist that penetrates the brain could lead to understanding. Cimetidine, an H2-receptor antagonist, which is now

being used in humans, does not normally enter brain (Ganellin 1978).

It has, however, produced rare and idiosyncratic responses that may

be revealing of central function. There are reports of mental confusion after cimetidine - patients described as agitated, delirious,

disoriented - all rapidly reversed when the drug was withdrawn

(Delaney and Ravey 1977; Robinson and Mulligan 1977; Menzies-Gow

1977; Nelson 1977; McMillen et al. 1978; Quap l978). Reports of

51
fever after cimetidine (Ramboer 1978; McLoughlin, Callender, and

Love 1978) also recall that LSD raises temperature in humans

(Gorodetzky and Isbell 1964; Klock, Boerner, and Becker 1975) and

rabbit (Carino and Horita 1977); in some animals, H2-receptor

stimulation causes hypothermia (Cox and Lomax 1977). Further

attesting to central effects is that increased plasma prolactin

levels have been observed especially when the drug is given by

intravenous injection (Della Pave et al. 1977; Carlson and Ippoliti

1977; Burland et al. 1978; Daubresse, Meunier and Ligny 1978);

animal studies have suggested that the histamine H2-receptor mediates events that inhibit prolactin release (Arakelian and Libertun

1977).

The H1-antagonists at suitable concentrations interact with both

H2- (Table 3) and cholinergic receptors (e.g., Nauta and Rekker

1978; van den Brink and Lien 1978). It is therefore risky to

attribute their behavioral effects to blockade of any one reeeptor.

It may be interesting to note, however, that H1-antagonists produce arousal in dogs (Shaw, Gershon, and Bentley 1957; Gershon and

Shaw 1958). In man, high dose of the H1-antagonists have produced

disorientation, euphoria (Gott 1968), visual hallucinations

(Soleymanikashi and Weiss 1970), and behavior characterized as

schizophrenic-like (Nigro 1968). Diphenhydramine, an H1-antagonist,

on intravenous administration (30 or 45 mg to each male subject)

produced disturbance of body image and thought. When administered

at the height of the LSD reaction (25 μg of LSD by mouth),

diphenhydramine intensified most of the LSD effects - especially

hallucinations, depersonalization, and disturbances of body image

and thinking - without prolonging the effects (Yamada, Tsunoda,

and Takashina 1957).

It is becoming increasingly clear that the complex behavioral and

neural effects of LSD or BrLSD cannot be explained on the basis of

any single effect on any single neurophysiological system or site.

The effect elicited at the lowest dose in the rat is a decreased

firing of the raphe nuclei (Aghajanian 1976). This effect cannot

explain the behavioral effect of LSD (Freedman and Halaris 1978;

Trulson, Ross, and Jacobs 1977; Pieri et al. 1978). In humans,

LSD - and, as quoted above, BrLSD - produce an array of neural

effects, which range from activation of the galvanic skin response

at the lowest dose (Greiner, Burch, and Edelberg, 1958). a sign

of neurophysiological arousal; to marked changes in perception,

affect, thought, body image; perseveration; heightened awareness;

depersonalization; illusions and hallucinations (Freedman, 1968;

Mandell and Geyer, 1976). One effect, hallucinations, is complex

enough (Editorial, 1977). It is very unlikely that all the effects can be attributed to action at one receptor at one site.

How the visual cortex processes a spot of light is still not

known, but it is clear that the

response properties of most neurones depend on subtle interactions within an intricate system of more

and less specific excitatory and inhibitory influences, whose cooperative details determine most aspects

of neuronal behaviour (Movshon 1978).

52
This complexity must apply to the action of drugs, which have access to all neurons and all receptors. Almost certainly the behavioral effects produced by any drug, e.g., LSD, BrLSD, are the

final consequences of a conflation of events occurring at different neural sites and by different mechanisms. The basis for the

neural and behavioral effects will be learnt after the receptor

sites at which these drugs act are explicitly and rigorously

delineated. Then will follow the concatenation of the neural

and behavioral events.

SUMMARY

Two aspects of the complexities of the mode of action of drugs

are described. One is the criteria and pitfalls of defining the

interaction with specific receptors. The other is the need to

consider each of the pharmacological effects of a drug as a

concatenation of receptor events, because it has become clear that

each drug may have substantial affinity for many specific receptors.

Illustrating these ideas is a characterization of the histamine

receptor linked to adenylate cyclase in brain. The activities of

a series of H2-antagonists and H2-agonists were shown to be the

same on the histamine receptor linked to adenylate cyclase as on

known H2-receptors. The KB values of antagonists and ED50 values

of agonists were not distinguishable among these receptors.

Notably, at high concentrations, the H1-antagonists are also competitive antagonists of the H2-receptor.

Cyproheptadine has especially high affinity for the H2-receptor.

It is the most potent H2-antagonist yet reported. Other published

results are reviewed to show the variety of receptors that cyproheptadine has affinity for. Its affinity for serotonin receptors

led us to examine other serotonin antagonists.

On this H2-receptor linked to adenylate cyclase in homogenates of

guinea pig hippocampus and cortex, D-LSD and D-2-bromo-LSD (BrLSD)

were shown to be competitive antagonists of histamine. L-LSD,

mescaline and psilocin were inactive.

Noting congruency in the molecular structure of D-LSD and known

H2-antagonists, we predicted a new H2-antagonist. This prediction

is shown to be correct: the compound has similar affinity to the

H2-receptor as has LSD.

The affinities of D-LSD and BrLSD for the H2-receptor are compared

with their affinities for other receptors. The pharmacology of

D-LSD and BrLSD is reviewed. Evidence is assembled that BrLSD has

considerable central effects.

ACKNOWLEDGMENTS

This work was supported by the National Institute on Drug Abuse

(DA 01875). The analysis of the results of this work were carried

out on the PROPHET System, a national computer resource sponsored

53
by the NIH. H.W. is recipient of a Career Scientist Award of the

Irma T. Hirschl Trust. We are grateful to Dr. Carl Kaiser for the

gift of SK&F 10856. We thank Don and Sanford Rosenthal for library

work.

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AUTHORS

Jack Peter Green, M.D., Ph.D.

Harel Weinstein, D.Sc.

Saul Maayani, Ph.D.

Department of Pharmacology

Mount Sinai School of Medicine

of the City University of New York

New York, New York 10029

59
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

The Nature of Opioid and LSD

Receptors: Structural Activity

Relationship Implications

William R. Martin

The structure activity relationships of the drugs under consideration are inexorably entwined with receptors and receptor

theory since they have been clearly demonstrated to have agonistic

and antagonistic activity. It also appears that their pharmacologic

effects are in some way related to neurotransmitters and neuromodulators. The discovery of the endorphins and enkephalins and the

similarities of some of their effects to those of the narcotic

analgesics has given rise to a most exciting chapter of pharmacology.

Over a period of nearly three decades, a pharmacology of the LSDlike hallucinogens has been emerging which suggests that they too

may interact with several receptors. In this presentation, I wish

to discuss some concepts that are emerging concerning receptor topology and drug selectivity which bear directly on receptor classification and which have relevance to SAR of these two groups of drugs.

NARCOTIC ANALGESICS

A number of investigators have studied the SAR of narcotic analgesics. For the most part, the data on which these studies have been

based have been generated in several species using different

measures of pharmacologic effects such as analgesia. Table 1

presents data on seven critical drugs that have great theoretical

significance in discussing their structural activity relationships.

Morphine is used as a standard drug against which the others are

compared. However, it should be recognized that morphine may have

several modes of action and interact with more than one receptor

(Martin et al. 1976; Gilbert and Martin 1976).

Normorphine is a weak analgesic in the mouse, is l/6 to l/3 as

potent as morphine in man and in binding assays, equipotent to

morphine on the guinea pig ileum but devoid of morphine like

activity in the dog.

Metazocine is an agonist approximately equipotent to morphine in the

muse, dog and man. It is clearly a strong agonist in the dog, but

has been characterized as an agonist-antagonist by Pert and Snyder

(1974) on the basis of data from binding studies having a relatively

low NaCl/no NaCl ratio (6).

Phenazocine is some 10 times more potent than morphine on measures

6 0
presented in Table 1, yet was only 1/5 as potent as morphine in

suppressing abstinence in the monkey.

N-allyl normetazocine (SKF 10047) is virtually devoid of analgesic

activity but produces delirium and hallucinations in man and

delirium in the dog. These effects can be antagonized by naloxone.

Meperidine is an effective analgesic in man and the mouse; is much

less potent relative to morphine on the guinea pig ileum and in

binding studies. In man it will partially suppress abstinence in

morphine dependent subjects (Himmelsbach 1942). Further, it has

a limited ability to produce physical dependence in man (Himmelsbach

1942, 1943). Of particular interest is the observation that

nalorphine was nearly ineffective in precipitating abstinence in

meperidine dependent patients (Isbell 1955). In the dog, meperidine

is devoid of morphine-like activity (Gilbert and Martin 1976) and

will not produce physical dependence (Carter and Wikler 1955).

Although it is relatively impotent in inhibiting the electrically

stimulated guinea pig ileum and in preventing binding of naloxone

to rat brain homogenate, it is qualitatively similar to morphine

in these preparations.

These data have been analyzed by calculating correlation coefficients

between the various measures and this analysis is presented in

Table 2. As can be seen, the only significant regression was between

the mouse and dog. Many of the correlation coefficients were

highly significant which stresses the weakness of this technique for

analysis of this type of data.

LSD-LIKE HALLUCINOGENS

There are both theoretical and practical problems of defining the

group of drugs considered LSD-like hallucinogens. The discussion of

these drugs will be restricted largely to data generated in the spinal

dog, man and rat. An LSD-like hallucinogen should, of course, have

pharmacologic activity like LSD. The effects of LSD can be divided

into several categories including: (1) Somatomotor and autonomic;

facilitation of the patellar (man) and flexor reflex (spinal dog),

evocation of the stepping reflex (spinal dog), tachycardia (dog and

man); increased blood pressure (man); mydriasis (man and dog),

tachypnea (man and dog) and increased body temperature (man and dog).

(2) Subjective and behavioral changes; euphoria, delusion and

hallucinations (man); arousal, restlessness, tracking and starting

(dog) and the alteration of operant behavior (rat). (3) The development of tolerance and the conferring of cross tolerance. (4) The

blocking of the effects with more or less specific antagonists.

Thus chlorpromazine and cyproheptadine will antagonize this effect

of LSD-like hallucinogens but not phenoxybenzamine.

As can be imagined the permutations of these attributes are large

and from a practical point of view resources allow only the study

of certain ones. Because of differences in biases of investigators,

certain aspects of the action of certain putative LSD-like

hallucinogens are focused on. For this reason, very little quantitative comparable data has been generated which allows hard

61
TABLE 1

POTENCY OF SEVERAL OPIOID AGONISTS AND ANTAGONISTS

ON DIFFERENT TEST SYSTEMS

62
TABLE 2

PRODUCT MOMENT CORRELATION COFFICIENTS (r), SLOPES (b) WITH STANDARD

ERROR OF ESTIMATE (sy,x) FOR DATA PRESENTED IN TABLE 1
comparisons which are useful for SAR considerations. At the

present time, it seems that LSD-like hallucinogens may act through

at least two mechanisms of action; a tryptaminergic and serotonergic

mechanism (Martin and Sloan 1977). The relative importance of these

two mechanisms in mediating different signs and systems has not

been ascertained at this time.

Table 3 and table 4 present comparisons of the effects of several

important drugs that are related in one way or another to LSD.

LSD is the prototypic drug against which the others are compared.

Psilocin and its phosphorylated congener, psilcybin, appear to be

very similar to LSD in their pharmacology. However, there is a

marked difference between the potency of psilocin in man and its

ability to prevent the saturable binding of 5HT and LSD in rat

brain homogenate on the one hand and in the dog on the other.

Further, LSD tolerant rats were not cross tolerant to psilocin. The

time course of brain concentrationhas not been studied (or even

plasma level) in these species so differences in distribution,

metabolism or excretion cannot be excluded as reasons for differences

in potency. Since both LSD and psilocin have a rapid onset and a

prolonged duration of action, differences in distribution, metabolism

or excretion probably do not explain the differences in potency in

different test systems. Differences in affinity between psilocin

and LSD for the receptor are another possibility which, if true,

could indicate a subtle species difference in receptor topography.

DMT is less potent in man than it is in the dog. Further, cross

tolerance to DMT in both LSD tolerant man and chronic spinal dogs

is not complete and is seen for certain signs and symptoms but not

for others.

A critical drug is BOL. It is questionable whether it can produce

LSD-like effects in man. Chronically administered BOL conferred

some cross tolerance to LSD in man (Isbell et al. 1959). In doses

up to 1 mg/kg intravenously BOL is devoid of LSD-like activity. In

the rat, BOL inhibits operant behavior and tolerance develops to

this effect with chronic administration (Appel and Freedman 1968).

Whether the BOL tolerant rat is cross tolerant to LSD cannot be

answered. Some tolerance was seen but it was not statistically

significant. The LSD tolerant rat may exhibit some tolerance to

BOL but it too was not statistically significant. BOL on the other

hand is nearly as potent as LSD in inhibiting the binding of both

LSD and 5HT to the brain. BOL is neither an agonist or antagonist

in man (Isbell et al. 1959).

DISCUSSION

The first point that these data suggest is that there are small but

significant differences in receptors between species. To illustrate

this point for both the opioid-like analgesics and LSD-like

hallucinogens the following contrasts are reviewed:

(1) Normorphine appears to resemble morphine in the mouse and

guinea pig ileum but is devoid of morphine activity in the dog.

64
TABLE 3

POTENCY OF SEVERAL LSD-LIKE HALLUCINOGENS IN MAN, DOG AND RAT

AS WELL AS THEIR ABILITY TO INDUCE TOLERANCE AND CROSS TOLERANCE
TABLE 4

PRODUCT MOMENT CORRELATION COEFFICIENTS (r), SLOPES (b) WITH

STANDARD ERROR OF ESTIMATE (sy,x) FOR DATA PRESENTED IN TABLE 3
(2) Metazocine is a typical morphine-like drug in man, dog, mouse

and guinea pig ileum but appears to resemble other agonists-antagonists in rat brain binding studies.

(3) Phenazocine is a very potent morphine-like agonist in man and

dog, but a much weaker and less effective agonist in the monkey.

(4) N-allylnormetazocine (SKP 10047) is devoid of morphine-like

activity in the mouse, dog and man, but is equipotent to morphine

on the guinea pig ileum.

(5) BOL produces effects similar to LSD in the rat but is nearly

if not totally devoid of LSD-like activity in the man and the dog.

The most probable explanation of these differences is that the

receptors differ in their intimate details from one preparation to

another. The critical dimension could be species or tissue (e.g.

brain vs. gut) or even different brain regions or different

functional systems. It may thus be important in SAR studies to

treat species or even varieties as an independent variable.

Perhaps the next level of receptor classification should be subspecies. Recently there has been increasing evidence that there

are subspecies of opioid receptors (Martin 1967; Martin et al. 1976;

Lord et al. 1977). I suspect that it will be demonstrated that

there are subspecies of the serotonin and tryptamine receptors also

and that these subspecies may also be in different functional systems.

Thus with regard to the opioid receptors, μ agonists produce one

type of analgesia as measured by the tail flick or hot plate while

κ agonists produce another which can be measured using the writhing

test or the flexor reflex. It is important for SAR consideration

that the data be homogeneous and indicative of one pharmacologic

action. This is important because many agonists and antagonists

are mixed, having several modes of actions.

Another important attribute of agonists is their intrinsic activity.

Partial agonists of the μ type (profadol, propiram and buprenorphine)

and κ type (nalorphine) have been identified. An important attribute

of partial agonists as they relate to SAR considerations is that

potency determinations will underestimate affinity.

As we attempt to understand the relationship of the chemical structure

of drugs, the nature of receptors and pharmacologic actions, our

theories have become more sophisticated which in turn necessitates

more precise and well designed experiments.

67
REFERENCES

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among psychotomimetic drugs. Psychopharmacologia, 13:267-274, 1968.

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Carter, R.L. and Wikler, A. Chronic meperidine intoxication in

intact and chronic spinal dogs. Fed Proc, 14:955, 1955.

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nalorphine-like drugs in the non-dependent,morphine-dependent,

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198:66-82, 1976.

Himmelsbach, C.K. Further studies on the addiction liability of

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Himmelsbach, C.K. Studies of the addiction liability of "Demerol"

(D-140). J Pharmacol Exp Ther, 75:64-68, 1942.

Isbell, H.: Withdrawal symptoms in "primary" meperidine addicts.

Fed Proc, 14:354, 1955,

Isbell, H., Miner, E. J., and Logan, C. R. Cross tolerance between

d-2-Brom-lysergic acid diethylamide (BOL-148) and the D-diethylamide

of lysergic acid (LSD-25). Psychopharmacologia, 1:109-116, 1959.

Kosterlitz, H.W. and Waterfield, A. A. In vitro models in the study

of structure-activity relationships of narcotic analgesics. Annu

Rev Pharmacol, l5:29-47, 1975.

Lord, J. A.; Waterfield, A. A.; Hughes, J.; and Kosterlitz, H. W.

Endogenous opioid peptides: multiple agonists and receptors,

Nature, 267:495-499, 1977.

Martin, W. R. Opioid antagonists. Pharmacol Rev, 19:463-521, 1967.

Martin, W. R.; Eades, C.G.: Thompson, J. A.; Huppler, R. E., and

Gilbert, P.E. The effects of morphine- and nalorphine-like drugs in

the non-dependent chronic spinal dog. J Pharmacol Exp Ther, 197:517-

532, 1976.

Martin, W.R., and Sloan, J. W. Pharmacology and classification of

LSD-like hallucinogens. In: Martin, W. R., ed. Handbook of Experimental Pharmacology. Drug Addiction II, Vol. 45/11. Heidelberg:

1977, pp. 305-368.

68
Martin, W. R.; Vaupel, D. B.; Nozaki, M.; and Bright, L. D., The

identification of LSD-like hallucinogens using the chronic spinal

dog. Drug Alc Dep, 3:113-123, 1978.

Pert, C. B. and Snyder, S. H. Opiate receptor binding of agonists

and antagonists affected differentially by sodium. Mol Pharmacol

10:868-879, 1974.

Pert, C. B. and Snyder, S.H. Properties of opiate-receptor binding

in rat brain. Proc Nat Acad Sci, USA, 70(8):2243-2247, 1973.

Pert, C. B., Snyder, S. H. and May, E. L. Opiate receptor interactions of benzomorphans in rat brain homogenates. J Pharmacol Exp

Ther, 196(2):316-322, 1976;

AUTHOR

William R. Martin, M.D., Professor and Chairman, Department of

Pharmacology, University of Kentucky College of Medicine,

Lexington, Kentucky 40506

69
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic. and R. Willette, eds.

National Institute on Drug Abuse, 1978

The Use of Rigid Analogues to

Probe Hallucinogen Receptors

David E. Nichols, Herschel J. R. Weintraub,

William R. Pfister, and George K.W. Yim

Over the past several years effort in our laboratory has been

directed toward the design and synthesis of hallucinogen analogues

which would be useful as probes of the hallucinogen receptor(s).

Although this has involved some investigation of aromatic substituent effects, major efforts have been directed toward the elucidation

of the stereochemistry of side chain binding for the phenethylamine

hallucinogens. We have tried to answer the question, "how can two

relatively dissimilar structures such as LSD and mescaline act at

the same receptor, if indeed they do?" The ability to identify

functional similarities between the indolealkylamines and the phenethylamines may ultimately lead to an understanding of possible

similarities between catecholamine and indoleamine receptors and to

underlying principles of receptor activation.

As an attack on this problem, we have used the rigid analogue approach. In this context rigid analogue is construed to mean any

compound which has fewer degrees of conformational freedom than its

open chain counterpart. The design of such molecules however, can

present considerable difficulties. For example, N-alkylation of

the phenylisopropylamine (amphetamine) hallucinogens generally leads

to loss of activity (Shulgin 1973; Ho et al. 1970). Extension of

the a methyl of the phenylisopropylamines to longer alkyl homologues

likewise abolishes hallucinogenic activity (Shulgin 1963; Standridge

et al. 1976). How then is one to design appropriate rigid analogues

which avoid these deleterious changes? One must have some confidence

that this is possible since, after all, LSD is a very rigid molecule.

The real question seems to be how to make appropriate changes in the

rigid molecule which compensate for loss of activity due to amine or

side chain alkylation. At the extreme, one is talking about the synthesis of new classes of hallucinogens where structure-activity

relationships (SARs) must be sufficiently developed to allow predictive arguments to be made.

Structure activity studies involve two separate inquiries. One

seeks to determine how changes in molecular structure effect (1)

mechanism of action and (2) intensity of action, or potency. With

respect to the first point it seems likely that hallucinogens may

interact with various monoamine pathways in the CNS. Unfortunately,

70
it is impossible to correlate chemical properties with particular

biological responses when varying mechanisms of action are involved.

Entirely different structure activity relationships may define the

different mechanisms.

On the other hand, one often may make simplifying assumptions.

Hansch (1969) began by assuming that for a congeneric series, one

particular biological reaction might be crucial and could act as a

"rate determining" step. The ability to identify such a critical

step can be a very useful asset. If this is an enzyme for example,

activity can be directly measured. At the level of the whole

organism, multiple steps may distort quantitative structure activity

relationships.

We have attempted to identify functional similarities between the

various type of hallucinogens which could be related to some critical biological interaction. Based on numerous pharmacological

studies, this would appear to be an interaction with central serotonin receptors (Brawley and Duffield 1972). However, as suggested

by many workers, the overall intoxication may result from additional

interactions with central pathways involving neurotransmitters other

than serotonin (Nichols 1976).

The use of carefully designed rigid analogues can be of great utility in developing structural comparisons. For example, studies of

2-amino-1,2,3,4-tetrahydronaphthalenes (2-aminotetralins; 2-ATs)

have led to important findings about the conformational requirements

of dopamine receptors (Goldberg et al. 1977). Similar studies have

not been as effective in elucidating requirements of hallucinogen receptors. The major difficulty in developing useful analogues has

been the catch words "carefully designed" and is a result of lack

of knowledge about the receptor. One can see that this is a cyclic

paradox where design initially plays a relatively unimportant role

and active compounds are the result of trial and error sequences.

Nevertheless, the rigid analogue approach has been used with a certain degree of success. As an important example of this approach,

early workers attempted to identify similarities between LSD and

mescaline by proposing that the side chain of mescaline was able to

assume a cisoid conformation which would resemble the indole nucleus

of LSD (Snyder and Richelson 1968). Although unlikely on theoretical

grounds, the proof against such a conformation came from studies by

Cooper and Walters (1972). These workers demonstrated mescalinelike activity in animals for trans-2-(3,4,5-trimethoxyphenyl)-cyclopropylamine 1. In contrast, the cis isomer would have been expected

to be more active on the basis of the indole mimicry hypothesis.

More recently, Aldous et al. (1974) examined a series of 2-phenylcyclopropylamines as potential hallucinogens. Notable in this study

were the results obtained for trans-2-(2,5-dimethoxy-4-methylphenyl)-

cyclopropylamine 2, an analogue of DOM. This compound was reported

to have about one-third the potency of DOM. These studies led to the

clear conclusion that the side chain of the phenethylamine hallucinogens must assume a transoid conformation.

71
At about the same time our group, and others, developed hypotheses

which attempted to relate the stereochemistry of the phenylisopropylamines to that of LSD. The basis for these proposals apparently had

its origin in earlier work by Baltzly and co-workers (1949), by

Bovet et al. (1951) and by Marini-Bettolo et al. (1952). In the

late 1940's and early 1950's these workers had proposed that the

oxytocic activity of the ergot alkaloids was related to the aminotetralin fragment, or A-C ring portions of lysergic acid, as

illustrated below. Kang and Green (1970) suggested extension of

this approach to consideration of LSD. Similarly, work was published from our laboratory which included recognition of the

possible relationship between LSD and the R enantiomer of phenylisopropylamines (Barfknecht and Nichols 1972). These predictions

were confirmed by clinical studies carried out by Shulgin (1973)

using the optical isomers of DOM. The R-(-) enantiomer showed

clear stereoselective activity at a dose level one-half that

required for the racemate. The development (Nichols et al. 1973)

of an asymmetric synthesis for the enantiomers of various substituted phenylisopropylamines allowed several subsequent studies

with the isomers. These clearly demonstrated that in animals and

in man the R-(-) enantiomer was the more active (Benington et al.

1973; Snyder et al. 1974). Certain in vitro serotonin receptor

preparations showed parallel stereo selectivity for the R

enantiomer (Dyer et al. 1973).

Concurrently, a number of aminotetralin derivatives were reexamined for potential psychotomimetic activity (Violland et al.

1971; Barfknecht et al. 1973; Green, Dressler and Khazan 1973).

The sympathomimetic action of 2-aminotetralin had been known

7 2
since 1889 (Bamberger and Filehne 1889) and later studies focused

on the potential oxytocic and pressor effects of 2-AT derivatives.

These studies invariably drew parallels to the structures of the

ergot alkaloids. It was not surprising therefore, that the possibility was explored that the aminotetralins potentially represented the hallucinogenic pharmacophore in LSD. To date however,

none of these attempts has led to compounds with clear-cut psychotomimetic activity. In animal models 2-aminotetralin and its 7-

hydroxy derivative do show certain effects characteristic of

hallucinogens (Barfknecht et al. 1973; Green, Dressler and Khazan

1973). There is no evidence however, that 2-AT or any of its

derivatives is hallucinogenic in man.

On the other hand, some interesting results have been obtained from

studies using aminotetralin derivatives. Cheng et al. (1974),

using a smooth muscle preparation, showed that aminotetralins are,

in general, about 20-30 times more potent in eliciting contractions

than are the correspondingly substituted phenylisopropylamines. A

direct comparison between DOM 5 and the rigid analogues 6 (DOM-AT)

and 7 (DOM-AI) showed 6 to be a more potent agonist at serotonin

receptors than either DOM or the indan 7. The contractions induced

by DOM or DOM-AT were completely blocked by cinanserin, a 5-HT

antagonist. In the rat fundus, DOM-AT was much more potent than

7 and the contraction was blocked by BOL (Nichols et al. 1974).

Behavioral tests in rats did not reveal a typical psychotomimeticlike profile for either 6 or 7. In cats, DOM-AT does elicit a sham

rage reaction and in rabbits produces excitement and hyperthermia

(unpublished results).

Although nothing definite can be extrapolated from these studies

to hallucinogenic effects in humans, it may be reasonable to conclude that there is a requirement for an extended planar side chain

conformation in order to obtain effective binding at peripheral

serotonin receptors and to elicit certain behavioral changes in

animals.

In view of recent data which indicate that LSD is able to act as an

agonist at central dopamine receptors, a potential structural similarity between LSD 3 and the dopamine agonist apomorphine 8 has

been proposed (Nichols 1976). This hypothesis and the implied

relationship between the structures of serotonin and dopamine led

us to reevaluate the theories relating the structure of LSD to

mescaline and other phenethylamine hallucinogens. Examination of

space filling (CPK) molecular models clearly seemed to indicate

73
that previously proposed aminotetralin-like conformations for

phenylisopropylamines were sterically unfavorable. Conformations

which seemed energetically favorable were not superimposable on

the structure of LSD using the A-C ring analogy. Since this was

the criterion used to develop the aminotetralin analogy, means

were sought to resolve this issue.

This proves to be a case where rigid analogues can be usefully

employed. Our first approach was to resolve and test the

enantiomers of 2-aminotetralin (Nichols et al. 1977). This was

partially prompted by the practical consideration of synthetic

availability. However, 2-AT does show a hallucinogen-like profile

in behavioral tests in rats (Barfknecht et al. 1973). We first

examined the effect of R-(+) and S-(-) 2-AT on spontaneous activity in mice. While current models predicted activity for the (+)

isomer, the data in figure 1 clearly show that the (-) isomer is

responsible for the previously reported depressant action of

racemic 2-AT. As an additional indicator of hallucinogenic

activity, we also carried out studies using rabbit EEG. The quantitative parameters total power, P, and median frequency in the

power spectrum, f50, were examined. Mescaline (20 mg/kg) in our

experiments produced a slight initial depression in f50 and an

initial increase in P. The S-(-) isomer of 2-AT produced nearly

identical changes at 4 mg/kg. On the other hand, the (+) isomer

of 2-AT produced a dramatic and long lasting decrease in f50.

While these studies cannot be judged to be conclusive, when combined with the stereochemical arguments they justified more

extensive efforts to test the validity of the A-C ring hypothesis.

In particular, in view of the proposed relationship between LSD

and the dopamine agonist apomorphine 8 we speculated that similar

logic might apply to the phenethylamine hallucinogens.

It was decided to examine a rigid analogue which more nearly resembled the structure of a known hallucinogen in man. For this we

undertook the synthesis and resolution of the two isomers of trans2-(2,5-dimethoxy-4-methylphenyl)-cyclopropylamine (IMCPA). As

indicated earlier, this is the DOM analogue which had been reported

as the racemic material by Aldous et al. (1974). It was reasoned

that the A-C ring analogy would predict activity for the 1S, 2R

isomer. In contrast, using reasoning based on our results with

isomers of aminotetralin, we predicted activity for the 1R, 2S

74
Figure 1. Effects of R-(+) and S-(-) 2-aminotetralin as compared

to saline controls. Aminotetralin isomers were administered intraperitoneally as the HCl salts (Nichols et al. 1977).

enantiomer. Effects of the isomers of DMCPA were examined in mice

(5 mg/kg) and in cats (0.125 mg/kg). The results are shown in figure 2. Figure 2A represents the cumulative spontaneous activity

for groups of three mice (n=4). Parallel studies were carried out

with the isomers of DOM. The reduction in activity in mice was

apparently not due to general depression but was a result of more

time spent in various stereotypic behaviors.

Figures 2B and 2C show the effects of the isomers of DMCPA and DOM

on the cat limb flick and head/body shake response reported by

Jacobs et al. (1976). These results are typical for several other

behavioral symptoms which were elicited. In all cases, the (-)

isomer selectively elicited effects which were characteristic of

hallucinogens (Jacobs et al. 1976).

We argue that the (-) isomer of DMCPA has the 1R, 2S absolute configuration (depicted in figure 3c) for the following reasons. Levoamphetamine is well-known to have the R configuration (Smith,

Cook and Warren 1964). The cyclopropyl analogue of amphetamine,

tranylcypromine, is also known to possess the (-)-1R,2S configuration (Riley and Brier 1972). Hence, the configuration at the

alpha carbon is R in the levorotatory isomer of both compounds.

Substituent introduction into the aromatic ring of amphetamine

does not affect sign of rotation (Nichols et al. 1973). For

example, DOM is basically a "substituted amphetamine" and has the

R-(-) configuration (Matin et al. 1974). We reasoned that con75
Figure 2. The effect of (+), (-), and racemic DOM and DMCPA on

(A) cumulative motor activity in mice, (B) the limb flick response

in the cat, and (C) head/body shakes in the cat. Motor activity

was measured for 60 min. Limb flicks and head/body shakes in cats

were measured for 60 min. Values are mean ± s.e.

version of DOM into the corresponding cyclopropyl compound would

parallel the amphetamine-tranylcypromine case.

Based on the results with (-) 2-AT and (-) DMCPA we present the

hypothesis that the phenethylamine hallucinogens correspond to the

structure of LSD as illustrated in figure 3. We have assumed that

binding occurs from the underside face of the molecule (Kang and

Green 1970) so that the relative side chain orientation allows the

a methyl of DOM or the methylene of DMCPA to project away from the

binding surface. This could explain the stereoselective activity

observed for hallucinogenic amphetamine derivatives. On the basis

of the earlier findings with 2-AT isomers, we propose correlation

of the ortho methoxy of the phenethylamines with the 2 position of

the tryptamines or LSD, rather than with the region at position 12

of LSD as suggested by earlier studies.

These arguments deal only with the ability of phenethylamines to

bind at serotonin receptors. This analogy obviously does not apply

to cases where the drug acts indirectly by the release of endogenous transmitter (Menon, Tseng and Loh 1976; Cheng et al. 1974).

76
Figure 3. A comparison of the structures of (a) 5R, 8R-(+)-LSD;

(b) R-(-)-1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM);

(c) lR,2S-2-(2,5-dimethoxy-4-methylphenyl)-cyclopropylamine and

(d) a framework superposition of a phenethylamine and tryptamine

moiety using the hypothesis discussed in the text.

© 1978, Pergamon Press, Inc.1

Although we initially suggested (Nichols et al. 1977) that the

aromatic ring of the phenethylamines corresponded to the pyrrole

region in the indole nucleus of LSD, a more general view is now

proposed in light of the following observations:

(1). The interatomic distance between the centers of the 02 and

05 atoms of DOM and the C2 and 05 atoms of serotonin is virtually

identical, being approximately 5.5-6

(2). Molecular orbital calculations have shown a high electron

density at C2 in LSD (Karreman, Isenberg and Szent-Györgyi 1959).

This has been cited as evidence for charge-transfer complex

formation. The 02 atom in the phenethylamines, with its unshared

electrons, could also interact with an electrophilic site at this

region of the receptor.

(3). The 05 atom in the phenethylamines probably binds at the same

site which accomodates the 5-hydroxy of serotonin. In the phenethylamines extension of the 5-methoxy to an ethoxy abolishes

hallucinogenic activity (Shulgin 1968). Similarly, homologation

of 5-methoxy tryptamine to 5-ethoxy or longer alkyl homologues

decreases 5-HT agonist activity (Arutyunyan et al. 1964; Buznikov

7 7
et al. 1965). It also seems possible that the 9,10 double bond of

LSD could interact with this site.

(4). The description of a lipophilic region on the receptor which

binds the para substituent of phenethylamine hallucinogens (Nichols,

Shulgin and Dyer 1977) is consistent with an area of the proposed

receptor which normally accomodates the C6-C7 edge of serotonin.

(5). The receptor is probably flexible. The cyclopropyl analogues

are relatively rigid and complete superposition of LSD is impossible. For a planar interaction, the amino group of IMCPA would

lie approximately 1 from N(6) of LSD.

The para substituent seems to play a unique role in defining

activity. As indicated above, this substituent may bind at an

area of the serotonin receptor which is "incompletely filled."

However, since metabolic deactivation appears to occur by oxidative attack at this position (Ho et al. 1971) the ability of the

substituent to resist oxidation probably influences activity. The

contrasting duration of action for DOB (4-bromine) and para-DOT

(4-methylthio) may reflect this.

THEORETICAL CALCULATIONS

We have also performed theoretical conformational calculations for

R-DOM and 1R,2S-DMCPA. The following structures indicate starting

orientation and direction of rotation for the major side chain

torsional angles. (Note that this does not correspond to the

standard convention for "torsional angles"). For this work we

employed the CAMSEQ software system developed by Weintraub (1975),

but using a revised solvation model for charged species in solution

(Weintraub and Nichols 1978). The conformational energy maps for

the two compounds are shown in figure 4. The active conformation

which we propose for DOM would lie in the region

0°. Although we cannot assign a strict value to τ1 we believe it

lies within ± 30° of this value. It will be noted from the conformational energy maps that this lies within a region approximately

1-4 kcal above the global minimum.

In DMCPA τ2 is fixed at about 328° (our convention). If one assumes

that in the active conformation this is an appropriate value of τ2

for flexible compounds, and that the amino group lies approximately

in the plane of the aromatic ring, as it does in LSD, a value for

7 8
Figure 4. A. Conformational isoenergy contour map for DOM in

solution and B. the conformational energy profile for rotation of

t1 in DMCPA.

79
τ1 of approximately 155° is required. This places τ1 in a relatively low energy region of the conformational energy map for both

DOM and DMCPA.

Similar calculations for the non-hallucinogenic a ethyl homologue

of DOM (Standridge et al. 1976) resulted in a conformational profile

very similar to DOM. This finding seems to indicate that the lack

of hallucinogenic activity for the a ethyl homologue is probably

not due to an inability to assume the active conformation. This

work is reported in detail elsewhere (Weintraub and Nichols 1978).

FOOTNOTE

1. Reprinted with permission, from Nichols, D.E., Pfister, W.R., and

Yim, G.K.W. LSD and phenethylamine hallucinogens: New structural

analogy and implications for receptor geometry. Life Sciences,

Vol. 22, No. 24. © 1978, Pergamon Press, Inc.

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Mittheilung uber hydronaphthylamine. Chem Ber, 22:777-778, 1889.

Barfknecht, C.F., and Nichols, D.E. Effects of S-(+) and R-(-)-

3,4,-dirnethoxyphenylisopropylamines in the rat. J Med Chem, 15:

109, 1972.

Barfknecht, C.F., Nichols, D.E., Rusterholz, D.B., Long, J.P.,

Engelbrecht, J.A., Beaton, J.M., Bradley, R.J., and Dyer, D.C.

Potential psychotomirnetics: 2-amino-1,2,3,4-tetrahydronaphthalene

analogs. J Med Chem, 16:804-808, 1973.

Benington, F., Morin, R.D., Beaton, J., Smythies, J.R., and

Bradley, R.J. Comparative effects of stereoisomers of hallucinogenic amphetamines. Nature (New Biology), 242:185-186, 1973.

Bovet, D., Bovet-Nitti, F., Sollero, L., and Marini-Bettolo, G.B.

Recherches sur les derives de la 1,2,3,4-tetrahydro-2-naphtylamine

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80
Brawley, P., and Duffield, J.C. The pharmacology of hallucinogens.

Pharmacol Rev, 24:31-66, 1972.

Buznikov, G.A., Zherebchenki, P.G., and Chudakova, I.V. Effect of

various indolylalkylamines on the motor cells of mollusk embryos

and the vessels of the rabbit ear. Byul Eksperim Biol i Med, 59:

59-63, 1965 (see Chem Abstr 63:8890, 1965).

Cheng, H.C., Long, J.P., Nichols, D.E., and Barfknecht, C.F.

Effects of psychotomimetics on vascular strips: studies of

methoxylated amphetamines and optical isomers of 2,5-dimethoxy4-methylamphetamine (DOM) and 2,5-dimethoxy-4-bromoamphetamine

(DOB). J Pharm Exp Ther, 188:114-123, 1974b.

Cheng, H.C., Long, J.P., Nichols, D.E., Barfknecht, C.F., and

Rusterholz, D.B. Effects of rigid amphetamine analogs on vascular

strips: studies of 2-aminotetrahydronaphthalene and 2-aminoindane

derivatives. Arch Int Pharmacodyn Ther, 208:264-273, 1974a.

Cooper, P.D., and Walters, G.C. Stereochemical requirements of the

mescaline receptor. Nature, 238:96-98, 1972.

Dyer, D.C., Nichols, D.E., Rusterholz, D.B., and Barfknecht, C.F.

Comparative effects of stereoisomers of psychotomimetic phenylisopropylamines. Life Sci, 13:885-896, 1973.

Goldberg, L.I., Volkman, P.H., Kohli, J.D., and Kotake, A.N.

Similarities and differences of dopamine receptors in the renal

vascular bed and elsewhere. In: E. Costa and G.L. Gessa, eds.

Advances in Biochemical Psychopharmacology. Vol. 16. New York:

Raven Press, 1977. pp. 251-256.

Green, J.P., Dressler, K., and Khazan, N. Mescaline-like activity

of 2-amino-7-hydroxytetralin. Life Sci, 12 (part I):475-479, 1973.

Hansch, C. A quantitative approach to biochemical structure-.

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and McIsaac, W.M. Analogs of amphetamine.5.studies of excretory

metabolites of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane

(DOM) in rats. J Med Chem, 14:158-160, 1971.

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Harris, R.T. Amphetamine analogs. II. Methylated phenethylamines.

J Med Chem 13:134-135, 1970.

Jacobs, B.L., Trulson, M.E., and Stern, W.C. An animal behavior

model for studying the actions of LSD and related hallucinogens.

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Kang, S., and Green, J.P. Steric and electronic relationships

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81
Karreman, G., Isenberg, I., and Szent-Gyorgyi, A. On the mechanism

of action of chlorpromazine. Science, 130:1191-1192, 1959.

Marini-Bettolo, B.G., Frediani, H.A., and Chiavaretti, S. Synthetic sympathoyltic agents in the ergotamine series. IX. Separation of the optical antipodes and the physicochemical properties

of N,N-diethyl-N'-(tetrahydro-2-naphthyl)glycinamide. Gazz Chim

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Matin, S.B., Callery, P.S., Zwieg, J.S., O'Brien, A., Rapoport,

R., and Castagnoli, N. Jr. Stereochemical aspects and metabolite

formation in the in vivo metabolism of the psychotomimetic amine,

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877-882, 1974.

Menon, M.K., Tseng, L.-F., and Loh, H.H. Pharmacological Evidence

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Nichols, D.E., Barfknecht, C.F., Long, J.P., Standridge, R.T.,

Howell, H.G., Partyka, R.A., and Dyer. D.C. Potential psychotomimetics 2: rigid analogs of 2,5-dimethoxy-4-methylphenylisopropylamine (DOM, STP). J Med Chem, 17:161-166, 1974.

Nichols, D.E., Barfknecht, C.F., Rusterholz, D.B., Benington, F.,

and Morin, R.D. Asymmetric synthesis of enantiomers of psychotomimetic amphetamines. J Med Chem, 16:480-483, 1973.

Nichols, D.E. Structural correlation between apomorphine and LSD:

involvement of dopamine as well as serotonin in the actions of

hallucinogens. J Theor Biol, 59:167-177, 1976.

Nichols, D.E., Pfister, W.R., Yim, G.K.W., and Cosgrove, R.J. A

new view of the structural relationship between LSD and mescaline.

Brain Res Bull, 2:169-171, 1977.

Nichols, D.E., Shulgin, A.T., and Dyer, D.C. Directional lipophilic character in a series of psychotomimetic phenethylamine

derivatives. Life Sci, 21:569-575, 1977.

Riley, T.N., and Brier, C.G. Absolute configuration of (+) and

(-)-trans.-2-phenylcyclopropylamine hydrochloride. J Med Chem,

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Shulgin, A.T. Mescaline: the chemistry and pharmacology of its

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8 2
Smith, H.E., Cook, S.L., and Warren, N.E. Jr. Optically active

amines. II. The optical rotatory dispersion curves of the Nbenzylidene and substituted N-benzylidene derivatives of some

open-chain primary amines. J Org Chem, 29:2265-2272, 1964; and

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Snyder, S.H., and Richelson, E. Psychedelic drugs: steric factors

that predict psychotropic activity. Proc Natn Acad Sci USA,

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Stereospecific actions of DOEt(2,5-dimethoxy-4-ethylamphetamine)

in man. Arch Gen Psychiat, 31:103-106, 1974.

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Shulgin, A.T. Phenylalkylamines with potential psychotherapeutic

utility. I. 2-Amino-1-(2,5-dimethoxy-4methylphenyl)butane. J

Med Chem, 19:1400-1404, 1976.

Violland, R., Violland-Duperret, N., Pacheco, H., Trouiller, G.

and LeBlanc, A. VIII. Psychotropes potentiels. Synthese et

activité phannacologique d'amino-2-tetralines apparentées a

divers psychotomimétiques. Bull Chim Ther, 196-202, 1971.

Weintraub, H.J.R. Applications of theoretical conformational

analysis in drug research. Ph.D. thesis. Case Western Reserve

University, 1975.

Weintraub, H.J.R., and Nichols, D.E. Conformational energy

calculations of some amphetamine analogs using a new solvation

model. Int J Quantum Chem, QB5:in press, 1978.

AUTHORS

David E. Nichols, Ph.D.

Herschel J. R. Weintraub, Ph.D.

Department of Medicinal Chemistry and Pharmacognosy

school of Pharmacy and Phannacal Sciences

Purdue University

West Lafayette, Indiana 47907

William R. Pfister

George K. W. Yim, Ph.D.

Department of Pharmacology and Toxicology

School of Pharmacy and Pharmacal Sciences

Purdue University

West Lafayette, Indiana 47907

8 3
Section II

Hansch Analysis and Other Empirical Methods

This portion of the meeting appeared to accomplish two

objectives. The papers highlighted several important

techniques of applying physicochemical parameters to the

problem of understanding the nature of the effects of

substituent variation upon the drug activity observed.

The areas where these parameters are weakest--most notably in stereochemical differences and in some steric

effects--were also pointed out. But perhaps the second

objective might, in the long run, prove more valuable to

the attendees. That objective was to clearly define

just what should be expected of this approach to QuaSAR;

i.e., an acceptable statement of philosophy.

In a paper given at an earlier session, Dr. Sydney

Archer put forth this challenge: Of the three levels of

drug design--(l) finding a totally new pharmacophore;

(2) circumventing a basic shortcoming of an established

one (e.g., eliminating dependence on drugs that have

analgesic action); and (3) improving potency--QuaSAR

methods addressed only the latter. Then in the discussion Dr. Jack Green added the further challenge that

the Hansch type of correlation analysis may not be appropriate to investigate basic questions of mechanism of

action but was rather intended to obtain "straight

lines" that have predictive value.

As was evident from the discussions clinicians do not

consider potency the "bottom line" in choosing which

drug to use, but rather look at the entire profile of

efficacy weighed against various side effects. And, of

course, "constructing straight lines with predictive

value" was not seen by those who gave papers utilizing

correlation analysis as the end and final goal of

QuaSAR. Each speaker came to present and defend some

rather narrow areas of their own expertise, and so the

challenge to defend the entire concept could not be met

84
with well-organized material prepared in advance. But

it was evident that the purpose of Dr. Archer and Dr.

Green was to force the Hansch analysis people to bring

out for open scrutiny some of the basic methods they had

established long ago and perhaps had taken for granted,

but which, for some of the conferees, would be crucial

in deciding how much credence should be given to the

results reported. If this was meant to be an application of the "gadfly" technique, it worked. The discussions were much more lively and enlightening than

they otherwise would have been.

In the general discussion periods there also appeared to

be a difference of opinion amongst the conferees as to

what tools or language one must be using before he

"really gets down to molecular mechanism." An example

was cited where the significance of an electronic effect

was appreciated through correlation analysis using a

sigma parameter. This prompted the investigators to do

a study with molecular orbital calculations which

enabled them to see where the sigma correlation came

from. Only then were they getting down to molecular

mechanisms. Naturally the question was raised: "Why,

if M.O. methods at their present level of feasibility

are more basic in describing molecular mechanisms, have

they not been used to better effect than sigma constants

in uncovering these electronic correlations?"

The session ended with a discussion of the various interpretations which could be given to a parabolic relationship between hydrophobicity and activity. Since

this is seen in some in vitro experiments, such as in

brain homogenates and even in semi-purified enzymes,

there is the strong possibility that part or all of the

parabolic dependency may not arise from transport

through cellular compartments, but, alternatively, the

drug may be involved in several competing reactions

which have different dependence upon hydrophobicity.

Occasionally these competing reactions can be studied

via Hansch analysis, and the knowledge put to use in

solving problems connected with area (2) in Dr. Archer's

scheme of drug design.

85
'QuaSAR' Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

QSAR: A Critical Appraisal

Sydney Archer

The pioneering work of Hansch on QSAR has led to an

avalanche of papers, as witnessed by the fact that the

first three papers in the January 1978 issue of the

Journal of Medicinal Chemistry deal with this subject.

QSAR has become so popular that two chapters in the

first three volumes of the series "Drug Design" edited

by E.J. Ariens are devoted to this subject (Hansch 1972;

Verloop 1972). In his Smissman Award Address, Alfred

Burger (1978) devoted the last several paragraphs of

this talk to QSAR. Because of the attention that this

aspect of medicinal chemistry is receiving, a critical

appraisal of the significance of the role played by QSAR

in drug aesign is the subject of this communication.

Before proceeding, it would be worthwhile to define what

is meant by drug design. Ariens (1972) defines the term

as follows: "Drug design is an effort to develop drugs

on as rational a basis as possible. This implies a

reduction of the trial and error factor in the procedure

to the absolute minimum. However, the fact that trial

and error is not completely excluded in the efforts to

develop a drug does not mean that drug design is not

involved." This writer finds this definition unsatisfactory for the following reasons: first, design is not

an effort; second, the implied goals in the definition

are far too modest; third, it does not conform to the

dictionary definition of the word design; fourth, it

involves the word rational, whose meaning is also

clouded with ambiguity; and fifth, it employs the word,

drug, which is to be defined in the definition.

What is a drug? According to Webster's 2nd International Edition a drug is "any substance used as a

medicine or in the making of medicines for internal or

external use." According to the Pure Food and Drug Act,

the term "drug" includes all medicines and preparations

recognized in the U.S.P. and N.F. for internal and

86
external use and "any substance or mixture of substances

intended to be used for the cure, mitigation or prevention of disease in either man or animals." The same

authority defines design as "a plan formed in the mind,

of something to be done or produced; a mental project or

scheme in which the means to an end are laid down..."

Finally, rational is defined as, 1) having reason or

understanding, reasoning; 2) of or pertaining to the

reason or reasoning process; of the nature of, based

upon, derived from, concerned with or characterized by

reason; as rational insight.

Since a drug is intended to cure or mitigate disease in

man or animals and design is a plan or scheme to produce

something, drug design may be defined as a mental process wherein plans are formulated to produce substances

which will cure or mitigate disease.

The problems facing medicinal chemists who are interestea in drug design may be classified into three

categories. Category I encompasses those diseases for

which there are no known or satisfactory treatments.

Here the medicinal chemist has no known models at his

aisposal so that drugs must be designed de novo if the

purely empirical approach is to be avoided. Black's

work on H2 receptor antagonists and the NachmansonWilson development of nerve-gas antagonists are examples

of solutions to problems which fit into this category.

Category II involves those diseases for which therapeutic agents are known but suffer from obvious clinical

disadvantages. The development of penicillinaseresistant penicillins and strong analgesics relatively

free of a morphine-like dependence syndrome may serve as

examples here. Category III is concerned with increasing potency of clinically satisfactory drugs. The

super-potent anti-inflammatory steroids, the highly

active thiazide diuretics, the ultrapotent morphine-like

analgesics fall into this class.

QSAR studies are for the most part concerned with the

last category. It is difficult to impress upon the

minds of many medicinal chemists and pharmacologists

that it is only in rare instances that potency per se is

of paramount concern. One example is in the development

of long-acting depot preparations. If a drug can be

milligram potency is of little concern to the clinician.

adminstered conveniently in a suitable dosge form.

Supporters of QSAR studies like to quote Spinks who is

reported to have estimated that one new drug arises out

of each 200,000 new compounds and that one anticancer

drug will emerge from 400,000,000 randomly tested compounds. The implication is that QSAR studies can reduce

these odds.

87
First, one must ask, what does Spinks mean by a new

drug? Does he mean one that belongs in Category I or II

or a simple molecular modification which would place the

compound in Category III for which QSAR may be of help?

If Spinks means by new drugs those which fit only in

Categories I and II, by far the more difficult to develop, then the reality of the situation belies these

estimates. Using antimalarial agents as examples of

Category I and II drugs, the therapeutic arsenal contains examples of the following: quinolinemethanols

(quinine), amino-quinolines (primaquine, chloroquine),

acridines (mepacrine), dihydrotriazines (cycloguanil),

aihydro-pyrimidines (pyrimethamine), sulfonamides

(sulphormethoxine), sulfones (DDS). If the 1:200,000

ratio is valid, then about 1.4 × 106 compounds would

have to be screened to yield this crop of drugs, a

number which is more than 1/3 of all known organic

compounds.

The fact of the matter is that truly random screening

rarely, if ever, takes place. In the antimalarial field

screening is limited, with rare exceptions, to N containing substances which also contain oxygen. Furthermore, the vast majority of compounds are either aromatic

(carbocyclic or heterocyclic) or combinations thereof.

Functionality such as COOH, SO3H and quaternary ammonium

are usually avoided to minimize absorption and distribution problems. Thus on an almost intuitive basis

true, random screening is usually avoided so that the

Spinks' estimate is at best a statistical exercise.

In one of the more important QSAR papers Hansch et al.

(1968) analyzed a large group of hypnotics, with special

emphasis on barbiturates. The biological data, obtained

from various literature sources, was amassed over the

period 1923-1949, hardly the golden age of modern

pharmacology. Not only were different methods of assessing hypnosis used but different species were

employed as test animals. Several equations were

developed and the most interesting conclusion that

emerged from this collection of inhomogeneous data was

that log Po for the oxybarbiturates equaled 2. The

authors concluded that the hypnotic activity of groups

of barbiturates depend almost entirely on the relative

lipophilic character as defined by their octanol-water

partition coefficients. In the case of the thiobarbiturates the mean log Po was 3.1. The authors state,

"The thiobarbiturates quite definitely do not fit into

the same pattern shown by other barbiturates or the

other hypnotics. Their maximum activity is attained

wnen their partition coefficients are about 10 times

that of the barbiturates. This strongly implies a

different overall mechanism of action. That the thiobarbiturates have quite different biological action from

88
the oxybarbiturates has been pointed out. by Aldridge and

Parker."

In 1960, eight years before the publication of the

Hansch paper, B.B. Brodie and his colleagues published a

paper (Brodie et al. 1960) entitled, "The importance of

dissociation constant and lipid solubility in influencing the passage of drugs into the cerebrospinal

fluid" wherein the penetration of drugs into the CNS was

carefully examined. This paper was not even mentioned

by Hansch (Hansch et al. 1968).

Broaie was able to determine penetration constants (P)

into the CSF using Fick's law for diffusion:

d(Cp-Cc)/dt = -P(Cp-Cc) (1)

where cp = concentration of drug in plasma

cc = concentration of drug in the CSF.

If Cp is kept constant as it can, by maintaining a constant rate of infusion, then integration of (1) gives

equation (2):

ln((Cp-Cc)/Cp) = -Pt (2)

Plotting log ((Cp-Cc)/Cp) vs. time gives a straight

line, the negative slope of which equals P. Using a

variety of drugs these authors developed a family of

curves from which the penetration constants were

calculated. Goldstein (Goldstein et al. 1974)

constructed table I using data generated earlier by

others. It is clear from inspection of this table that

lipophilicity as measured by the effective partition

coefficient (column e) correlates well with P and the

penetration half-time. Thus the effects of lipophilicity on penetration into the CNS was well appreciated some time before the QSAR treatment and it would

have been surprising if Hansch's conclusions differed

from those of Brodie.

More aisturbing is Hansch's interpretation of the

results obtained with the thiobarbiturates. On the

basis of the different log Po values for oxybarbiturates

and thiobarbiturates it is claimed that this result

"strongly implies a different mode of action" and cites

Aldridge and Parker (1960) as support. What these

authors do say is: 1) "The oxybarbiturates...inhibit

but do not uncouple oxidative phosphorylation of liver

mitochondria with pyruvate as a substrate." 2) "The

thiobarbiturates inhibit and to a certain extent uncouple oxidative phosphorylation. The uncoupling

appears to be correlated with their ability to activate

mitochondrial ATPase." 3) "It is possible that thio89
TABLE 1
barbiturates have two unrelated mechanisms of action,

one inhibiting respiration by a similar mechanism to

that of the oxybarbiturates and the other activating

ATPase." (Emphasis added).

The last two statements are a far cry from Hansch's

interpretation of the Aldridge-Parker paper, "that the

thiobarbiturates have quite different biological action

from the oxybarbiturates."

Another questionable interpretation of the literature is

the statement, "The possibility that more lipophilic

centers in the brain are involved in the case of the

thiobarbiturates can be inferred from the work of

Goldstein and Aronow (1960) and Roth and Barlow (1961).

Their efforts have shown that the concentration of the

tniobarbiturates rises in the brain considerably above

that in blood plasma in a rather short time." Actually

Goldstein and Aronow showed that the brain and plasma

concentrations of thiopental are equal within a minute

or so after drug administration as a result of rapid

equilibration. Roth and Barlow agreed that whole brain

levels and those in plasma were about equal but they

believed the concentration of thiopental in the cerebral

cortex was twice that in the plasma at very early times.

The Goldstein-Aronow experiments were carried out to

account for the ultrashort duration of thiopental in

comparison with pentobarbital. It was known that thiopental and pentobarbital were equipotent, that is to

say, that equal brain concentrations of the two drugs

produced the same degree of anesthesia. It was known

that thiopental concentrates in fat depots, but the

kinetics of the fat depot plasma interchange could not

account for the ultrashort action of the hypnotic.

Owing to the favorable lipid solubility pentothal

rapidly penetrates into the brain and brain levels and

plasma levels are essentially identical in a very short

time (figure 1). The brain level of the drug is above

the threshold level required to produce anesthesia so

that the pharmacological response is very rapid. As

plasma levels decay, brain levels follow so that at the

end of about 5 minutes brain levels of drug are insufficient to maintain the hypnotic state. In contrast

pentobarbital equilibrates relatively slowly and in the

Goldstein-Aronow experiment this oxybarbiturate did not

reach a sufficiently high concentration in brain to

produce anesthesia. The experiment showed why a higher

dose of pentobarbital is needed to produce anesthesia,

why it is slow in onset and why it is protracted in

duration. The conclusions drawn from this study are at

variance with those indicated by QSAR studies, in the

91
FIGURE 1
sense that no difference in mechanism of action between

the thiobarbiturates and oxybarbiturates is implied.

II. QSAR AND STRONG ANALGESICS

The reason for the intense interest in research in this

field was well stated by Eddy and May (1966): "The ever

present stimulus to the recurring search for new synthetic analgesics has been the hope that new active

compounds might be found free of the undesirable side

effects of morphine itself." By far the most important

side effect of this classical opiate is its ability to

produce physical dependence.

Burger (1978) has cautioned that "Quantitative structure

activity relationships are only as good as the biological test methods against which physicochemical parameters are plotted." There is no better illustration of

this point than in the field of strong analgesics.

In the mid-1950's the laboratory models for testing

strong analgesics were the D'Amour-Smith test or some

slight modification thereof in rats and the EddyLeimbach "hot plate" test in mice. Both procedures were

capable of detecting morphine-like analgesic activity

and were extremely accurate predictors of milligram

potency in man. At that time, thanks largely to the

efforts of Beecher and his collaborators, assays of

analgesic activity in man could be carried out accurately with a relatively small patient population.

Investigators at the Addiction Research Center in

Lexington were devising ways to determine addiction

liability in man in a quantitative manner. With all the

proper bioassays in place, the stage was set to find a

potent agonist with little or no addiction liability.

Unfortunately the goal remained elusive despite the fact

that a number of structurally distinct series of compounds, some members of which showed strong'analgesic

action in animals and man, were synthesized. These

included compounds in the methadone, meperidine, levorphanol, benzomorphan and etonitazene series. Despite

the aiversity of chemical types the similarity in

biological effects was extremely frustrating and

disappointing. Compounds several hundred times as

potent as morphine were synthesized but the pharmacological profiles were essentially identical with

morphine.

The partial solution to this problem stemmed from two

related serendipitous observations. The first was that

replacement of the N-methyl group of morphine resulted

in a compound (I) which antagonized the pharmacological

effects of morphine. Nalorphine was essentially inactive in the D'Amour-Smith and Eddy-Leimbach tests and

thus was considered not to be an analgesic. The second

93
important observation was made by Beecher and Lasagna in

1954, who found that in man nalorphine was a strong

analgesic equal in milligram potency to morphine. Yet

when given after morphine the agonist effects of the

latter were abolished. Unfortunately, the drug produced

such bizarre effects in man that it was not considered

suitable for clinical use. Preliminary studies on its

ability to induce physical dependence were negative.

Clinical studies on other narcotic antagonists were

carried out by Arthur Keats. A few were found to be

good analgesics but those that were also produced unacceptable CNS side effects. The significance of the

Keats work lay in the fact that it clearly demonstrated

that the clinical behavior of nalorphine was not unique

and that a careful investigation of other narcotic antagonists might lead to a useful drug.

In our laboratory, analysis of the available animal and

clinical data led to the conclusion that the laboratory

models for evaluating strong analgesics were more reliable predictors of addiction liability than of clinical analgesic potency.

The design of potential analgesics involved the synthesis of putative narcotic antagonists in the benzomorphan

series, testing for analgesic antagonism in rats and

also for activity in the D'Amour-Smith test. A positive

result in the latter test resulted in rejection of a

compound from further study. At that time the only

reliable assessment of analgesia was trials in humans.

Fortunately the severe restrictions on early clinical

investigations which followed in the aftermath of the

passage of the Kefauver-Harris bill were not yet in

force so that clinical research could be carried out

with far less toxicological, biochemical and metabolic

data than are required now. It should be added that our

collaborating clinical pharmacologists employed all the

necessary clinical safeguards and there were no greater

dangers to the patient than we have now. Several narcotic antagonists were tested in a relatively short

period of time. One of these, pentazocine, a weak

antagonist was found to be a clinically acceptable drug

and is presently marketed. It was also found that

94
TABLE 2

95
TABLE 2 (cont.)

TABLE 3

96
powerful antagonists could also be potent clinically

active analgesics. Cyclorphan and cyclazocine are two

such examples. These drugs did not produce a morphinelike physical dependence in monkeys and even more important, in man. These developments stimulated other

workers to look at other narcotic antagonists as possible analgesics and table II shows some results of

their efforts.

In light of this history it was surprising to see the

recent paper by Katz et al. (1977) on a QSAR study of

narcotic analgesics in which the assay method was the

Eady-Leimbach test procedure. One could safely predict

that none of the compounds in table II would show significant activity in that test. Table III reports some

results from the Eddy-Leimbach test and clinical potency

of some benzomorphans which are known to be mixed

agonist-antagonists.

It should be clear from the few examples shown in table

III that the Eddy-Leimbach test is virtually useless as

a predictor of useful analgesic activity.

Since the early studies on narcotic antagonists, pharmacological tests have been devised which can detect the

agonist potency of this group of analgesics. However,

one must be careful in the choice of assays because of

difficulties with internal correlations. For example,

it is well known that quaternary ammonium salts of

strong analgesics generally are inactive in whole animal

tests. Yet in isolated systems such as the guinea pig

ileum and isolated receptors Opheim and Cox (1976) have

shown that the methiodide of levorphanol (III) was about

1/3 as active as levorphanol (II) using the myenteric

plexus of the longitudinal muscle of the guinea pig

ileum, a preparation popularized by Kosterlitz

(Kosterlitz and Waterfield 1975). The activity in this

preparation was reversed by naloxone.

Using guinea pig brain homogenates the methiodide was

able to depress the stereospecific binding of (

3

H)-

etorphine. The dextro isomer was far less active.

97
Recently Kosterlitz (Lord et al. 1977) found a new

opiate receptor in the mouse vas deferens. As can be

seen from table IV there is a difference in potency of

Met-enkephalin analogs in these two preparations.

TABLE IV

Another striking difference between these two preparations is the amount of naloxone necessary to antagonize the action of some opioid peptides as shown in

table V.

TABLE V

98
The guinea pig ileum assay results for agonist potency

correlate well with clinical results, but some discrepancies could surface if compounds such as quarternary salts are ever tested in man, a highly unlikely

situation. It was pointed out earlier that potency is

not of primary concern to clinicians--nor should it be

to medicinal chemists. There are many unanswered

questions in the field of strong analgesics, the answers

to which should be of greater concern than problems

involving maximizing potency. Some of these are: 1)

Why is it that, in this field, the structural requirements for agonist activity are relatively few while

those for antagonists are highly restrictive? In other

areas of pharmacology such as adrenergic, serotoninergic and cholinergic systems, the reverse is usually

true, that is, that the agonists are represented by few

structural types whereas greater diversity can be found

in the antagonists. 2) Why is it that lengthening the

chain on nitrogen in the morphinan and benzomorphan

series converts a compound which is a pure agonist to

one that is either a pure antagonist or a mixed agonistantagonist? Explanations based on conformational differences are not convincing since it is difficult to see

why the methyl and amyl analogs should be conformationally similar and both differ from the n-propyl

compound (table VI). 3) How can one account for the

unusual effects of 14-OH substitution in the morphinans

and the corresponding 11-OH substitution in the benzazocines? For example nalorphine is a mixed agonistantagonist while the structurally similar agent, naloxone, is devoid of agonist action but is essentially a

pure antagonist. Similarly naltrexone, a 14-OH-Ncyclopropylmethyl derivative, is a pure antagonist

whereas other non-14-hydroxylated derivatives such as

cyclorphan and cyclazocine are highly potent agonists

and antagonists. Replacement of the N-cyclopropylmethyl

radical with the cyclo-butylmethyl radical in the 14-

hyaroxy-morphinans reduces antagonist action but agonist

activity reappears as in the case of nalbuphine and

butorphanol.

Even if one accepts that the modest goal of QSAR studies

is to reduce the trial and error approach to the maximization of potency then such investigations must have

some heuristic value; otherwise QSAR will become a

sterile exercise in data manipulation. However, Verloop

(1972) states "Examples of predictions of bioactivity

from regression equations are still rare. As far as the

present author is aware, there are no examples of cases

in which the prediction made by one author was later

substantiated in another paper, with the exception of

the 'thyroxine case' quoted by Hansch."

99
TABLE VI

A report (Humber et al. 1975) prepared by an ad hoc

Committee of the Section on Medicinal Chemistry of the

IUPAC entitled, "'Predicted' Compounds with 'alleged'

Biological Activities from Analyses of StructureActivity Relationships" lists 15 such cases of compounds

whose activity was predicted on the basis of QSAR

studies, but which were never synthesized and tested for

the pharmacological activity indicated. It would seem

to this writer that it would be useful to put these

predictions to the test. Positive results of such an

exercise could give greater credence to the implicit and

explicit claims that QSAR have an important place in

drug design.

ACKNOWLEDGMENT

We wish to thank the National Institute on Drug Abuse for its

support under a grant, DA-01674.

100
REFERENCES

Aldridge, W.N., and Parker, V.H. Barbiturates and

oxidative phosphorylation. Biochem J, 76:47-56, 1960.

Ariens, E.J. A general introduction to the field of

drug design. In: Ariens, E.J., ed. Drug Design Vol.

I. New York: Academic Press, 1972. pp. 2-270.

Brodie, B.B., Kurz, H., and Shanker, L.S. The importance of dissociation constant and lipid-solubility

in influencing the passage of drugs into the cerebrospinal fluid. J Pharmacol and Exp Therap, 130:20-25,

1960.

Burger, A.J. Drug design and development. A realistic appraisal. J Med Chem, 21:1-4, 1978.

Eddy, N.B., and May, E.L. Synthetic Analgesics Part IIB.

New York: Pergamon Press, 1966. p. 71

Goldstein, A., and Aronow, L. The duration of actions of

thiopental and pentobarbital. J Pharmacol and Exp

Therap, 128:1-6, 1960.

Goldstein, A., Aronow, L., and Kalman, S.M. Principles

of Drug Action, ed 2. New York: John Wiley and Sons,

1974. p. 194.

Hansch, C. Quantitative structure-activity relationships. In: Ariens, E.J., ed. Drug Design Vol I. New

York: Academic Press, 1972. pp. 271-342.

Hansch, C., Steward, A.R., Anderson, S.M., and Bentley,

D. The parabolic dependence of drug action upon

lipophilic character as revealed by a study of

hypnotics. J Med Chem, ll:l-11, 1968.

Humber, L.G., Albert, A., Campaigne, E., Cavalla, J.F.,

Anand, N., Protiva, M., Rachlin, A.I., and Sensi, P.

"Predicted" compound with "alleged" biological

activities from analyses of structure-activity relationships. IUPAC Information Bulletin, 12-20, 1975.

Katz, R., Osborne, S.E., and Ionescu, F. Applications

of the Free-Wilson technique to structurally related

series of homologues. Quantitative structure-activity

relationship studies on narcotic analgesics. J Med

Chem, 20:1413-1419, 1977.

Kosterlitz, H.W., and Waterfield, A.A. In vitro models

in the study of structure-activity relationships. Ann

Rev Pharmacol, 15:29-47, 1975.

101
Lord, J., Waterfield, A.A., Hughes, J., and Kosterlitz,

H.W. Endogenous opioid peptides: multiple agonists and

receptors. Nature, 267:495-499, 1977.

Opheim, K.E., and Cox, B.M. Stereospecific interaction

of the quaternized opiate, N-methyllevorphanol with

opiate receptors. J Med Chem, 19:857-858, 1976.

Roth, L.J., and Barlow, C.F. Drugs in the brain.

Science, 134:22-31, 1961.

Verloop, A. The use of linear free energy parameters

and other experimental constants in structure-activity

studies. In: Ariens, E.J., ed. Drug Design Vol. III.

New York: Academic Press, 1972. pp. 133-188.

AUTHOR

Sydney Archer, Ph.D.

Professor of Medicinal Chemistry

Cogswell Laboratory

Chemistry Department

Rensselaer Polytechnic Institute

Troy, New York 12181

102
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

QSAR of Agents Involved in

Serotonin and LSD Binding Sites

Y.L. Chan, E. J. Lien, and J. C. Shih

INTRODUCTION

Using cerebral cortex membrane preparations of rat brain, Bennett

and Snyder (l976) studied the displacement of specifically bound

[

3

H]-serotonin (5-HT) and d-[3H]-lysergic acid diethylamide (LSD)

by various drugs. It was concluded that both binding sites

have similar structural specificity, with a more crucial role played

by the 5-hydroxy substituent in the binding of various tryptamines

for the [3H]-5-HT binding sites than for the d-[3H]-LSD sites.

The compounds studied by Bennett and Snyder include LSD analogues;

tryptamines and anti-serotonin drugs. Since lipophilicity (Lien

1975; 1974), type of amines (Hansch and Lien 1968) and molecular

size (Lien 1974; Lien 1976) have been shown to be important factors

in drug uptake, distribution and drug action, it was felt worthwhile to apply multiple regression analysis to the data of Bennett

and Snyder (1976) using 1-octanol/water partition coefficient as a

measure of lipophilicity and molecular weight as a measure of the

molecular size, and nH to represent different types of amines

(Hansch and Lien 1968).

METHODS

The biological data were converted to log 1/IC50 (moles/1) (Table 1).

Whenever available the experimentally determined log P(octanol,w)

values of the undissociated forms were used. When the log P values

were not available, they were calculated from the log P of structurally related congeners and the π values of the substituents

103
TABLE 1

Displacement of [3H]-Serotonin and d-[3H]LSD from Rat Cerebral Cortex

Membranes by Various Drugs and Their Physicochemical Constants
Table 1. (Continued)
(Leo, Hansch and Elkin 1971; Hansch et al. 1973; Hansch 1971).

For example: log P (2-Br-LSD) = log P (LSD) + π Br = 2.95 + 0.86 =

3.81. Log P (d-Isolysergic acid amide) = log P (LSD) -

2.95-2.00 = 0.95. log P (methysergide) = log P (d-lysergic acid

amide + log P (butanol) + πbranch ing + = 0.95 + 0.88 - 0.20 +

0.71 = 2.34. log P (5-methoxytryptamine) = log P (5-HT - πOH +

= 0.21 - (-0.67) + (-0.02) = 0.86.

log P (tryptamine) =- log P (5-HT) - πOH = 0.21 - (-0.67) = 0.88

log P (5-OH-N, N-dimethyltryptamine) = log P (5-HT) - +

= 0.21 + 0.15 - (-1.04) = 1.10.

log (5,6-dihydroxytryptamine) = log P (5-HT) + log P (catechol) -

log P (phenol) = 0.21 + 0.80 - 1.46 = -0.45

log P (haloperidol) = 2 log P (benzene) + π F + + +

log P + log P (cyclohexanol) - log P (cyclohexane) +

π Cl = 2 × 2.13 + 0.14 + 1.00 + (-0.55) + 0.85 + 1.23 - 3.44 +

0.71 = 4.20.

The logarithmic scale of the molecular weight was used in order to

be in line with the biological data. The method of least squares

was used in deriving the equations using an IBM 370/155 computer.

The number of hydrogens of the protonated amino group is represented by nH, i.e., nH = 3 for a primary amine and nH = 1 for a

tertiary amine. Cyproheptadine, methiothepin, mianserine, psilocybin, and psilocin were not included in the regression analysis

since no partition data were available for these molecules or their

analogs. Also excluded from the analysis was LSD, since a fair

number of both enantiomorphs would be needed to allow a meaningful

analysis of optical isomers (Lien 1976). 

RESULTS AND DISCUSSION

The equations correlating the displacement data with the physicochemical paramters are summarized in Table 2. For the displacement of [3

H]-5=HT, a parabolic equation of log P gives a

statistically highly significant correlating (Eq. 3, r = 0.873,

s = 0.766, F1, 11 = 34.05; F1, 11.9995 = 23.6). About 76 percent

(r2 = 0.76) of the variance in the data can be "accounted for" by

this equation.

106
TABLE 2

Equations Correlating the Displacement Data with the Physicochemical Constants
Addition of the log M.W. term does not improve the correlation

significantly (Eq. 4). The ideal lipophilic character log Po for

maximum binding derived from Eq. 3 is 2.11 with a 95 percent confidence interval of 1.76 to 2.52 (see Figure 1).

For the displacement of d-[3H]-LSD, log M.W. gives a slightly better

correlation than log P (Eq. 6 vs. Eq. 5). A parabolic equation of

Log P again gives a highly significant correlation (Eq. 7). About

82 percent (r2 = 0.82) of the variance in the data is "explained"

by this non-linear equation. An F-test indicates that the (log P)2

term is significant at 99.9 percentile level (F1,13 = 20.05;

F1,12 = 18.7). Addition of the log M.W. to Eq. 7 yields Eq. 8,

which is significant at a level between 90 and 95 percentile.

(F1,12 = 3.65; F1,12.90 = 3.18). The log P0 from Eq. 8 is 2.87

(2.08-4.08), while that from Eq. 7 is 3.36 (2.77-4.83), significantly highers than that of Eq. 3 for the displacement of d[3

H]LSD. Furthermore,

(see Figures 2 and 1), indicating greater degree of hydrophobic

interactions involved in the displacement of d[3H]LSD. Furthermore,

the dependence on log M.W. is positive in Eq. 8 while that in Eq. 4

is negative. This reflects that while large molecules like chlorpromazine and promethazine will be effective in displacing d[3

H]LSD

from the binding site but not so effective in displacing [3H]5-HT.

This is also partly due to the difference in log P0 values as well

as the qualitatively different dependence on log M.W. Addition of

nH term does not result in any significant improvement in correlation for both sets of data.

It is interesting to note that log P0 values obtained are very much

in line with the log P0 values observed for the CNS activities of

various drugs observed in vivo (Lien 1976; Hansch and Clayton

1973). Since the displacement of binding data were obtained from

membrane preparations, the difference in log P0 values reflects

most likely the different hydrophobic characters of the binding

sites of serotonin and LSD.

It is important to note that for unlabelled 5-hydroxytryptamine

displacing [3H]5-HT (see Table 1) the observed log 1/IC50 value is

1.54 units higher than the predicted value, while for the displacement of d[3

H]LSD the difference between the observed and the calculated value is only 0.62 unit. The 5-methoxytryptamine, on the

other hand is well predicted for both binding sites. This is in

agreement with the original qualitative conclusion of Bennett and

Snyder concering the crucial role of the 5-OH group in the binding

site. Similar activity enhancement by the OH group has also been

observed in series of anticholinergic compounds (Lien, Ariëns and

Bled 1976).

The important of the side chain amino group in binding with both

sites is clearly indicated by the extremely low binding activities of

108
FIGURE 1

109
FIGURE 2

110
5-hydroxytryptophol and 5-hydroxyindole acetic acid (IC50 > l05

 nm).

It is conceivable that an anionic site is involved in the binding of

the protonated form of the side chain amino group under physiological pH.

The positive dependence on the log M.W. and the higher log P0 for

the displacement d H LSD suggest a greater area of accessory

binding site available for less specific type of intermolecular

interactions (e.g., van der Waals forces)(Ariëns and Simonis 1967).

It is quite conceivable that the two different conformations with the

critical binding site (an anionic site) being surrounded by two

different hydrophobic environments. The one for the agonist (e.g.,

5-HT) then appears to be less hydrophobic than the one for the

antagonist (e.g., methysergide, cyproheptadine, etc.). It is well

known that the log P0 for anticholinergics and antihistaminergics

are also higher than that of the respective agonists (Lien, Ariëns

and Beld 1976; van den Brink and Lien 1977). LSD having an

intermediate lipophilicity can occupy both receptors and behave as

agonist-antagonist.

Shih and Rho (1977) have shown that the serotonin-binding protein

obtained from affinity chromatograph (Shih et al. 1974) caused a

bathochromic shift in the fluorescence and the excitation wavelength of LDS, while bovine serum albumin did not cause such a

change. This was intepreted an extensive delocalization of

the LSD molecular orbital electrons when LSD was bound to the

serotonin-binding protein.

ACKNOWLEDGEMENTS

This study was supported in part by BRSG Grant RR-05792 awarded by

the Biomedical Research Support Grant Program, Division of Research

Resources, National Institute of Health.

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drugs, do they act on common receptors? Ann N Y Acad Sci, l44:842-

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111
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Europ J Pharmacol, 35:245-252, 1976.

Shih, J.C. and Rho, J. The specific interaction between LSD and serotonin-binding protein. Res Commun Chem Path and Pharmac, 16(4):637-

647, 1977.

Shih, J.C., Eiduson, S., Geller, E., and Costa, E. Serotonin-binding

proteins isolated by affinity chromatography. In: Costa, E., Jessa,

G.L., and Sandler, M., edits. Advances in Biochemical Psychopharmacology. Vol. 11. New York:Ravens Press, 1974, pp.101-104.

Snyder, S.H. Neurotransmitter and drug receptors in the brain. Biochem

Pharmacol, 24:137l-1374, l975.

Van den Brink, F.G., and Lien, E.J. pD2-, pA2- and pD2- values of a

series of compounds in a histaminic and a cholinergic system. Europ J

Pharmacol, 44:251-270, 1977.

112
AUTHORS

Y. L. Chan, E. J. Lien, and J. C. Shih

School of Pharmacy, University of Southern California

Section of Biological Sciences and

Section of Biomedicinal Chemistry

1985 Zonal Avenue, Los Angeles, CA. 90033

113
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Structure Activity Studies by Means

of the SIMCA Pattern Recognition

Methodology

J. Dunn and Svante Wold

INTRODUCTION

Since the publication of the linear models by Free and Wilson (Free

and Wilson 1964) and Hansch (Hansch 1969) there have been many

published examples of QuasAR using these methods. Such examples

illustrate that biological-structure activity data can behave in a

continuous manner and can give consistent results when analyzed

by appropriate statistical methods. The method of Hansch can be

very helpful in attempts to determine how subtle changes in structure

can effect changes in the level of biological response of structurally

similar substances.

This method has limitations, however, and cannot be used, for example,

to classify compounds according to their type of biological response.

For solutions to such problems one must look to methods of classification such as linear discriminant analysis and the various methods

of pattern recognition (PaRC). In the past few years the use of

such methods in the solution and development of QuaSAR has been

reported (Martin et al. 1974; Ting et al. 1973; Kowalski and Bender

1974; Chu et al. 1975; Soltzberg and Wilkens 1977). Such methods also

have limitations and their use in developing QuaSAR has been

criticized (Perrin 1972; Mathews 1975).

In a recent report (Dunn, Wold and Martin 1977) we have described

the use of the SIMCA method of PaRC in the classification of

phenethylamine beta adrenergic agonists and antagonists of general

structure (I).

In this report we describe the SIMCA method, briefly compare it to

other methods of PaRC and illustrate its utility in classifying the

above mentioned agonists and antagonists. The data analyzed are

those reported by Lefkowitz (Lefkowitz et al. 1976).

114
PHILOSOPHY OF THE USE OF PaRC IN THE DEVELOPMENT OF QuaSAR

The basic assumption underlying the application of PaRC methods to

the development of QuaSAR is that structurally similar substances

can be described mathematically to be similar. If it is assumed

that the type of pharmacological response that a compound elicits

is a highly specific function of its structure and that if seemingly

similar substances give different (opposite) responses, then mathematical rules based on the chemical structure of the two types

(pharmacological) of compounds should be derivable which could be

used to distinguish or classify these compounds. The use of PaRC,

therefore, requires two considerations: 1) a description of the

compounds by appropriate structural parameters and 2) a method which

will derive rules based on these parameters that will distinguish

one pharmacological type from the other.

The compounds of known pharmacological type, in this case, agonists

and antagonists are placed in sets called training sets. From

each training set the methodology detects patterns of similarity

which exist between the compounds and derives the mathematical

ruleswhichdefine eachclass. In a second step, compounds of

unknown class assignment, the test set, are then compared with

the rules of each class: They are assigned to the class which they

are calculated to be similar to. From the calculation the probability of the prediction should also be obtained.

The description of the compounds by structural parameters is

usually the most critical aspect of a PaRC study. If the compounds

to be classified are not described as completely as possible the

method chosen to generate the mathematical rules might not be able

to distinguish the different classes. For use with the linear learning machine and linear discriminant methods it has been suggested

that molecular descriptors derived from the structure, such as

the presence or absence ofcertainfragments or functional groups

be used (Brugger, Stuper and Jurs 1976). The generation of such

descriptors from connection tables and their use in PaRC have been

discussed (Brugger, Stuper and Jurs 1976).

For the description of each compound in this report, physicochemically

based substituent parameters such as the Hansch p constant, the

Hanmett s constant and the Verloop steric constants were used. These

were chosen because it is assumed that agonist and antagonist activity

result from specific drug receptor interactions and we believe

that parameters derived from systems which model these interactions

should be used as the basis of differentiation. Quantum mechanically

based descriptors might also be useful since they, along with the

other descriptors mentioned above, are theoretically derivable and

their use precludes synthesizing compounds and making measurements

of them. Measured physicochemical parameters such as pKa, log P,

pKi, etc., can also be included as descriptors if available. This

overall approach to compound description has the additional advantage

of using variables which are derived from continuous functions.

The separation of classes of pharmacological substances on the basis

of such descriptors is illustrated graphically in Figure l. Here in

115
3-dimensional variable space two classes of compounds are well

separated. Each compound is represented by a point (vector) in the

3-space.

FIGURE 1

This brings us to the second consideration in the use of PaRC

methods and that is the selection of a method which can be used to

separate the two or more classes in a study. The commonly used

linear learning machine and linear discriminant analysis methods

classify on the basis of a hyperplane which is inserted between the

classes as shown in Figure 1. These methods are sensitive to the

number of variables (N) relative to the number of compounds (M)

since if N > M a separation plane will always exist. In addition

if N approaches M the probability of finding such a plane increases

which can lead to the observation of artifacts. This limitation,

which also applies to multivariable regression methods, requires

that M > N/3 where N is the initial number of variables in the analysis.

The SIMCA method, differently from the linear learning machine or

linear discriminant analysis, classifies by deriving a classification

function that contains each class in a closed mathematical structure,

or 'hyperbox'. This is shown in Figure 2. A more detailed discussion of the method will be presented later. At this time a comparisonofthe two approaches will be presented in the context of

different levels of classification which are possible in QuaSAR.

116
FIGURE 2

FORMULATION OF A PROBLEM AS ONE OF CLASSIFICATION

Once it has been recognized that a problem can be treated as one of

classification, there are different levels of classification which

can be achieved (Dunn, Wold and Martin 1977). The levels are very

much method dependent and the ambition of a classification study can

determine the method of classification required. Four levels of

classification can be recognized (I-N).

Level I

At this level the objective is to classify an unknown as a member of

one class or another. All methods of data analysis can classify

at this level.

Level II

At this level the objective is to classify an unknown as a member

of one class or the other with the possibility of the unknown being

a member of none of the defined classes. If the unknown is classified as being a member of neither class it is considered to be an

outlier and may be a member of a third, as yet undescribed class.

Since SIMCA confines the classes in closed mathematical structures

it always works on at least level II.

117
Level III

At level III classification includes that of level II accompanied

by the observation of a relationship between the position of the

compounds in the class structure and the level of activity of the

compounds. Such a relationship, if observed, would constitute a

QuaSAR. Therefore, at level III one would not only be able to tell

what type of activity an unknown will have but also one can predict

the level of response expected from the unknown.

Level IV

On this, or highest, level sweral effect variables such as measures

of side effects, may be available for objects in the classification

study. One might find, for example, that toxicity of certain canpounds could be related to their position in the class structure

derived from classification of the agents as agonists of the beta

receptor. The more toxic substances may be positioned differently

from the more active substances and one could conclude that the

processes have different structural requirements and specificities.

Since SIMCA encloses the classes in the aforementioned mathematical

structures or 'hyperboxes' it can operate at the higher levels of

classification. For the classification of agonists and antagonists

of the beta adrenergic receptor we formulate the problem at level III

in which a closed structure is defined for each class. After classification the position of each compound in each class is related to

its activity as agonist or antagonist.

DESCRIPTION OF SIMCA

The basis for the separation of classes by SIMCA is that the

descriptor variables for the compounds which compose each class can

be described by a principal components (PC) model. This is based

on the assumption that the function generating the variables can

be Taylor expanded (Wold 1976; Wold and Sjöstrom 1977). The PC

model is shown in equation 1. Referring to Figure 2 this corresponds

to an A-dimensional plane of closest fit of the data points in the

space chosen to describe the compounds. The indices i and k refer

to variables and compounds, respectively, while y refers to a specific

variable. a refers to the specific component in the PC model for the

g-th class. mi is the mean value for variable i, b is a term which

is model specific and u is a compound specific term. eik are the

residuals for the variables y. The compound specific term, u,

is a measure of the pisition of the compound in the variable-space.

For each class a standard dwiation can be calculated relative to

the plane of closest fit. A similar standard deviation can be

calculated for each object in each class. Fit to the class is

determined by this parameter which is approximately F-distributed.

If a compound has a standard deviation for its residuals which is

118
less than that for the class, it is a member of that class and it

falls within the "hyperbox" for that class.

In classification studies a complete description as possible of

the compounds in each class is desirable. Therefore, a large

number of variables, some of which may be irrelevant, can result.

A rationale must be devised by which the irrelevant variables can be

eliminated from the class description as they can lead to "noise" in

the analysis. SIMCA uses information from the residuals, eik, in

assessing variable significance. Variables which contribute to the

determination of class structure are said to have modelling power

while those which contribute to class separation are said to have

discrimination power. In SIMCA analyses variables with low modelling

and low discrimination power are deleted. This has the advantage of

selecting variables which optimize class description rather than those

which optimize class separation.

As in most methods of PaRC, SIMCA can use variables which are regularized. In this study data are regularized by placing them on a scale

such that each variable has zero mean and variance of unity. This

prevents the masking of variables with little variation by those with

large variation. Other variable transformations such as conversion

to logs, etc., can be used.

DATA SET OF BETA ADRENERGIC AGONISTS AND ANTAGONISTS

The biological activities and descriptor variables for the compounds

in this study are given in Tables I and II. The compounds are classified in the training sets according to that of Lefkowitz (Lefkowitz et

al. 1976). The biological measurements were made on racemic mixtures

with the affinities (binding constants) for the beta receptor determined by displacement of -(3

H)-alprenalol from the receptor binding

sites of partially purified frog erythrocytes. The agonist activity

was determined as the ability to stimulate beta adrenergic coupled

adenylate cyclase while antagonist activity was determined as the

ability to inhibit agonist stimulation. The agonist used was isoproterenol.

The positions of variation in structure (I) were described by Rekker

hydrophobic fragment constants (Nys and Rekker 1974), Es constants

(Taft 1956) and Verloop steric constants (Verloop, Hoogenstraaten and

Tipker 1976). Electronic effect of the amino group was characterized by σ- star (Taft 1956) and the estimated pKa (Clark and Perrin

1964). In addition the binding constant to the beta receptor was

included as a variable for each compound.

RESULTS

Using the regularized data as previously discussed, SIMCA was applied

to these data. Analysis with all variables included led to a PC

model with 2 (A=2) components describing the agonists and one with

1 component describing the antagonists. On the basis of low modelling

and low discrimination power the variables σm, fφ , Lm and Bm were

deleted for the compounds in both classes. SIMCA application then

led to a PC model with 3 components (A=3) describing each class.

119
TABLE I
TABLE II

Substituents and descriptor variables
TABLE III
On the basis of these models, the compounds in the training sets were

classified with 15/15 (100%) of the agonists being correct and 15/17

(88%) of the antagonists being correct. These results were validated

by omitting every fourth object from the training set for each

class and from the 3 component model calculated for each class, the

deleted compounds were classified. This process was repeated until

all compounds had been deleted one and only one time. The results

of the validation was correct classification of 30/32 (94%) of the

compounds in the classes. The results of the classification are

given in Table II and the statistical parameters in Table III.

On the basis of the PC models objects 33-37 of the test set were

classified. It can be seen in Table II that compound 33 (norepinephrine) is correctly classified as an agonist while compounds 34-36,

tyramine, octopamine and dopamine were also classified as agonists.

Dopamine is classified by Lefkowitz as an agonist even though its

intrinsic activity could not be determined. Its affinity was determined from the assay for antagonists. Tyramine and octopamine were

both classified as antagonists by Lefkowitz. Compound 37, phenethylamine, had no detectable activity in either assay; SIMCA classifies

it as an antagonist. Classification, in itself, does not provide

sufficient information for identification of these compounds.

GRAPHICAL ANALYSIS OF THE ui PARAMETERS

The ui values for the PC models which describe each class are

measures of the position of the objects in the variable space for

that class. It is reasonable to assume that those objects with

similar levels of activity will cluster in the variable space as do

the objects with similar types of activity. This is shown in Figures

3 and 4 in which u1 vs u2 is plotted for the classes 1 and 2, respectively. For class 1 the less active substances are in the region

with positive u1 and u2 coordinates while the more active compounds

have negative u1 and positive u2 coordinates. For the agonists

coordinates A plot of (u1 + u2) vs activity for antagonists is

(class 2) the less active analogs cluster with negative u1 and u2

given in Figure 5 while Figure 6 shows a plot of (u1 - u2) for the

agonists. Inclusion of the test compounds shows that they are predicted to be weekly active.

PaRC, as a predictive tool, should be trained using variables which

are theoretical in the sense that they can be derived for a compound without the necessity for synthesizing and making measurements

on it. The experimentally determined receptor binding constant was

included in this study as a descriptor in the training sets. The

relevant case would be that in which a hypothetical molecule is

created in order that its type and level of activity could be

predicted. In this circumstance, to test our PC models derived with

the binding constant included the models were applied to the training sets with pKb set to zero. The classification that resulted

was significant with 88% (two compounds from each class incorrect)

classification. The result of a similar graphical analysis as

discussed above are given in Figures 7 and 8 and it can be seen that

the graphical analysis holds. The antagonists are well predicted

while the agonists are not quite as well fit.

123
FIGURE 3

FIGURE 4

124
FIGURE 5

FIGURE 6

125
FIGURE 7

FIGURE 8

126
The identification of the test objects can be better understood

in terms of the graphical analysis. Their classification was based

on pKb values set as upper limits of 3.70 since they could not be

experimentally determined to be above this value. Their positions

in their predicted class are shown in the Figures 5 and 6 and it can

be seen that they are estimated to have very low activities.

SIMCA as shown by this graphical analysis can lead to significant

information further than that of classification. The objective

of some PaRC studies has been classification at level I in our

proposed scheme. Such studies may sometimes be trivial to the

trained pharmacologist. Level II and III studies are usually

less trivial. In this first application of SIMCA to study the QuaSAR

of biologically significant compounds, we are pleased with its

performance and its predictive ability, not only of pharmacological

type but of the level of activity for each type.

REFERENCES

Brugger, W.E., Stuper, A.J., and Jurs, P.C. Generation of descriptors

from molecular structure. J Chem Info Computer Sci, 16(2): 105-

110, 1976.

Chu, K.C., Feldmann, R.J., Shapiro, M.B., Hazard, G.F., and

Geran, R.I. Pattern recognition and structure-activity relationship studies: Computer-assisted prediction of antitumor activity

in structurally diverse drugs in an experimental mouse brain

tumor system. J Med Chem 18(6): 539-545, 1975.

Clark, J. and Perrin, D.D. Bacisity constants for organic bases.

Quart Rev, 18: 295-320, 1964.

Dunn, W.J., Wold, S., and Martin, Y.C. A structure-activity study

of beta-adrenergic agents using the SIMCA method of pattern

recognition. J Med Chem, submitted for publication, 1977

Free, S.M. and Wilson, J.W. Quantitative structure activity relationships. J Med Chem, 7(2): 395-404, 1964.

Hansch, C. A quantitative approach to biochemical structure-activity

relatioships. Accts Chem Res, 2(2): 232-239, 1969.

Kowalski, B.R. and Bender, C.F. Application of pattern recognition to screening prospective anticancer drugs, adenocarcinoma

755 data. J Amer Chem Soc, 96(3): 916-917, 1974.

Lefkowitz, R.J., Mukherjee, C., Carom, M.G, and Mullikin, D.

Structure-activity relationships of adenylate cyclase-coupled betaadrenergic receptors: determination by direct binding studies.

Mol Pharmacol, 12(1): 16-31, 1976.

127
Martin, Y.C., Holland, J.B., Jarboe, C.H., and Plotnikov, N.

Discriminant analysis of relationships between physical properties and inhibition of monoamine oxidase by aminotetralins and

aminoindans. J Med Chem 17(4):409-414, 1974.

Mathews, R.J. A comment on structure-activity correlations

obtained using pattern recognition methods. J Amer Chem Soc,

97(4):935-936, 1975.

Nys, G.G. and Rekker, R.F. The concept of hydrophobic fragmental

constants (f-values) II. Extension of its applicability to the

calculation of lipophilicities of aromatic and heteroaromatic

structures. Eur J Med Chem 9(4):361-375, 1974.

Perrin, C.L. Testing of computer-assisted methods for classification

of pharmacological activity. Science, 183:551-552, 1972.

Soltzberg, L.J. and Wilkens, C.L. Molecular transforms: a potential

tool for structure-activity studies. J Amer Chem Soc, 99(2):

439-443, 1977.

Taft, R.W. Separation of polar, steric and resonance effects in

reactivity. In: Newman, M.S., ed. Steric Effects in Organic Chemistry

New York: John Wiley, 1956. pp. 556-676.

Ting, K.H., Lee, R.C.T., Milne, G.W.A., Shapiro, M., and

Guarino, A.M. Applications of artificial intelligence: relationships

between mass spectra and pharmacological activity of drugs. Science,

180:417-420, 1973.

Verloop, A., Hoogenstraaten, W., and Tipker, J. Development and

application of new steric substituent parameters in drug design.

In: Ariens, E.J. ed., Drug Design, New York, Academic Press, 1976.

Wold, S. Pattern recognition by means of disjoint principal

components models. Pattern Recog, 8:127-139, 1976.

Wold, S. and Sjöstrom, M. SIMCA: a method for analyzing chemical

data in terms of similarity and analogy. In Kowalski, B.R., ed.

Chemometrics: Theory and Application. ACS symposium series no. 52.

1977, pp. 243-282.

AUTHORS

W. J. Dunn, III, PhD, Department of Medicinal Chemistry, University

of Illinois at the Medical Center, Chicago, Illinois and Visiting

Professor, Research Group for Chemometrics, Department of Organic

Chemistry, Institute of Chemistry, Umeå University, Umeå, Sweden.

Svante Wold, PhD, CIC Fellow, Research Group for Chemometrics,

Department of Organic Chemistry, Institute of Chemistry, Umeå

University, S-901-87 Umeå, Sweden.

128
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Quantitative Relationship Between

Antinociceptive Activity and Opiate

Receptor Affinity: The Importance

of Lipophilicity

Arthur E. Jacobson

Medicinal chemists have synthesized a multitude of

opioids over the past century and a half. The term

"opioids" has been defined as substances which have

morphine-like pharmacological effects. I will use

"opioids" and "narcotic agonists" as interchangeable

terms. There are, then, a considerable number of these

compounds of diverse structural type, all of which have

at least one pharmacological property in common. The

number of compounds, and the defined biological effect

which can be easily measured in small animals, should

lead theoretically inclined medicinal chemists to the

opioids in their search for the relationships between

molecular structures and a biological property.

Unfortunately, apparently the very diversity of these

molecules, and their rather peculiar pharmacologic

effects, have militated against successful QuaSAR.

OPIATE RECEPTORS

The discovery of the opiate receptor in verterbrate

brain has given us a tool with which we can measure

receptor mediated events. These receptors may be there

to interact with endogenous ligands, the enkephalins

(Hughes et al. 1975) and endorphins (Ross et al. 1977).

The endogenous ligands are small, or large, peptides,

and presumably the opioids mimic at least one of their

pharmacological effects, that of analgesic action. The

opioids, on interaction with the opiate receptor, can

produce several effects in vivo. Almost all opioids

have been shown (Kosterlitz, Waterfield, and Berthoud

1973) to have some antagonist effect, as well as

narcotic agonist or opioid effect. They also have "side

effects" in vivo, such as tolerance, dependence of the

opiate type, some nausea, cough suppressant effect,

respiratory depressant effect, etc. The "side effects"

129
and the narcotic antagonist activities, may be mediated

by the same receptor.

Multiplicity of Receptors

There has been discussion in the literature on the

possibility of a number of receptors with which the

opioids and their antagonists can interact. Several

scientists (Martin et al. 1976; Lord et al. 1977)

believe that there are mu receptors for morphine types

of opioids, sigma receptors for agonist antagonist types

like N-allylnormetazocine, kappa receptors for

ketocyclazocine-like compounds and, perhaps, other types

of receptors as well. It should be noted that Martin's

work is-based on symptomatology from in vivo dog work,

and Kosterlitz (Lord et al. 1977) has compared the rank

order of binding affinity of a series of compounds in

the mouse vas deferens and in the guinea pig ileum. Our

recent work on a few "nonclassical" narcotic antagonists

(Jacobson and Klee 1978) may have some bearing on the

concept of multiple receptors. I will return to that

subject at the end of the paper. It is important,

howerer, to maintain the idea that we have the

possibility of multiple receptors, and that opioids tend

to have more than one effect on interaction with the

receptor or receptors, if we are going to speak of the

relationship of molecular structure to biological

action. My topic is necessarily limited to the

relationship of receptor binding to antinociceptive

action, since I have not succeeded in relating structure

to biological effect quantitatively.

Receptor Interaction And Biological Activity

The main point which I should like to make is that the

assumption of parallelism between receptor interaction

and biological activity can be misleading. I do not

believe that the measurement of receptor binding

affinities always relates directly to the biological

effect of the opioids in vivo. This is a very simple

point, but one which I believe has been unfortunately

ignored by many pharmacologists, and perhaps by some

medicinal chemists. In order to relate receptor binding

to biological activity, a series of compounds should be

chosen which have mostly the same pharmacological

activities. The type of pharmacological effect that

will occur when a drug interacts with the opiate

receptor cannot be presumed a priori, but must be

ascertained from in vivo experiments.

The compounds which I will discuss have, overuhelmingly,

antinociceptive effects in vivo, in the Eddy hot plate

test (Eddy and Leimbach 1953; Jacobson and Nay 1965).

Most of these compounds have been examined in the rhesus

130
monkey, under the aegis of the Committee on Problems of

Drug Dependence (CPDD), Inc., and have been found to

have little or no narcotic antagonist activity. That

is, they do not appear to exacerbate the opiate

dependence syndrome in single dose suppression studies

in the Rhesus monkey, or precipitate abstinence in

nonwithdrawn monkey studies. They do have "side

effects", which I shall ignore, and they all presumably

interact with the classical mu receptor. Thus, we will

discuss compounds which have a measurable

antinociceptive effect (in one laboratory, by the same

technician), and which, hopefully, interact with mainly

one type of receptor.

QUALITATIVE RELATIONSHIP OF BINDING AND ACTIVITY

The initial attempts to relate receptor binding to

antinociceptive activity were done by pharmacologists

who were merely interested in showing that the opiate

receptor interacted stereospecifically with opioids and

their antagonists. These were qualitative relationships

established to indicate that there actually were

receptors for the opioids in vertebrates.

LINEAR DEPENDENCE IN HOMOLOGOUS MOLECULES

The first quantitative relationship which was published,

that I am aware of, was from my laboratory. This was

the work of Yilson et al. (1975). They determined the

antinociceptive activity of a series of homologous

ketobemidones and related that activity to the receptor

binding affinity. I have replotted their data, in

figure 1, so that the log of the activity, in nM/kg, can

be seen to be directly related to the binding constant.

The binding constant is the concentration of drug, in

nM/L, which is required to inhibit stereospecific

binding of (3H) dihydromorphine, or (3H) naloxone, or

(3H) naltrexone, etc., in 1.0 nM concentration, by 50

percent, to the opiate receptors in rat brain

homogenates. They obtained a correlation coefficient of

0.95, and 90 percent of the variance was "explained" by

this direct relationship, in the absence of sodium.

Effect of Sodium on Binding

A reasonable correlation in this, and in other work

(Stahl et al. 1977), could not be obtained in the

presence of added sodium. Sodium salts have been noted

to effect the binding affinity of antagonists

preferentially. They had an inhibiting effect on

narcotic agonists (Pert, Pasternak, and Snyder 1973:

Simon 1975). The suggestion was made that this is due

to a conformational change in the receptor. If the NaCl

to no NaCl (+Na/-Na) ratio is indicative of antagonist

131
FIGURE 1

Relationship Between Antinociceptive Activity and

Opiate Recptor Affinity for N-Alkylnorketobemidones

132
activity, as it has been said to be, then in the

presence of NaCl some of the ketobemidones should have a

small +Na/-Na ratio. That is, a few of the several

ketobemidones tested in rhesus Monkeys by the CPDD

showed antagonist activity, or, at least, caused

behavioral signs in the monkey which might be

interpreted as a physical effect of a narcotic

antagonist.

The ratio did change for some of these compounds, since

some affinities decreased less than others, and none

showed increased binding affinity in the presence of

NaCl. The +Na/-Na ratios predicted that the hexyl and

higher homologs would be antagonists. Of these, only

the hexyl and heptyl homologs mere shown to have

antagonist activity in the monkey, and the N-amyl

compound was noted to have "atypical" antagonist

activity (Swain, Villarreal, and Seevers 1973).

However, the N-amyl compound could not be shown to have

antagonist activity by rat infusion techniques (Dewey

and Harris 1977). The N-hexyl and heptyl compounds are

presently being examined for antagonist activity by rat

infusion. These new binding affinities, in the presence

of NaCl, apparently aid not reflect antinociceptive

activity in vivo. The correlation was excellent only in

the absence of NaCl.

Hot Plate Test for Antinociception

It was noted some time ago that the hot plate test for

antinociceptive activity fails with agonist antagonist

molecules. That is, the antinociceptive activity of

agonist antagonists will generally be underestimated by

that test, However, the hot plate assay, evidently, did

not underestimate the antinociceptive activity of these

ketobemidones, since the correlation with binding in the

absence of sodium was successful. This suggests that

the ketobemidones do not behave as agonist antagonists

in the hot plate test, or had very little antagonist

activity compared with their antinociceptive activity,

in partial disagreement with the results from monkeys

and with the +Na/-Na binding ratio.

Further Quantitative Relationships

In that same year in our laboratory, Rogers, Ong, and

Nay (1975) found a quantitative correlation between

receptor binding affinity and antinociceptive activity

in a set of homologous N-alkyl-3-benzazocines. A replot

of their data is shown in figure 2. The correlation

coefficient obtained was 0.95, the proportion of

variance "explained" by the regression was 89 percent.

The third example of quantitation is from a series of

133
FIGURE 2

Relationship Between Antinociceptive Activity and

Opiate Recptor Affinity for N-Alkylnorbenzazocines

134
diastereoisomeric prodine analogs by Iorio and Klee

(1977): Iorio prepared the series of prodine analogs

shown in figure 3. Their correlation coefficient was

0.99. About 98 percent of the variance was rationalized

by the direct correlation of antinociceptive activity

obtained from hot plate assay, and the receptor binding

from rat brain homogenate data. The data was

transformed into log units for consistency with the

other sets.

CORRELATION IN STRUCTURALLY DIVERSE ANALGESICS

These three examples of quantitative correlation between

activity and binding affinity, in three different

subsets of compounds, certainly appear to indicate that

the antinociceptive activity of opioids is directly

related to their binding to the opiate receptor,

presumably of the mu type. We thought, however, that

before the concept became too firmly established with

medicinal chemists, as it had become with

pharmacologists before any quantitative work had been

completed, we should examine the question of whether the

relationship would be valid over a much wider range of

structurally different opioids (Jacobson, Klee, and Dunn

1977).

lie chose compounds from'the endoethenooripavine,

propionamide, methadone, pethidine, morphinan, and

morphine families of opioids, which had previously had

their partition coefficients determined by Kutter et al.

(1970). The molecular structures of the ten compounds

are shown in figure 4. The data for the regression

analyses, and the experimental and calculated

antinociceptive activities are shown in table 1. A

direct relationship between antinociceptive activity

determined by hot plate assay, and receptor binding

affinity did not exist among these heterogenous

analgesics.

Importance of Lipophilicity

The equations determined by the regression are shown in

table 1. Only 41 percent of the variance could be

accounted for by a direct correlation tetween binding

and activity. A direct relationship also did not exist

between antinociceptive activity and lipophilicity

(equation 2, table 1), nor does a correlation exist

between receptor binding and lipophilicity. In the

latter case, the correlation coefficient uas 0.26. In

the correlation between antinociceptive activity and

binding affinity, the explained variance was increased

to 82 percent, with a correlation coefficient of 0.91,

when the partition coefficient (log P) was included in

135
FIGURE 3

Relationship Between Antinociceptive Activity and

Opiate Recptor Affinity for Prodine Related Compounds

136
FIGURE 4

137
TABLE 1

Data and Equations From Regression Analyses
Table 1 continued
the regression (equation 3, table 1). The use of a

Squared log P term did not improve the correlation

(equation 4, table 1). We concluded from these data

that when a number of different types of opioids are

examined it is necessary to account for their

differential ability to reach the receptors in the

brain. This transport phenomenon can, apparently, be

reasonably simulated by partition coefficients.

Possible Contradiction

In contrast to this work, if not in apparent

contradiction to it, is a paper by Stahl et al. (1977).

They measured antinociceptive activity through

intravenous application in a tail withdrawal reflex

assay of a number of disparate analgesics (including

etotphine, levorphanol, fentanyl, morphine and a number

of neuroleptics and antidiarrheal agents). They

obtained a good correlation, directly, with the binding

affinities for the compounds in rat brain homogenates,

without sodium. The correlation coefficient which they

reported was 0.907, with 82% of the variance explained

by the correlation. In the presence of sodium, the

correlation was not as good, which would be in agreement

with the data from the ketobemidones.

If the data from Stahl et al. (1977) are closely

examined for four of the compounds which have had their

partition coefficients determined by Kutter et al.

(1970), morphine, etorphine, fentanyl and levorphanol,

it can be seen that incorporation of partition

coefficients might be advantageous. In the paper of

Stahl, these four compounds were not predicted to be in

their correct order for antinociceptive potency when

compared with their binding affinities. For a quick

estimation of the effect of the incorporation of

partition coefficients into their data, the coefficients

shown in equation 3, table 1 were utilized, since it was

not possible to determine new coefficients based on only

four data points. The effect on the rank order of

antinociceptive potencies is shown in table 2. When

partition coefficients are included, the correlation is

improved. The calculated and observed activities agree

nicely, if fortuitously, by the incorporation of a

constant. With these four compounds, in any case, the

antinociceptive activity did not parallel receptor

binding, unless partition coefficients were included, in

agreement with my data. Overall, with their large

number of compounds, the statistics proved to be

acceptable. It is possible, however, that if the

partition coefficients of the compounds were determined

and incorporated in the regression, a greater proportion

of the variance would be accounted for.

140
Thus, although a direct correlation between binding

affinity and in vivo opioid activity exists within

limited homologous series of opioids, the inclusion of

partition coefficients is advantageous, and sometimes

necessary, to achieve reasonable correlations among

opioids with radically different structures. It is

likely that if we knew more about the metabolism of the

various types of opioids, and could incorporate these

data in the regression, we would obtain a further

increase in "explained" variance.

Table 2. Relationship Betweeen Antinociceptive Activity

And Receptor Binding Affinity After Incorporation of

Partition Coefficients

Compound Log 1/IC50aLog 1/ED50a

b,c

Obs. Calcd.

Etorphine 0.40 2.42 2.49

Pentanyl -1.40 1.48 0.86

Levorphanol -0.60 0.07 0.69

Morphine -1.43 -0.92 -1.17

a

 Original data from Stahl et al. 1977, transcribed

into log units; b from equation 3 in table 1; c includes

a constant (+2.0), added to obtain correspondence with

observed data.

BIOLOGICAL TESTS FOR NARCOTIC ANTAGONISTS

The work which has been examined merely relates

biological activity to receptor binding. Although we

have shown that partition coefficients should not be

neglected when a disparate group of opioids are

examined, the molecular structures of these opioids have

not been quantitatively related to either binding

affinity or to antinociceptive activity. The

relationship of the molecular structure of agonist

antagonists to in vivo antagonist biological activity

may also prove to be difficult. Although there are

141
several assay systems which can quantitatively relate in

vivo antinociceptive activity of opioids, and these are

generally relatable to each other, there are relatively

few assay systems available for determining the

antagonist activity of agonist antagonists.

Assay Systems

Ue obtain qualitative antagonist activities from studies

in monkeys, through the Committee on Problems of Drug

Dependence, Inc. Also, Aceto et al. (1977) have

reported their quantitative results from a tail flick

antagonist assay vs. morphine to the Committee on

Problems of Drug Dependence, Inc. In a rapid review of

the last three years, I have found at least twenty very

different antagonists for which they have determined

antagonist activity. It should be noted that a number

of these have standard error limits an order of

magnitude greater than we normally obtain in the hot

plate assay for antinociceptive activity, and that some

compounds which show antagonist activity in the monkey

do not show antagonist activity in the tail flick assay.

I do not know which is more dependable, when compared

with the effect of the compound in man. Unfortunately,

few partition coefficients, or other physicochemical

data, and little binding affinity data are knoun for

most of these compounds,

Another set of compounds, some of which overlap those

obtained by Aceto et al. (1977), have been examined by

Kosterlitz, Waterfield, and Berthould (1973) in the

guinea pig ileum assay and in the mouse vas deferens.

These two sets of data are the most comprehensive

available in the literature. It would be of interest,

as more compounds are added to both sets, to see whether

these quite different assays correlate well with each

other.

Atypical Narcotic Antagonists

We have noted that odd results can be obtained from the

guinea pig ileum and the mouse vas deferens assays with

a new type of antagonist. We have prepared opioid

antagonists based on substitution in the 9-alpha

position of the benzomorphans, rather than on the

nitrogen atom (Rice et al. 1977). Classical, potent,

opioid antagonists had been prepared by replacing the

N-methyl group of potent, structurally rigid, opioids by

certain, moieties.

our non-classical type of opioid antagonist appears to

interact with the receptors in the ileum, and even more

so in the mouse vas deferens, to give above normal

increases in the amplitude of the twitch after naloxone

142
treatment (Jacobson and Klee 1978), unlike the classical

antagonists which do not produce this supranormal

increase. The experiments were run by first obtaining a

decrease in twitch tension by normorphine (10-7 M),

followed by reversal (but not quite back to baseline

levels) with the non-classical antagonist (10-6 to 10-7

M). Introduction of naloxone (10-7 M) at that point

resulted in a supranormal increase in twitch tension.

These experiments can be interpreted as an interaction

of these "non-classical" antagonists with receptors

other than the mu receptor or, more simply, by assuming

that these antagonists have stimulant activity as well

as opioid antagonist activity. We may be seeing a

complex mixture of biological actions which would not be

easy to dissociate, rendering QuaSAR with these

compounds much more difficult. Thus, QuaSAR with

structurally disparate antagonists should be critically

examined when biological activities are obtained from

ileum or vas deferens measurements. Some of the binding

which is measured may not be due to the antagonist

activity of the molecule.

QuaSAR IN THE FUTURE

If QuaSAR with disparate opioids or agonist antagonists

are attempted, they should include the newer, and

apparently quite potent, peptides which have recently

been reported in, for example, the Basel Zeitung. Some

of these peptides, from the Sandoz Pharmaceutical Co.,

are said to have ten thousand tines the activity of

morphine in vivo. It would be of great interest to

obtain physicochemical constants of different kinds for

these and other antinociceptive agents with such

remarkable structural diversity, so that we might, one

day in the future, try to relate structure to activity

with a greater hope for success.

ACKNOWLEDGMENTS

I would like to thank Mrs. Louise Atwell (NIAMDD, NIH)

for the biological assays which she determined, using

the hot plate method. I would also like to thank Dr. C.

Hansch (Pomona College, California) for allowing the use

of his computer program, and Dr. Frank Quinn (National

Cancer Institute, NIH) for his conversion of the Hansch

program to the IBM 370/165 computer system.

REFERENCES

Aceto, M.D., Harris, L.S., Dewey, W.L., and Balster,

R.L. Dependence studies of new compounds in the rhesus

monkey. In: Problems of Drug Dependence. Washington,

D.C.: Committee on Problems of Drug Dependence, Inc.,

1977. Addendum. pp. 586-613.

143
Dewey, W.L. and Harris, L.S. "Rat infusion studies with

ketobemidones." Unpublished data, 1977.

Eddy, N.B. and Leimbach, D. Synthetic analgesics. II.

Dithienylbutenyl- and dithienylbutylamines. J Pharmacol

Exp Ther, 107:385-393; 1953.

Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill,

L.A., Morgan, B.A., and Morris, H.R. Identification of

two related pentapeptides from the brain with potent

opiate agonist activity. Nature, 258:577-579, 1975.

Iorio, M.A. and Klee, W.A. Relationships between opiate

receptor binding and analgetic properties of

prodine-type compounds. J Med Chem, 20:309-310, 1977.

Jacobson, A.E. and Klee, W.A. "Assays for the

antagonist activity of non-classical narcotic

antagonists. Manuscript in preparation, 1978.

Jacobson, A.E. and Hay, E.L. Structures related to

morphine. XXXI. 2'-substituted benzomorphans. J Med

Chem, 8:563-566, 1965.

Jacobson, A.E., Klee, W.A., and Dunn, W.J. A

quantitative relationship between antinociceptive

activity, opiate receptor affinity and lipophilicity.

Eur J Med Chem, 12:49-52, 1977.

Kosterlitz, H.W. Waterfield, A.A., and Berthoud, V.

Assessment of the agonist and antagonist properties of

narcotic analgesic drugs by their actions on the

morphine receptor in the guinea pig ileum. In: Braude,

M.C., Harris, L.S., Hay, E.L., Smith, J.P., and

Villareal, J.E., ed. Narcotic Antagonists, Advances in

Biochemical Psychopharmacology. Vol. 8. Hew York: Raven

Press, 1973. pp. 319-334.

Kutter, E., Herz, A., Teschemacher, H.-J., and Hess, R.

Structure-activity correlations of morphine-like

analgetics based on efficiencies following intravenous

and intraventricular application. J Med Chem,

13:801-805, 1970.

Lord, J.A.H., Waterfield, A.A., Hughes, J., and

Kosterlitz, H.W. Endogenous opioid peptides: multiple

agonists and receptors. Nature (London), 267:495-499,

1977.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler,

R.E., and Gilbert, P.E. The effects of morphine- and

nalorphine-like drugs in the nondependent and

morphine-dependent chronic spinal dog. J Pharmacol Exp

Ther, 197:517-532, 1976.

144
Pert, C.B., Pasternak, G., and Snyder, S.H. Opiate

agonists and antagonists discriminated by receptor

binding in brain. Science, 182:1359-1361, 1973.

Rice, K.C., Klee, W.A., Aceto, M.D., Swain, H.H., and

Jacobson, A.E. Potential long acting opiate

antagonists: preparation, pharmacological activity, and

opiate-receptor binding of N-substituted

2'-hydroxy-5-methyl-9 alpha-propyl-6,7-benzomorphans. J

Pharm Sci, 66:193-197, 1977.

Rogers, M.E., Ong, H.H., and Bay, E.L. Analgetic

activity and in vitro binding constants of some

N-alkyl-3-benzazocines. J Med Chem, 18:1036-1038, 1975.

Ross, M., Dingledine, R., Cox, B.M., and Goldstein, A.

Distribution of endorphins (peptides with morphine-like

pharmacological activity) in pituitary. Brain Res,

124:523-532, 1977.

Simon, E.J. Opiate receptor binding with 3H-etorphine.

Neurosciences Res Prog Bull, 13(1):43-50, 1975.

Stahl, K.D., Van Bever, W., Janssen, P., and Simon, E.J.

Receptor affinity and pharmacological potency of a

series of narcotic analgesic, anti-diarrheal and

neuroleptic drugs. Eur J Pharmacol, 46:199-205, 1977.

Swain, H.H., Villarreal, J.E., and Seevers, M.H.

Evaluation of new compounds for morphine-like physical

dependence in the Rhesus monkey. In: Problems of Drug

Dependence. Washington, D.C.: Committee on Problems of

Drug Dependence, National Academy of Sciences, National

Research Council, 1973. Addendum. pp. 701-702.

Yilson, R.S., Rogers, M.E., Pert, C.B., and Snyder, S.H.

Homologous N-alkylnorketobemidones. Correlation of

receptor binding with analgesic potency. J Med Chem,

18:240-242, 1975.

AUTHOR

Arthur E. Jacobson, Ph.D.

Medicinal Chemistry Section, Laboratory of Chemistry

National Institute of Arthritis, Metabolism, and

Digestive Diseases

National Institutes of Health

Bethesda, Maryland 20014.

145
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Quantitative Stereo-StructureActivity Relationships I. Opiate

Receptor Binding

Howard Johnson

One of the central problems in the design of safer and more

effective drugs is that of better defining physicochemical properties and structural attributes which contribute to selective high

affinity for specific receptors. While modern methods of analysis

of quantitative structure-activity relationships (QSAR) effectively

address the relationship of structure and bulk physical properties

of molecules (such as partition coefficierits) to macromolecular

binding and transport phenomena these methods are generally deficient in not adequately addressing steric relationships of molecular

geometry in relation to specific receptor binding and biological

activity. Yet it is to be expected that the geometrical distribution of physicochemical properties is of paramount importance with

regard to binding to specific receptors. Indeed this is well substantiated by many examples of stereospecificity in drug action and

in receptor binding as illustrated by the narcotic analgesics

(Goldstein 1974).

The present study represents a reasonably successful attempt to

correlate opiate receptor affinities of a rather diverse group of

narcotic drugs with their three dimensional structures in terms of

the geometrical distribution of additive components of various

physical properties. The structures considered are too diverse for

a simple substituent constant approach, and it is shown that an

adequate correlation is not obtained by considering bulk properties

as represented by molecular parameters such as log P, molecular

wieght, and molar regraction (Hansch 1968). However, the advantages

of multiple linear regression methods are retained in the method

described which is conceptually similar to pattern recognition

approaches. Significant correlations were obtained for forty two

compounds whose affinities for the opiate receptor span a range of

more than then logs (-11.5 to 0.69).

METHODS

Experimental Data. For purposes of multiple regression analysis,

in vitro affinity for the opiate receptor was defined as log 1/IC50

Values for opiate concentrations which cause a 50% displacement of

146
tritiated naloxone in stereospecific binding assays (IC50 values)

in the presence of 100 mM NaCl are those of Pert and snyder (1974),

Pert, Snyder and portoghese (1976), Pert, Snyder, and May (1976),

and Wilson et al. (1975). Although several studies were involved

in the generation of such data, duplication of results indicated

sufficient consistency to justify treatment of the combined results

as a single database. Results from binding studies conducted in

the presence of 100 mM NaCl were selected for analysis because

receptor conformation is believed to best mimic the normal physiological state under such conditions (Pert and Snyder 1974; Creese

and Snyder 1975). Furthermore, receptor affinities with and without 100 mM NaCl were found to be highly correlated: log (1/IC50) =

1.103, log (1/IC50) = 1.628 (IC50 = IC50 in the absence of 100 mM

(NaCl);

R= 0.9196; F= 219.02.

Type structures for the compounds included in the present study are

shown in figures 1 and 2.

FIGURE 1

PRIMARY STRUCTURAL TYPES

147
FIGURE 2

ORIPAVINE GENERAL STRUCTURE AND MOLECULAR REGION DEFINITIONS

Analytical Procedures. The oripavine general structure (figure 2)

was used as a basis for selecting regions in molecular space for

assignment of structural fragments. The fragments which are common

to all of the compounds examined are the phenyl ring(J) and the

propylamine moiety (R) shown in heavy lines.

Because most of the structures examined contain the phenylpiperidine

moiety, their superimposition on figure 2 is relatively nonarbitrary

(coincidence of R and J detemines the locations of other portions

of the molecule). In the case of methadone and propoxyphene,

however, the propylamine chain could be aligned with either R or C

in figure 2. Alignment with R was arbitrarily chosen on the basis

of receptor interaction concepts discussed by Feinberg et al. (1976).

This resulted in the assignment of one of the N-methyl groups to the

C region with the second phenyl group of methadone and the benzyl

group of propoxyphene then distributed between the C and F regions.

In methadone the phenyl moiety was assigned to the C region;

in proxphene the entire phenyl ring was assigned to the

F region.

Subsequent to such alignment and regional assignment of structural

fragment to the various regions in molecular space as shown in

figure 2, various parameter values were then calculated for each of

these regional fragments.

The parameters used were hydrophobicity (log P), molar refraction

and molecular weight. Regional hydrophobicity values (f) were

calculated using the fragmental constants of Leo et al. (l975).

148
Similarly, regional fragment molar refraction values (m) were

obtained or calculated from the compilation by Hansch et al. (1973).

Fragment molecular weight values are designated as w.

The summation of regional fragment parameter values (A+BtC...+K)

yields the calculated molecular parameter values log P, MR (molar

refraction) and MW (molecular weight) (table 1):

Σ f = log P;Σ m = MR; Σw = MW.

These molecular parameters were used both alone and in combination

with the corresponding fragmental values in stepwise multiple

regression amputations using log (1/IC50) as the dependent variable.

Computations were performed both within and across parameter classes.

Regressions were performed stepwise interactively using a combination

of arbitrary and program-guided insertion and deletion of terms.1

There are numerous possibilities with regard to the manner in which

the oripavine structure could be segmented for designation of

geometrical regions. It should be noted that a number of conditions

implicit in the regional designations chosen for this study (figure 2)

are important in interpretation of results. The value of J, for

example, is inversely proportional to the extent of substitution of

the phenyl ring because such substitution eliminates ring protons.

Thus, J is smallest in the morphinoid compounds due to their fused

rings and is greatest in the meperidine analogs. Values of J,

however were used only for summation purposes to obtain molecular

parameter values. For multiple regression purposes J was considered

a constant. Selection of R as a constant neglects

any differences in nitrogen basicity: In the structurally less complicated compounds, values for a number of regions are zero: H>O

only when region H is part of the ring defined by B, H, I, C and R;

K>O only when it includes substituents attached to the BHICR ring;

thus K>O only in the oripavines of which only two are included in

the present study. Fragment G was considered identical in all compounds included in this study and R differs only in the case of

propoxyphene and methadone. Regions G, J and R were omitted from

regression equations.

RESULTS

Parameter coefficients and statistical data for correlations

involving bulk molecular parameters (log P, molecular weight, and

molar refraction) are shown in table 2. Rather poor correlations

were obtained with these parameters and even the inclusion of all

three accounted for only 25% of the affinity variance (R2 = .25).

Even with the inclusion of second order terms the best correlations

obtainable (in terms of R) accounted for no more than 36% of the

variance. Furthermore, parameter coefficient values indicated

variable qualitative (+ or -) contributions by each parameter

depending upon which of these were included in the equation.

In contrast to the use of molecular parameters, regional parameters

yielded correlations accounting for 65-80% of the variance in

receptor affinity (R2 = 0.65 to 0.80). Furthermore, such

149
TABLE 1

OPIATE RECEPTOR AFFINITIES AND CALCULATED

MOLECULAR PARMETER VALUES

Log 1/IC50

Compound 100mMNACL Log P

Mol. Wt.

x10 - 2

Mol. Ref.

x10 - 1

Etorphine

Diprenphine

Naltrexone

Naloxone

Normorphine

Oxymorphone

Dihydromorphine

Morphine

Nalorphine

Levallorphan

-1.79 1.92 4.12 11.49

.69 2.09 4.24 11.74

.69 -.01 3.41 9.39

-.41 -1.07 3.27 9.08

-6.55 -.75 2.71 7.51

-3.40 -1.26 3.01 8.14

-4.94 1.12 2.87 7.92

-4.70 .11 2.85 7.98

-1.39 .30 3.11 8.86

-.69 4.25 2.83 8.72

Levorphanol -2.70 4.06 2.57 7.84

Metazocine -5.30 3.65 2.31 7.1

Etazocine -4.94 4.73 2.59 7.09

Propylnormetazocine 4.38 4.73 2.59 8.03

Allylnometazocine -1.71 3.84 2.57 7.96

Phenylnormetazocine

Ketocyclazacine

Et-Ketocyclazacine

Pentazocine

Cyclazocine

Phenazocine

Meperidine

Ethylnormeperidine

Propylnormeperidine

Butylnormeperidine

Phenethynormeperidine

Hexylnormeperidine

Heptylnormeperidine

Octylnormeperidine

Nonylnormeperidine

-7.82 4.66 2.93 9.54

-4.09 3.24 2.85 8.47

-4.08 3.78 2.99 8.93

-3.91 4.79 2.65 8.5

-.41 4.90 2.7l 7.88

-2.08 5.32 3.07 8.6

-10.82 3.04 2.47 7.45

-10.82 3.58 2.62 7.91

-11.51 4.12 2.76 8.38

-10.30 4.66 2.9 8.85

-9.62 5.20 3.04 9.32

-7.61 5.74 3.18 9.79

-6.48 6.28 3.33 10.26

-6.69 6.82 3.47 10.73

-8.99 7.36 3.61 11.2

Ketobemidone

Ethylnorketobemidone

Propylnorketobemidone

Butylnorketobemidone

Pentylnorketobemidone

Hexylnorketobemidone

Heptylnorketobemidone

Octylnorketobemidone

Nonylnorketobemidone

Decylnorketobemidone

Propoxyphene

Methadone

-4.25

-7.60

-6.91

-6.55

-3.40

-3.69

-3.91

-5.29

-6.55

-6.55

-9.39

-5.30

1.98 2.47

2.50 2.61

3.04 2.75

3.58 2.89

4.12 3.03

7.39

7.85

8.32

8.79

9.26

4.66

5.20

5.74

6.28

6.82

3.17

3.32

3.46

3.6

3.74

3.39

3.09

9.73

10.2

10.67

11.14

11.61

150

5.14

4.54

10.46

9.95
correlations could be obtained with inclusion of a minimum of three

or a maximum of six regional terms in the parameter class equations

(table 3) and the qualitative contribution of a given region remained

constant among the various correlation equations for a given parameter type.

The similarities among equations suggest that f, m, and w are

highly colinear. That this is true was evident from examination of

the correlation matrix. Fragment weights and refractions were

highly colinear except for regions A and I. Fragment hydrophobicities

were highly colinear with weight except for region A and were highly

colinear with refractions except for regions A and I. Furthermore,

regions D and I were highly colinear with respect to weight and

refraction. Also evident in the correlation matrix was the generally

high colinearity between compound molecular weights and molar

refractions.

The inclusion of region R values (constant except for propoxyphene

and methadone) in regression equations was discontinued early in the

study because it had little influence on correlation statistics.

The inclusion of molecular parameters (Σ f, Σw, Σm) in the parameter

class equations (table 4) did not result in better correlations.

It did, however, result in substantial changes in regional coefficients. If it is true that regional properties are more important

in stereospecific binding than total molecular properties, inclusion

of the latter would be expected to dampen the sensitivity of correlations to regional influences.

The inclusion of second order terms produced some improvement in

correlation (R and F values) but such improvements required a

greater number of terms.2 Similarly, regressions which were not

restricted to individual parameter classes produced somewhat better

correlations in terns of values of R and F, but generally with

greater numbers of terms (table 5).

DISCUSSION

The use of molecular hydrophobicity and steric bulk parameters

without regard to the steric distribution of these properties

(log P, molecular weight, molar refraction) did not result in satisfactory correlations (table 2). In general, one might expect such

parameters to suffice for a simple homologous series because parameter changes would then reflect fragmental changes which are

restricted sterically to a small number of regions in molecular

space. In such a case the use of log P, for example, is quite

analogous to the use of π for the substituents. Similarly, log P

should suffice when the dependent variable is primarily a reflection

of some phenomenon which in turn is dependent primarily upon bulk

hydrophobicity; presumably this would be the case where drug transport

by passive diffusion is of major importance. For binding to specific

receptors, however, bulk hydrophobicity could hardly be expected to

adequately define affinities which are based upon steric distribution

of molecular binding regions. The validity of this reasoning appears

to be substantiated by the fact that considerably better correlations

151
TABLE 2

CORRELATION EQUATIONS BASED ON MOLECULAR PARAMETERS
TABLE 3

CORRELATION EQUATIONS BASED ON REGIONAL FRAGMENT PARAMENTERS
TABLE 4

CORRELATION EQUATIONS BASED ON REGIONAL FRAGMENT PARAMENTERS
TABLE 5

CORRELATION EQUATIONS BASED ON REGIONAL FRAGMENT PARAMENTERS
were obtained using fragmental parameter values for sterically defined

molecular regions.

The regional fragmentation pattern chosen for this study was intended

to specify major fragments which are absent or altered in various

opiate classes while simultaneously minimizing the number of separate

fragments to be considered in regression analysis. Aside from such

considerations, the pattern chosen was quite arbitrary. It is

possible that better correlations could be obtained by using a different pattern. The fact that inclusion of all fragments (except

J, G and R) in regression equations did not result in any vanishingly small fragment coefficients suggests that the regional pattern

chosen was reasonable.

It should be noted that the present molecular model method for steric

specification of regional parameters neither implies nor requires

that the compounds with high degrees of conformational freedom must

bind to the receptor in a conformation closely approximating that of

the model. Such conformtional modeling is merely a methodological

tool for steric description. However, the extent to which such

binding conformations are involved among the compounds included may

indeed determine how good a correlation is obtained. Thus, the

inclusion of a "conformational probability" term might result in

improved correlations.

When all variable regions are included in equations the resulting

regional coefficients provide both a qualitative and quantitative

indication of the relative importance of regional properties to

opiate receptor binding. The results obtained in this manner are

generally in agreement with common concepts of opiate structureactivity relationships. Thus hydrophobicity appears to be of little

value or even disadvantageous in regions A and B which frequently

contain either no substituents or polar oxygen functions. The

presence of polar steric bulk appears to be important in region A.

Similarly, hydrophobicity appears to be desirable, though of modest

importance, at E (the N-substituent), and undesirable at F which is

the 14-hydroxyl group in some morphinoids. The importance of hydrophobicity in the K region coincides with the high activities and

receptor affinities of the oripavines. Similarly, the importance

of region H hydrophobicity is quantitated.

The relatively minor importance of E region hydrophobicity in spite

of the great variation in N-substituents among the compounds included

in this analysis is perheps unexpected. However, this result is

consistent with the possibility that the N-substituent is primarily

of importance in determining agonist-intrinsic activity versus

antagonist properties rather than receptor affinity. On the other

hand, the K region appears to contribute substantially to both

receptor affinity and agonist/antagonist properties.

The apparent importance of both steric bulk and hydrophobicity in

region H suggests an area for synthetic exploration among nonmorphinoid compounds such as the benzomorphans, meperidine analogs, etc.

156
These conclusions are further emphasized in the minimum fragment

equations (table 3) which indicate that the regions most determinant

of receptor affinity are A, B, E, H, and K.

In addition to addressing the problem of steric relationships to

biological activity, the present method combines the quantitative

and statistical advantages of multiple linear regression with the

broader applicability and power of pattern recognition approaches.

As such it is not unrelated to the Free-Wilson method and, in some

respects, is a further extension of the substituent constant and

dummy parameter variations of the basic Hansch analysis. The steric

modeling aspect is only broadly defined but provides ample opportunity for the preception and creativity of the individual investigator. Work currently in progress involves completely computerized

superimposition and comparison of structures in three dimensional

molecular space with computer assignment of regional fragment parameter values; such assignments are made on the basis of parent

model defined regional substructure searches wherein the regional

search limits are defined by the investigator as a function of atomic

radii.

FOOTNOTES

1. "The organization and analysis of the data base associated with

this investigation were carried out using the PROPHET System, a

unique national computer resource sponsored by the NIH. Information about PROPHET, including how to apply for access, can be

obtained from the Director, Chemical/Biological InformationHandling Program, Division of Research Resources, National

Institutes of Health, Bethesda, Maryland 20014."

2. For example: log 1/IC50 = 5.97 fA + 10.70 fE + 2.65 fE -

0.31 fE

2

 + 42.30 fH - 55.86 fH

2

 + 3.44 fI -20.0

R = 0.893 F= 19.12

ACKNOWLEDGMENT

The author is indebted to Ms. Kay Leibrand of SRI International

for Implementation of the computer programs required for this

investigation.

REFERENCES

Creese, I. and Snyder, S.H. Receptor binding and pharmacological

activity of opiates in the guiuea pig intestine. J Pharmacol Exp

Therap, 194:205-219, 1975.

Feinberg, A.P., Creese, I., and Snyder, S.H. The opiate receptor:

a model explaining structure-activity relationships of opiate

agonists andantagouists. Proc Natl Acad Sci USA, 73:4215-4219,

1976.

Goldstein, A. Minireview: Opiate receptors. Life Sciences, 14:

615-623, 1974.

157
Hansch, C. The use of substituent constants in structure-activity

studies. In: Ariens, E.J., ed. Physico-Chemical Aspects of Drug

Action. New York: Pergamon Press, 1968. pp. 141-167.

Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D., and

Lien, E.J. "Aromatic" substituent constants for structureactivity correlations. J Med Chem, 16:1207-1216, 1973.

Leo, A., Jow, P.Y.C., Silyso, C., and Hansch, C. Calculation of

hydrophobic constant (log P) from p and f constants. J Med Chem,

18:865-868, 1975.

Pert, C.B. and Snyder, S.H. Opiate receptor binding of agonists

and antagonists affected differentially by sodium. Molec Pharmacol,

10:868-879, 1974.

Pert, C.B., Snyder, S.H., and May, E.L. Opiate receptor interactions

of benzomorphans in rat brain homogenates. J, Pharmacol Exp Therap,

196:316-322, 1976.

Pert, C.B., Snyder, S.H., and Portoghese, P.S. Correlation of opiate

receptor affinity with analgetic effects in meperidine homologues.

J Med Chem, 19:1248-1250, 1976.

Wilson, R.S., Rogers, M.E., Pert, C.B., and Snyder, S.H. Homologous

N-alkylnorketobemidones. Correlation of receptor binding with analgesic potency. J Med Chem, 18:240-242, 1975.

AUTHOR

Howard Johnson, Ph.D., SRI International, 333 Ravenswood Avenue, Menlo

Park, California 94025.

158
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National lnstitute on Drug Abuse, 1978

Progress with Several Models for

the Study of the SAR of

Hallucinogenic Agents

Lemont B. Kier and Richard A. Glennon

At the present time, the studies in our laboratories are directed.

toward several facets of the SAR of hallucinogenic agents. It is

desirable to take this opportunity to disclose the broad objective

of our efforts and, at the same time to record the progress in each

aspect of our research.

We can study the action of hallucinogenic agents by the use of

models, each one providing a framework for analysis and research

into the structure activity relationships (SAR) governing observable phenomena.

Using as a model just the conservative molecule, one considers

properties and events arising directly as a consequence of molecular structure. At this molecular level, we ignore interactions with other molecules, in a milieu or with receptors. The

model here arises from a reduction of variables until the molecule in the isolated state remains as the governing structure

dictating the magnitude of observable phenomena. The extraction

of SAR from this model leads to information which is necessarily

limited by certain exclusions of reality, but which is frequently

the most attainable kind of a relationship. The expectation is

that modest success in this category of SAR study may lead to

prediction of alternate structures with predictable experimental

properties.

SAR studies in this molecular framework involve the definition of

structure using molecular orbital theory or topological descriptions such as molecular connectivity. In its simplest form, structural influence on activity may be defined by a count of carbon

atoms in a normal alkane series (Kier and Hall 1976).

From successful studies with this model, we may presume to have

some insight into molecular structure influencing activity. Denied to us is the ability to reflect on intermediate mechanisms

involving aggregates of molecules.

A second model for SAR studies involves some attempt to simulate

the dynamic intermolecular events taking place as a biological

agent encounters a tissue-based receptor molecule. In the absence of any real understanding of the structure of a receptor,

surrogate molecules may be used to simulate the in vivo part Of

an interacting molecular pair.

A presumption inherent in this stochastic model is that the in159
SAR MODELS
teraction energy emerging from the molecular pair bears some relationship to the magnitude of the observed biological phenomena.

Theoretical calculations using molecular orbital theory or empirical calculations quantitating categorized forces lead to energy

values as a function of intermolecular distance and model structure.

This general approach to SAR may lead to information about molecular structure and possible molecular level mechanisms of action.

The information is much harder to obtain than from conservative

molecule SAR considerations since all of the spatial aspects of

interacting pairs must be probed and a suitable receptor model

established.

A third model of SAR analysis involves the development of quantitative relationships between suspected parallel properties or actions of molecules. This general approach relies heavily upon the

quantitation of pharmacological, biochemical, and physicochemical

properties. The interplay of these properties in the search for

relationships presumes the existence of models depicting mechanisms of action at the level of bulk solutions, tissues and even

organs.

Structural information arising from successful correlations using

this general model is limited. The real benefit evolves from a

greater possibility of mechanistic interpretation.

Because of facilities and capabilities it is possible in our laboratories to utilize all three of these general SAR models. Thus

we have utilized molecular structural description, stochastic modeling and pharmacological and physicochemical measurement, to probe

on a broad front the phenomenon of hallucinogenic activity. We

describe here some of the results in each category.

CONSERVATIVE MOLECULE SAR

The pioneering work of Karreman, Isenberg and Szent-Gyorgyi (1959),

and of Snyder and Merril (1965) ushered in theoretical studies of

the influence of molecular structure on the hallucinogenic potency

of LSD (1), phenylisopropylamines (2) and tryptamines (3). Using

primarily the human data of Shulgin, Sargent and Naranjo (1969) a

relationship with molecular orbital energy parameters was reported.

Later studies by Green and collaborators (Green 1967; Kang and

Green 1970a; Green and Kang 1970; Kang and Green 1970b) sought

to improve electronic correlates of hallucinogenic potency by using

more sophisticated molecular orbital methods, and by examining additional parameters.

An electronic parameter to which hallucinogenic activity was initially related was the energy of the compounds' highest occupied

molecular orbital(EHomo), an index thought to reflect the compounds' ability to donate electrons in a charge-transfer type of

interaction. More recently this relationship between EHomo and

activity has been questioned with respect to LSD analogs (Kumbar

and Siva Sankar 1973; Green, Johnson and Kang 1974)

and in respect to N, N-dialkyl tryptamines (Glennon 1974). Green

161
has commented that such a molecularly gross characteristic as EHOMO

probably cannot explain so specific a biological activity. To date,

no direct electronic correlate relating the hallucinogenic activity

of phenylisopropylamines, tryptamines and LSD analogs has been reported.

Topological structure of hallucinogens has been studied using molecular connectivity (Kier and Hall 1977). A direct correlation

was found between connectivity indices and hallucinogenic potency,

which was of a quality at the upper limit of the value of the experimental data. Thus for 23 ring substituted phenylisopropylamines

(amphetamines) (table 1), an equation was found:

Two important consequences of this work emereged. The first was that

statements, comprehensive to the medicinal chemist, could be made

about molecular structure and its influences on hallucinogenic potency. The equation delineates structural fragments which are of

significance in governing the experimental potency.

No understanding of intervening mechanisms or biopharmaceutical

events is possible from the equation. The Information from molecular connectivity calculations is purely structural. Nevertheless, the Information makes it possible to make predictions of

potency, the second important consequence of this work. Table 2

lists six satisfactory potency predictions reported from the equation, compounds 27 to 32 (Kier and Hall 1977), plus six additional

predictions, compounds 33 to 38 (Glennon and Kier,unpublished

data) since that publication. The potency of LSD (1) can also be

162
TABLE 1

Substituted Phenylisopropylamines and Predicted hallucinogenic Activity a

163
predicted quite well (table 2) using Equation I. It might be

pointed out that the molecular connectivity method does not consider stereochemistry and that it cannot account for variation

in hallucinogenic potency resulting from alteration in the route

of administration.

In a more recent study, a series of phenethylamine (mescaline)

analogs was analyzed using molecular connectivity (Glennon, Kier

and Shulgin, unpublished data). The hallucinogenic activity of

phenethylamines was found to relate with two connectivity indices

according to the following expression:

Again, with this equation, a higher percent of the variation in

potency is found, than is probably warranted by the experimental

data.

A number of structural conclusions are possible from these findings

(table 3). From the relating equation, several generalizations can

be derived about the impact of structural variation on activity.

The (term suggests that increasing the number of nuclear substituents will generally result in an increase in potency; compare

the activities of 39 and 40 with, for example, those of 41 and

42. This is also accord, biologically, with the findings of

Clarke, Bennington and Morin (1965) that those phenethylamines with

high degrees of methoxylation serve less well as substrates for enzymic degradation. In addition, the presence of the term implies that the lower the δ

v

 in the substituent, the higher the potency, i.e. Br > CH3 > 0CH3; compare 42, 45 and 46.

The term is related to the substitution pattern of the phenethylamine ring. Because the coefficient for this term in the relating

equation is negative, the greater the value of the term, the lower

the potency of the compound. In the monosubstituted phenethylamines, an ortho substituent contributes two additional terms and

a meta substituent one additional term as compared to a para substituent. Therefore the position of the stistituent would be expected to enhance activity in the order: ortho < meta < para.

Considering phenethylamines with more than one ring substituent,

the term suggests that activity is enhanced when two substituents are para to one another (para > meta > ortho). Such substitution can only be realized, in disubstituted molecules for example, when the phenethylamine is 2,5-disubstituted.

The above statements, which can be quantified using the chi terms,

appear to echo those structure activity relationships, which have

evolved, in a qualitative manner, by inspection of a series of

164
TABLE 2

Predictions of Hallucinogenic Potency for

Compounds Not Use in Generating Equation I

165
TABLE 3

Chi Terms and Activities of Various Phenethylamine Analogs

166
phenethylamines. Shulgin (1978) has reported that if the paramethoxy group is replaced with another substituent (higher alkoxy,

methyl, halo) there is an unquestioned Increase in potency. Shulgin (1978) has further commented that, in general, it appears that

2, 4, 5-orientation is more effective than 3, 4, 5-orientation although there are two few examples to establish this generality.

Investigations of SAR can serve to highlight those structural features which Influence activity. However, because hallucinogenic

activity can not be measured accurately, one can not expect to accurately predict hallucinogenic potency. This is especially true

with those compounds that are only weakly active as hallucinogens

and is reflected by the standard deviation of equation II. Application of the relating equation (equation II) should, nevertheless, afford a relative measure of predictable activity, particularly when activity is moderately high. Smythies and co-workers

(Smythies et al. 1967) employing a modified Bovet-Gatti profile,

have found that 2, 3, 4, 5, 6-pentamethoxyphenethylamine (4 9)

possesses an approximate activity of eight mescaline units in animal models. Using the relating equation (equation II), 49 is

predicted to have a potency of 4.5 MU. As another example, an

iodinated derivative, 4-iodo-2,5-dimethoxyphenethylamine ( 5 0 ),

has recently been titrated In humans although effective intoxication levels have not yet been explored (Shulgin 1978). The iodo

group, which is para to the side chain, possesses a δ

v

 which is

even lower than that of a bromo group. In addition, this molecule

possesses two methoxy groups which are para to one another (i.e.

2,5 dimethoxy substitution). On this basis, 50 is predicted by

the relating equation to be approximately 45 times more active than

mescaline. Shulgin (1978) has found that threshold effects of

50 are clearly noted at a total dose of 8 mg.; this corresponds

to an activity of about 44 MU.

INTERACTION MODEL SAR

The goal in this endeavor is to deduce a model of interacting species,

to estimate the energy of interaction at reasonable distances of

separation and then to attempt to correlate the energies for a

series of such pairs with an observed biological property. The hypothesis that the energetics so computed govern the measured biological response underlies this approach.

In practice, the drug molecule structure is known while the receptor must be found. By elimination and speculation, this frequently is taken to be a portion of a polypeptide chain, usually an amino

acid side chain.

We have reported such a study on 17 hallucinogenic phenylisopropylamines (DiPaola, Kier and Hall 1978). Calculations of interaction energy using the Claverie and Rein (1969) procedure have

deduced a receptor model, an interaction distance and orientation

and a relating equation for activity in log mescaline tits:

167
Log MU= 0.28 ET+ - 0.26 I3,4 - 0.63 Eqn III

where ET is the total calculated interaction energy computed for

the model and orientation mode optimizing the relationship and I3,4

an indicator variable depicting the presence of substituents in

the 3 and 4 ring positions.

The optimized model, shown in figure 1, is based on the amphetamine

ring separated from an indole receptor surrogate by 4.5A.

A further sophistication of this approach involves the estimation

of the electrostatic potential field around each atom. The interaction calculations thus consider a changing charge mosaic as the

molecules approach. Current work considers these charge fields.

MECHANISTIC MODEL SAR

The probing of potential pharmacological mechanisms is the objective of structure activity studies using this general model.

Determination of the mechanism of action of agents whose biological effect is essentially subjective in nature, i.e. a psychopharmacological response, is inherently fraught with the problems of measuring this effect. Much of the comparative human

data on the hallucinogenic activity of various substances has

been accumulated by Shulgin and co-workers who have estimated a

variation in activity data of about 25% (Shulgin, Sergeant and

Naranjo 1969). While the use of so-called “Mescaline Unit" data

is necessitated by the lack of any other type of comparative human

data, Shulgin is quick to point out that such data do not reflect,

for example, route of administration, time of onset, duration of

action, and, are extrapolated from dose ranges. What is needed is

a model or surrogate measure of the hallucinogenic response, or

rather, a model which might reflect or yield results which reasonably parallel hallucinogenic activity. Though such an idea is

not new,a study of drug/model-receptor interactions might still

be useful inasmuch as similar interactions might be responsible

for the hallucinogenic response. Regarding the validity of using

model receptor systems, Steinsland and Hieble (1978), for example,

have recently reported on a rabbit ear artery dopamine receptor

model. The affinities determined for various neuroleptic agents,

using their model system, closely parallel the dopamine receptor

binding affinities which were determined using calf brain homogenates.

In these studies, then, pharmacological and physicochemical correlates are sought; these, in turn,can be analyzed using previously described SAR methods, i.e. molecular orbital theory or

molecular connectivity. Various mechanistic models might be constructed which relate hallucinogenic activity to familiar drug

receptor systems or to measurable physicochemical phenomena known

to occur in vivo.

What model systems might be useful? Tryptamine, phenalkylamine

168
FIGURE 1

Optimum mode of interaction predicted for amphetamine rings

and the methylindole receptor model.

169
and lysergamide hallucinogens are known to interact with various

neurotransmitter systems, for example, serotonergic and dopaminergic systems. Because of the close structural similarity between

these hallucinogens and serotonin (5-hydroxytryptamine, 5-HT),

we have chosen the 5-HT receptors for our initial. investigations.

This does not preclude the possibility that hallucinogens might

produce some facets of their activity by interaction with other

types of receptors.

With this as a working hypothesis we have examined various model

5-HT receptor interactions of hallucinogenic agents. Woolley initialy suggested that 5-HT/hallucinogen interactions might be implicated in their mechanism of action (Wolley and Shaw 1954;

Woolley 1962). Later, others (Gaddum et al. 1955; Vane 1959; Earlow and Khan 1959; Offermeier and Ariens 1966; Winter, Gessner and

Godse 1967) examined the ability of tryptamines and related compounds to interact as agonists or antagonists with the 5-HT receptors of various isolated tissue preparations. These investigations, however, were not solely directed toward finding a correlate of hallucinogenic activity but were, rather, studies of

drug-receptor interactions. One of the first studies,using model

receptors, designed expressly for investigating hallucinogenic activity was that of Green and Kang (1970). Employing nitrogen-unsubstituted tryptmines and using an isolated rat stomach fundus

preparation, several potential quantum chemical correlates of agonistic activity were implicated.

Using a somewhat different approach, Glennon and Gessner (1975;1977)

investigated the binding affinities (p A2's) of N, N-dialkyltryptamines and related analogs for the 5-HT receptors of this same

model system (table 4). Although further work is indicated from

the results, several interesting findings have emerged: (a) increasing the steric bulk on the terminal amine decreases affinity,

(b) methoxyl-substitution is most favorable at the 5-position,

moving this methoxy group to the 4- or 6-position results in about

a 10-fold decrease, (c) replacement of the indole nitrogen by a

sulfur atom has no apparent effect on affinity while replacement

by an sp

3

-hybridized carbon atom results in a two-fold decrease in

affinity.

The electronic properties of a number of these compounds were examined by various molecular orbital methods (Glennon and Gessner

1977). No significant correlation was observed between affinity

and EHOMO or between affinity and any other electronic parameter,

for that matter. However, if the 7-methoxy analog is deleted, a

correlation is observed between affinity and the electrophilic

frontier orbital electron density at the 4-position (r = .96,

n= 8). Only eight compounds were examined; because the variation

in small, the significance of this correlation is not clear

and may even be fortuitous. A larger series of compounds should

be examined. Such correlations between electronic parameters and

5-HT receptor binding affinities remain to be fully developed, Interestingly, using different compounds and a different procedure to

measure activity, Green and co-workers have found relationships be170
TABLE 4

Binding Affinities of N,N-Dialkyltryptamines and Related

Compounds a,b

171
tween activity and various electronic parameters, including both

in their 1970 study (Green and Kang 1970) and in later investigations

(Johnson and Green 1974; Weinstein et al. 1976).

Two questions emerge from these studies. First, is there a receptor related parameter which might be used to investigate the hallucinogenic phenalkylamines as well as the N,N-dialkyltryptamines,

and secondly, are agents within these two,classes of hallucinogens

capable of producing similar behavioral effects? In other words,

is it justifiable to examine these compounds as one large group

as opposed to examing each class separately. Though each of

these classes may elicit unique behavioral responses, an investigation of a common component of their activity might afford the

most useful data. Evidence that such an approach might be warranted comes from molecular connectivity studies. Using the equation relating hallucinogenic activity to the structure of a series

of phenylisopropylamines, satisfactory predictions were made

(table 2) concerning the hallucinogenic potency of several N,Ndialkryltryptamines (which were not used in generating the equation).

In addition, in collaboration with Dr. J. Rosecrans of MCV, we are

examining the behavioral effects of various hallucinogens employing

a discriminative stimulus model. Rats are trained to discriminate between a drug and non-drug (saline) state until a high level

of performance is attained. These animals are then challenged

with a new drug to detemine if the challenge-drug is perceived

by the animals as producing; an effect similar to the test-drug.

If generalization occurs,that is, if the animals cannot discriminate between the effects of the challenge-drug and the test-drug,

this is an indication that both agents are producing similar behavioral effects and, furthermore, that they might be operating

via a similar mechanism (Kuhn, White and Appel 1977).

The discriminative stimulus properties of several hallucinogens

have been recently reviewed (Kuhn, White and Appel 1977). LSD,

for example, will serve as a discriminative stimulus in animals.

Though generalization will occur with psilocybin, it does not occur with morphine,amphetamine, PCP, or phenobarbital. Rosecrans

and co-workers (Hirschhorn, Hayes and Rosencrans 1975) have also

shown that LSD will generalize to electrical stimulation of midbrain raphe.

We have found that 5-OMe DMT possesses discriminative stimulus

properties and we have trained rats to discriminate between this

compound and saline. When challenged with DOM,the animals are

unable to discriminate between DOM and the 5-OMe DMT stimulus.

Thus, generalization is occurring (table 5) and both compounds

are presumed to be producing similar behavioral effects. Additional studies involving other hallucinogens are currently underway, to determine if there are certain common components of their

behavioral effects.

Phenylisopropylamines are know to affect serotonergic systems both

in vivo and in vitro. There is an obvious structural similarity

172
TABLE 5

Generalization of DOM to 5-OMe DMT stimulus a

Drug n

5-OMe DMTc 1mg/kg 48

Saline 0.9 1ml/kg 42

DOM c

0.25 mg/kg 6

0.50 mg/kg 12

0.75 mg/kg 5

1.00 mg/kg 6

2.00 mg/kg 6

% 5-OMe DMT

Correct Responsesb

85%

20%

39%

53%

60%

85%

d

a

b

Glennon, Rosecrans and Gaines; unpublished data

Average responses of n animals

c In 0.9% Saline

d Behavior disrupted, animals did not respond

173
between these structures and the structures of 5-HT and the N,N-dialkyltryptamines. Superimposition of these structures, as shown in

figure 2 may be conceived as one way (or conformation) in which

these compounds might interact with 5-HT receptors. Others have

FIGURE 2

Phenylisopropylamine structure superimposed

over a tryptamine structure

also noted these structural similarities. Therefore it might be

expected that the phenylisopropylamines might possess binding affinity chacteristics similar to those of the N,N-dialkyltryptamines. We have examined this possibility (Glennon, Liebotitz and

Mack 1978) and some of the results are presented in table 6.

Though this study is far from complete, several generalizations

might be made.

2,5-Dimethoxyphenylisopropylamine (6 ) possesses a pA2 of 6.83.

Para-methoxylation does not appear to alter affinity (compound 11 ).

This in line with the suggestion that p-methoxylation increases

potency by blocking metabolism at that position. Para-methylation,

on the other hand, results in a two-fold increase affinity (compound 22 ). Nichols, et al. (1977) have recently demonstrated

a hydrophobic site in the region of this para-position, in the

isolated sheep umbilical artery 5-HT receptor model. A similar

site in the rat fundus model would explain the observed enhancement in affinity upon methylation of the 2,5-dimethoxy compound.

If such is the case with our model and if the interaction is Similar to that shown in figure 2, then the hydrophobic region would

be in the vicinity of the tryptamine 7-position. Green et al. noticed, as we did, the detrimental effect that charge-related terms,

involving the 7-position of the tryptamines, had on attempted activity correlations employing the rat fundus preparation (Green,

Johnson and Kang 1974). One explanation which they offered was

that this position might be coming into contact with a hydrophobic

Site. On this basis and because p-methylation doubled the affinity

of 2,5-dimethoxyphenylisopropylamine, the 7-methyl analog of DMT

was synthesized (Glennon et al. 1978) and evaluated. As expected,

7-methylation of DMT resulted in a two-fold increase in affinity

(compound 65)

Another finding is that there is no direct relationship between the

affinity of the phenalkylamines for 5-HT receptors of the model and

their hallucinogenic potency. This is demonstrated by the similar

174
TABLE 6

Binding Affinities of Phenylalkylamines a

175
affinities of 6 and 60 , while it is known that removal of the αmethyl group of 6 decreases its potency. So, while affinities

may be similar, other explanations, such as relative rateS of oxidative deamination in vivo might be necessary to account for

differences in activity. It might be noted that in those compounds where stereoisomers exist, the racemic mixtures were used.

What other support is there for similarities in phenylisopropylamine and N,N-dimethyltryptamine binding? Demethylation of 5-OMe

DMT, to yield 5-OH DMT, results in a two-fold increase in binding

affinity. Evoking our structure in figure 2, this would correspond to the 2-position of the phenylisoproylamines. As shown in

table 7, demethylation of the appropriate methoxy groups results

in an increase in binding affinity in each case(Glennon and Liebowitz 1978). Not enough data have yet been collected to examine

the results by molecular orbital or molecular connectivity methods,

yet the affinity data appear interesting and informative in their

own right.

However, as these data were being collected, Nichols, Shulgin and

Dyer (1977) reported their results on the agonistic activity of

seventeen phenalkylamines on the 5-HT receptors of the isolated

umbilical artery preparation (table 8). They suggested that such

activity, coupled with lipophilicty, might be predictive of hallucinogenic potential in man. We explored the structural aspects

of these seventeen phenalkylamines by use of the molecular connectivity method (Kier and Glennon 1978). Agonistic activity was

found to relate to three molecular connectivity indices according

to the following expression:

The predicted activities, using equation IV, are shown in table 8.

Of the seventeen compounds for which activity was reported, variation in structure divides these compounds into several structural

types. The compounds (a) either possess (phenylisopropyl analogs) or are devoid (phenethylamine analogs) of an α-methyl group

and (b) possess either a 2-methoxy or a 3-methoxy substituent. The

major structural alteration is at the 4-position, which includes

bromo,alkoxy,methlthio and alkyl derivatives and one example of

a nitro derivative. It has been demonstrated that the 3, 4, 5-

trisubstituted compounds are about as active as 2, 4, 5-trisubstituted compounds of comparable log P in this model system (Nichols,

Shulgin and Dyer 1977). For this reason, and because the major

structural variation is at the 4-position it might be expected

that molecular connectivity indices will focus on these 4-position

substituents. Both and can be related to the α-methyl

The index also increases by one subgraph as the length of the

and suggest an enhanced actitity when this group is present.

substiiuent in the 4-position increases. Because, in the second

term, this index is negative and squared, increasing the length

of the chain will result in a rapidly diminishing effect on ac176
TABLE 7

Binding Affinities of Methoxy Derivatives and Their

Corresponding Hydroxy Analogs a

177
TABLE 8

Serotonin Receptor Agonist Activity

178
tivity. Thus, this second term governs a maximum potency

value with an intermediate length side chain. This appears with

44 in the phenethylamine series with 24 in the phenylisopropylamine series. The great size of the benzyloxy side chain of

compound 73 contributes adversely to activity.

The emergence of valence chi terms in the equation indicates that

the nature of atoms is of more than passing importance. That means

that heteroatoms are playing a role more than just space filling

comparable to carbon isosteres. The valence chi terms indicate

that ring substituents with heteroatoms are going to differ in potency from alkyl substituents of the same length. The magnitude

of the difference depends on the overall size of the substituent

and whether its length is less than or exceeds the maximum value

governed by the two indices.

Nichols, Shulgin and Dyer (1977) have discussed the importance of

lipophilicity and have found that this parameter correlates with

activity if an indicator parameter, necessary to limit the steric

length of the 4-position substituent, is additionally introduced

into the equation. It would thus appear that this model might be

useful in predicting hallucinogenic potency, and that the hydrophobic region of the receptor will need to be considered. Molecular connectivity can be used to describe the structure of molecules

in an SAR study of these compounds and so might also prove to be

a useful tool in predicting agonistic activity.

In another study by Glennon and Kier (l978) a modelwas invoked in

which hallucinogenic potency of LSD analogs was presumed to arise

from interaction with 5-HT receptors. Cerletti and Doepfher (1958)

have examined the 5-HT antagonistic activity of a series of lysergamides (table 9) which differ only in respect to their amide

substituents. Molecular connectivity studies on these molecules

produced an equation correlating potency (log relative biological

response) with three connectivity indices:

A number of conclusions are possible concerning the important structural features contributing to activity. The 5-HT antagonistic activity of the lysergamides is influenced by their amide substituents.

Molecular connectivity indices quantify the effect of these substituents which might otherwise have been revealed,only in a qualitative manner, by inspection of the structures. The 2

x term suggests that branching of the amide substituents will have an unfavorable effect on activity. An even greater unfavorable effect

results from the incorporation of the amide substituent into a cyclic structure as revealed not only by the 2

x term but also by the

6

xp term. In addition, the 6xp indicates that the diethyl amide

structure of LSD is optimal for 5-HT antagonism, and that lengthen179
TABLE 9

Observed and Calculated Activities of Lysergamides as

5-HT Antagonists

180
ing both amide substituents will have an increasingly detrimental

effect on activity. Shortening both diethyl substituents by one

carbon atom, to yield the dimethyl homolog, is even more deleterious than lengthening the diethyl substituents to the dipropyl or

dibutyl homlogs. The valence term, ox

v

, indicates that the incorporation of nonaliphatic cyclic substituents, such as those containing a heteroatom or uhsaturation, further reduce activity. In

this manner,the influence of each structural change can be quantitated.

Most of the studies now underway in our laboratories are still in

their infancy. The object of these investigations is to find biological and/or physicochemical correlates of hallucinogenic activity and then to examine these correlates by means of molecular

orbital or molecular connectivity methods. Though hallucinogenic

potency can be studied by these methods directly, the use of models

might enhance precision and might also yield information on the

nature of the binding of these compounds to various receptors.

The direction of our future research, then, will be to accumulate

such data by synthesizing and examining various compounds, particularly, modifications of tryptamines and phenylisopropylamines.

For example, if both classes of compounds interact with 5-HT receptors in the manner shown in figure 2, then the optimal stereochemistry about the carbon atom bearing the terminal amine might

be expected to be opposite in comparing a phenylisopropylamine

with an α-methyptamine.

In addition, the behavioral studies will continue, to determine if

compounds with high 5-HT receptor affinities will generalize to

5-OMe DMT and DOM stimuli.

It should be stressed that we have not implicated serotonergic

mechanisms as playing a role in the hallucinogenic response.

Rather, we are using a 5-HT receptor model to investigate the

SAR of hallucinogens agents. With such data in hand, molecular

orbital, and in particular molecular connectivity investigations should not only provide us with SAR, but will allow us to

quantitate these data.

CONCLUSION

These studies can be discussed at two levels. At the level of results, we can review, as we have just done, a mosaic of studies

ranging across several molecular types and several biological responses. The mosaic, as yet, is incomplete. We cannot conclude

that the 5-HT receptor is the hallucinogenic receptor, for example, and yet these studies point to trends of information which

will surely form a basis for definitive conclusions about mechanism and structure activity relationships.

At another level, we can comment on the broad spectrum of SAR methodology utilized in these studies. It should be clearly noted that

we have not confined ourselves to only one model in our research.

181
We have, for example, not relied solely on isolated molecule studies

using sophisticated M. O. theory. Neither have we looked exclusively at affinity data.

The entire gamut of SAR models must be utilized, conservative, dynamic, and mechanistic, in order hopefully to approach the goal of

understanding of the complex processes at work in hallucinogenesis.

ACKNOWLEDGMENTS

We wish to acknowledge the U.S. Public Health Service

(grants DA-02046 and DA-01642) for their support of this work. In

addition we would like to express our appreciation to NIDA and the

Psychopharmacology Research Branch of NIMH for supplying us with

certain compounds, to Dr. Max von Strandtmann of WIRI for compound

56 and to Drs. P.K. Gessner, J.A. Rosecrans and J.F. Stubbins for

their co-operation and/or helpful discussions. Use of the Data

Acquisition facilities at MCV/VCU are greatly appreciated.

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Clark, L.C., Bennington, F., and Morin, R.D. The effects of ringmethoxy groups on biological deamination of phenethylamines. J

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DiPaola, T., Hall, L.H., and Kier, L.B. Structure-activity studies

on hallucinogenic amphetamines using model interaction calculations.

J Theor Biol, in press, 1978.

Gaddum, J.H., Hameed, K.A., Hathway, D.E., and Stephens, F.F. Quantitative studies of antagonists for 5-hydroxytryptamine. J Exp Physiol,

40: 49-74, 1955.

Glennon, R.A. A quantum chemical investigation of the π -electronic

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Glennon, R.A., and Gessner, P.K. Correlations between the activity of

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Glennon, R.A., and Gessner, P.R. The electronic and serotonin receptor

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Glennon, R.A., Liebowitz, S.M., and Hack, E.C. Serotonin receptor binding

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Glennon, R.A., Martin, B., Johnson, K.M., and End, D. 7,N,N-Trimethyltryptamine: A selective inhibitor of synaptosomal serotonin uptake.

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Green, J.P. The-application of quantum mechanics to pharmacology.

Some substances active in the nervous system. Molecular Basis of

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N.Y., 1967. pp 95-109.

Green, J.P., and Kang, S. The correlation of electronic structures

of indole derivatives with their biological activities. Molecular

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Green, J.P., Johnson, C.L., and Kang, S. Application of quantum

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319-342, 1974.

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Kang, S., and Green, J.P. Correlation between activity and electronic state of hallucinogenic amphetamines. Nature, 226: 645, 1970b.

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Kuhn, D.H., White, F.J., and Appel, J.B. Discriminative stimulus

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Morin, R.D., and Clark, L.C. Structure-activity relationship studies

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Reactivity characteristics of large molecules and their biological

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indenealkylamines and 3-benzo [b] thiophenealkylamines with their

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184
Wooley, D.W., and Shaw, E. A biochemical and pharmacological suggestion about certain mental disorders. Science, 119: 587-588, 1954.

Vane, J.R. The relative activities of some tryptamine analogs on

the isolated rat stomach strip preparation. Brit J Pharmacol, 14:

87-98, 1959.

AUTHORS

Lemont B. Kier, Ph.D.

Richard A. Glennon, Ph.D.

Department of Pharmaceutical Chemistry

Medical College of Virginia/VCU

Richmond, Virginia 23298

185
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

QSAR of Narcotic Analgetic Agents

E. J. Lien, G. L. Tong, D. B. Srulevitch, and

C. Dias

INTRODUCTION

Analgetics are drugs which alleviate pain without significantly

imparing consciousness. Lim et al. (1964) have proposed that analgetics groups into (a) peripherally acting, nonnarotic (e.g.,

salicylates); (b) centrally acting, nonnarcotic (e.g., d-propoxyphene); and (c) centrally acting, narcotic (e.g., morphine, meperidine, etc.).

The major sites of action of centrally acting narcotic analgetics

are the cerebrum and medulla (Korolkovas and Burckhalter 1976);

therefore, their ability to pass the blood brain barrier is extremely important. It is now well recognized that this ability is intimately related to the lipophilicity of the drug as measured by

lipoid/water partition coefficient (Lien 1975).

Although the precise mechanism of drug-receptor interaction for

analgetics has not been elucidated several common structural features

have been recognized:(a) a quaternary carbon atom;(b)a phenyl or

isostere ring attached to this carbon atom; (c) a tertiray amino

group two carbon atoms removed, and (d) a phenolic hydroxyl group in

meta position relative to the point of attachment to the quaternary

carbon atom if the tertiary nitrogen is part of a six-membered ring

(Korolkovas and Burckhalter 1976).

It becomes apparent that both electronic and steric parameters are

important besides the lipophilicity of the drug molecule for selective

drug-receptor interaction. The main purpose of this report is to

examine how the physicochemical properties of the compounds, namely,

partition coefficient, molecular weight, molar refraction, and steric

constants may affect the analgetic potency or their binding with the

receptor.

METHODS

The first set of data was from the work of Bukckett (1964) on esters

of 14-hydroxycodeinone. The relative activity (morphine = 1) was converted to the logarithmic scale in order to be in line with the

linear-free energy related parameters like log P.

186
The partition coefficients of the uuprotonated forms were calculated

from the experimental value of morphine and codeine, and the π constants of the substituents (Leo, Hansch and Elkins 1971). The log MW

and log MR of the side chain (Hansch et al. 1973) were also included

in the regression analysis (see Table 1).

The second set of data was from the excellent work of Pert, Snyder

and Portoghese (1976) on meperidine homologues. The apparent partition coefficients in octanol-phosphate buffer pH 7.4 (Table 2)

(Larson and Portoghese 1976) were used in the correlation. Since

the pKa's of the homologues do not vary substantially because of the

close σ* values of the R group, the correlations obtained will differ

from those using true partition coefficient only by a constant term

(Hansch and Lien 1968).

The method of least squares was used in deriving the equations using

an IBM 370/155 computer executed through a Tektronix 4012 terminal.

RESULTS AND DISCUSSION

The equations correlating the analgetic activities of esters of 14-

hydroxycodeinone are summarized in Table 3. The equations correlating

the analgetic activities and the receptor binding of N-alkylnormeperidine homlogues are given in Table 4.

From Equations 1 and 4 of Table 3 one can see that there is a parabolic dependence of the analgetic activity on the lipophilicity as

represented by 1-octanol/water partition coefficient (log P). The

(log P)2 term in Equation 4 is statistically highly significant as

indicated by an F-test (F1,10=76; F1,10 .9995=25.5).

Combination of log P with log MW or log P with log MR also gives

highly significant correlations (Equations 2 and 3). Addition of

log MW to Equation 4 yields Equation 5 which is significant at 90

percentile level (F1,9 =4.3; F1,9 .90 =3.4).

The optimum lipophilicity (log Po) derived from Equation 4 is 3.23

with a 95 per cent confidence interval of 2.92 to 3.46. The log Po

for Equation 5 is 1.45 with a wider 95 per cent confidence interval.

The positive dependence on log MW and log MR probably reflects fairly

non-specific Van der Waals interactions with the receptor in addition

to hydrophobic interactions. Several possible explanations of the

parabolic dependence of activity on log P have been proposed by Hansch

and Clayton (1973) and by Lien (1975). It is gratifying to see that

the relative analgetic activities of all 13 compounds with side chain

R ranging from one to 12 carbon atoms and even aromatic side chains

were well correlated with Equation 5 (see Table 1).

For the analgesic activities of N-alkylnormeperidine homologues

Equations 6-8 were obtained (Table 4). The correlation coefficient

of the parabolic equation (Eq. 7) is only 0.824. This is primarily

due to the apparent "biphasic" curve of the log 1/C vs. log P plot

as indicated by Figure 1. Meperidine, the lowest member in the

series, appears to have activity higher than expected from the

167
regression. However, when the method of least squares was applied,

meperidine did not appear as an outlier. The deviation was only

slightly higher than the standard deviation of the regression (0.264

vs. 0.239). Pert, Snyder, and Portoghese (1976) have attributed the

relatively high analgetic activity of meperidine to its ability to

penetrate the brain (600-fold higher brain level relative to morphine).

From Figure 1 one can also see that meperidine also has relatively

high affinity in the inhibition of naloxone binding, especially in

the absence of NaCl. Addition of the Hancocks corrected steric

constant (1961) to the parabolic equation improved the correlation

significanrly as indicated by an F-test (F1,5 = 124.96; F1,5 .9995 =

63.6). Positive dependence on suggests that the bulk

tolerance oh the N-substituent is very small.

Figure 2 clearly shows that the constant changes quickly from C1

to C3 but remains fairly constant from C3 and above. This suggests

that perhaps in addition to greater penetrability to the brain,

meperidine, having the least hindered N-CH3, will bind more tightly

to the receptor site (presumably a negative site). This interpretation is in agreement with the finding that the receptor affinity

of meperidine in the absence of sodium is more drastically enhanced

than in the presence of sodium ion. The terms in Equations 12

and 14 are statistically significant at 99 and 27.5 percentile

levels, respectively, (F1,5 = 17.76 for Equation 12 and 12.96 for

Equation 14). Similar "biphasic" dependence of the analgetic

activity of N-alkylnorketobemidones on the length of the N-R side

chain has been observed by several investigators (Oh-ishi and May,

1973; Wilson, Rogers, Pert and Snyder, 1975). This again can be

attributed to the difference in constants .

The log Povalues for maximum analgesia obtained from Equations 7 and

8 are around 3. This also agrees with the Peak for inhibition of

naloxone binding in the presence of NaCl as seen from Figure 1, as

well as from Equations 12 and 14.

From the above-mentioned analysis, it may be worthwhile studying the

modification of meperidine and ketobemidone on the benzene ring or

the ester or keto side chain to reach a log P of 3 (octanol/buffer

pH 7.4) but keep the N-CH group unchanged in order to minimize the

steric effect.

188
TABLE 1

Physicochemical Properties and Analgetic Activities of

Esters of 14-hydroxycodeinone in Mice.
TABLE 2

Physicochemical Properties and Analgetic Activities of

N-Alkylnormeperidine Homologues
TABLE 3

Physicochemical Properties and Analgetic Activities of

Constants of Esters of 14-Hydroxycodeinone in Mice
TABLE 4

Physicochemical Properties and Analgetic Activities of

Constants of N-Alkylnormeperidine Homologues
FIGURE 1

Dependence of the Analgesic Activity and Receptor Binding

Affinity on the Apparent Partition Coefficient

193
FIGURE 2

Dependence of the Electronic and Steric Constants

on the Chain Length, where + 0.306 (n-3)

194
ACKNOWLEDGMENT

This study was supported in part by BRSG Grant RR-05792 awarded by

the Biomedical Research Support Grant Program, Division of Research

Resources, National Institutes of Health.

REFERENCES

Buckett, W.R. The relationship between analgesic activity, acute

toxicity and chemical structure in esters of 14-hydroxycodeinone.

J Pharm Pharmacol, 16:Suppl 68T-71T, 1964.

Hancock, C.K., Meyers, E.A., and Yager, B.J. Quantitative separation

of hyperconjugation effects from steric substituent constants. J Am

Chem Soc, 83:4211-4213, 1961.

Hancock, C.K., and Falls, C.P. A Hammett-Taft polar-steric equation

for the saponification rates of m- and p-substituted alkyl benzoates.

J Am Chem Soc, 83:4214-4216, 1961.

Hansch, C., and Clayton, J.M. Lipophilic character and biological

activity of drugs II: The parabolic case. J Pharm Sci 62(1):1-21,

1973.

Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D., and Lien,

E.J. "Aromatic" substituent constants for structure-activity correlations. J Med Chem, 16(11):1207-1216, 1973.

Hansch, C., and Lien, E.J. An analysis of the structure-activity relationship in the adrenergic blocking activity of ß-haloalkylamines.

Biochem pharmacol, 17:709-720, 1968.

Korolkovas, A., and Burckhalter, J.H. Essentials of Medicinal Chemistry. New York: John Wiley and Sons, 1976, Pp. 113-129.

Larson, D.L., and Portoghese, P.S. Relationship between ED50 dose

and time-course brain levels of N-alkylnormeperidine homologues.

J Med Chem, 19(1):16-19, 1976.

Leo, A., Hansch, C., and Elkins, D. Partition coefficients and their

uses. Chem Rev, 71(6):525-616, 1971.

Lien, E.J. Structure-activity-distribution relationship: Significance

for drug design. In: Ariens, E.J., ed. Drug Design, Vol. V. New York:

Academic Press Inc., 1975. pp. 81-131.

Lim, R.K.S., Guzman, F., Rodgers, D.W., Goto, K., Braun, C., Dickerson,

G.D., and Engle, R.J. Site of action of narcotic and non-narcotic

analgesics determined by bradykinin-evoked visceral pain. Arch int

Pharmacodyn, 152(1-2):25-58, 1964.

Oh-ishi, T., and May, E.L. N-Alkylnorketobemidones with strong agonist

weak antagonist properties. J Med Chem, 16(12):1376-1378, 1973.

195
Pert, C.B., Snyder, S.H., and Portoghese, P.S. Correlation of opiate

receptor affinity with analgetic effects of meperidine homologues.

J Med Chem, 19(10):1248-1250, 1976.

Wilson, R.S., Rogers, M.C., Pert, C.B., and Snyder, S.H. Homologues

of N-alkylnormeperidines. Correlation of receptor binding with analgesic potency. J Med Chem, 18(3):240-242, 1975.

AUTHORS

E. J. Lien, G. L. Tong, D. B. Srulevitch, and C. Dias

School of Pharmacy, Section of Biomedicinal Chemistry

University of Southern California

1985 Zonal Avenue, Los Angeles, California 90033

196
Section III

Molecular Mechanics

The presentations using quantum mechanical calculations

are primarily intended to predict drug activity. The

rather large number of papers using this method of approachillustrates the extent to which this is a current

tool in QuaSAR studies. These calculations can be classified into two categories: The isolated molecule

model, which provides information on the early stages of

the drug-receptor interaction; and molecule-receptorcomplex models, which are designed to obtain insight on

relevant features of the drug molecule at the receptor

site. In both cases the quantum mechanical models explore "intrinsic" activity at the receptor site rather

than overall in vivo activity.

One special application of the isolated model approach

is tne calculation of molecular conformations. Similarities in conformation, charge distribution and size

of atoms or groups of atoms may suggest similar binding

to the receptor. However, inferences from calculated

conformations should be made cautiously: the molecule

may possess several close lying conformations and

varying aegrees of flexibility; the active conformation(s) is (are) not known; and the calculated geometries are "method dependent." This last point is

actually true for any quantum mechanical index but the

situation is nevertheless not hopeless: ab initio

Hartree-Fock calculations are generally reliable and

f'ull configuration interaction calculations, when possible to carry out from a practical standpoint, give the

exact non-relativistic energies; on the other hand, the

simple semi-empirical procedures (e.g., IEHT), carefully

calibrated and judiciously applied can be well correlated to experimental data. It was pointed out by Dr.

Kaufman that perhaps the greatest caution is necessary

in using the intermediate calculational schemes (e.g.,

CNDO). The paper of Katz et al. (this monograph) is an

illustration of this point.

197
An alternative method of calculating molecular geometries via classical potential functions has also been

used on isolated and solvated molecules. While this

method has the advantage of being relatively simple to

use, it does have the limitation of being an empirical

approach.

Some concern was expressed about conclusions that are

based on properties of individual molecular orbitals

(e.g. HOMO and LUMO). Within the Hartree-Fock functional form for a molecular wave function, the orbital

energies and orbital symmetries are both invariant quantities and thus quantum mechanical observables. The

shape of a molecular orbital is not an invariant property

and in fact is strongly dependent on the orbital basis

set used in the calculations. However, the careful use

of individual molecular orbitals in predicting chemical

behavior is clearly justified by its practical success

over the years.

198
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Quantitative Structure-Activity

Relationships in the 2,4,5-Ring.

Substituted Phenylisopropylamines

George M. Anderson III, Neal Castagnoli, Jr.,

and Peter A. Kollman

ABSTRACT

The potency of a series of 2,4,5-ring substituted phenylisopropylamines was examined using the rabbit hyperthemia assay. An excellent

correlation (r=0.99) was found between the rabbit hyperthermic and

human psychotomimetic potencies. In the hyperthermic model, the

4-X-substituted-2,5-dimethoxyphenylisopropylamines were found to be

one to two orders of magnitude more potent than the 2-X- or 5-X-substituted positional isomers (X=-H,-CH3,-SCH3,-Br). Conformational

perturbations induced by substituents ortho to the ethylamine side

chain were studied with the PCILO and ab initio molecular orbital

methods. The variations in the biological activities could not be

rationalized in terms of the ability of the ortho substituents to

stabilize conformations which mimic LSD. The electronic structures

of the positional isaners were examined in the corresponding toluene

analogues using the CNDO/2 method. A reasonable correlation (r=0.98)

was found between the Highest Occupied Molecular Orbital (HOMO) energy

and the ionization potentials reported from photoelectron spectroscopy

studies. In the case of the positional isomers, the HOMO energies

were ordered as follows: 4-X>5-X>2-X. However, the regression analysis of the relationship between these orbital energies and Log Biological Activity (B.A.) was not impressive. Examination of the

partitioii coefficients (octanol/water) of the positional isomers

indicated that the 4-X- and 5-X-substituted compounds have almost

equivalent Log P's, but that the 2-X-substitute-4,5-dimethoxyphenylisopropylamines are unusually hydrophilic. The regression of Log H.P.

to the HOMO energies resulted in a marginally significant relationship; addition of the Log P's resulted in no significant improvement.

Qualitative models based on both regiospecific lipophilicity or

electron densities and also metabolic conversion to reactive intermediates are presented.

INTRODUCTION

Structural manipulation of the aromatic moiety of amphetamine has

provided numerous methoxy-substituted analogues and a variety of

4-substituted-2,5-dimethoxyphenylisopropylamines. Attempts to relate

199
the psychotomimetic potency of these compounds to physical properties

have elaborated three "themes." The first approach is based on the

basis of physical properties suggested by Karreman et al. (1959) and

further elucidated by Snyder and Merrill (1965) and Kang and Green

(1970) proposing that potency is related to the electron donor

properties of the aromtic system. In the second scenario, addressed

by Snyder (1970), Kang and Green (1970), Baker et al. (1973), and

Nichols et al. (1978), the potency of various classes of agents

having the arylethylamine structure is related to the ability of the

side chain to assume conformations which mimic the potent psychotomimetic LSD. Finally, Barfknecht et al. (1975) and Nichols et al.

(1978) have pointed to the correlation of activity with lipid

solubility and implicated the importance of distribution effects in

modulating the psychotomimetic potency of the ring substituted

phenethylamines and phenylisopropylamines.

In the intact animal, psychotomimetic activity appears to be a

complex function involving all of these variables. Since this is

the case, it is difficult to draw conclusions about molecular events

from the structure activity relations. We have attempted to simplify

the structure activity relations by designing a novel series of

"rearranged"' phenylisopropylamines bearing substituents at the 2,4,

and 5 positions:

The logic behind our approach is that positional isomers might be

expected to be isolipophilic. We had therefore hoped to factor the

electronic and conformational aspects of the activity equation from

distribution considerations. With these expectations in mind, we

began our theoretical investigation into the physical parameters of

conformation and electronic structure using molecular orbital methods.

Specifically, we have studied the side chain conformation of various

ortho-substituted phenethylamines and attempted to relate this information to the biological activities of 2-X-substituted-4,5dimethoxyphenylisopropylamines. In parallel with this, we present rabbit

hyperthermic potencies of these rearranged isomers and demonstrate

a good correlation between these numbers and human psychotomimetic

potencies. Next, we have analyzed the effects of substituents on

the orbital energies and electron densities of the aromatic ring.

We have evaluated the validity of the assumption that positional

isomers are isolipophilic and have attempted to explain the deviations

in the partition coefficients in terms of electronic effects. The

data have also been interpreted in terms of how structural effects

may influence the metabolic fate of these compounds. Finally, our

studies have led us to propose potentially interesting analogues for

further analysis.

200
METHODS AND PROCEDURES

Synthesis of the "rearranged" positional isomers was afforded by

condensation of the appropriately substituted benzaldehydes with

nitroethane followed by reduction of the phenylnitropene with

lithium aluminum hydride. Details of the synthetic scheme will be

described elsewhere.

The biological activities of compounds (1 )-(14) were measured using

the rabbit hyperthermia assay and the potencies are reported in

Standard Rabbit Units (SRU) in which racemic DOM (5) is taken as the

reference. Details of the assay have been reported by Jacob III

et al. (1977).

The PCILO semiempirical molecular orbital method (Diner et al. 1967)

was used to study the conformational structure of the ethylamine side

chain in a variety of ring substituted phenethylamines. Standard

bond lengths and angles were used and parameters for the bromine atom

were taken from the previous assignments of Kollman et al. (1973).

Extensive applications of this method to problems of biological interest have been made by Pullman et al. (1973), who have demonstrated

the reliability of the calculated conformational minima. Rotations

were made about the T1 and T2 axes of the phenethylamine side chain

in increments of 30°, and the energies of the localized Kekúle

structures were averaged. The energy of several of the PCILO minima

were also calculated with the ab initio program Gaussian 70 (Hehre

et al. 1974) using the STO-3G basis set

The electronic structure of the variously substituted analogues was

examined in the corresponding substituted toluenes using CNDO/2. The

Highest Occupied Molecular Orbital energies were taken at the conformational minima with respect to 30° rotation of the methoxy substituents. Calculated HOMO energies have been shown by Domelsmith, et

al. (1977) to correlate with ionization potentials measured by photoelectron spectroscopy. The technique is also appropriate for

describing rotations about a bond with partial bond character as

occurs with methoxy substituted benzenes.

The distribution characteristics of compounds (1 )-(14) were modeled

by partition coefficients (octanol/water). Substituent contributions

to the overall molecular lipophilicity were derived from the measured

Log P of the substituted phenylisopropylamine or from a study of model

compounds. When partition coefficients were determined in solvents

other than octanol, both the substituted and reference values were

taken from the same solvent, converted into the Log P in octanol

according to the equations of Leo et al. (1971), and subtracted to

give the substituent π values.

RESULTS AND DISCUSSION

Rabbit Hyperthermia

The rabbit hyperthermia assay has been employed in the pharmacological

evaluation of a series of LSD analogs (Cerletti 1959), substituted

tryptamines (Brimblecombe 1967), and phenylisopropylamines (Aldous

201
et al. 1974). The applicability of this animal model to psychoactive

agents can be measured in terms of the correlation between the animal

and human potencies. The hyperthermic potencies of compounds (1 )-(14)

are summarized in table 1; an excellent correlation exists between

the hyperthermic and human potencies (n=8, r=0.99). As can be seen,

the most potent compounds were the 4-X-substituted-2,5-dimethoxyphenylisopropylamines. In this series, substitution of the para-H with a

-OCH3, -SCH3 -CH3 and -Br group results in large increases in hyperthe 5-X-substituted-2,4-dimethoxyphenylisopropylamines which all have

thermic potency. Similar increases in activity are not achieved in

202
comparable potencies, except in the case of the methoxy analogue

which clearly is more potent. In the 2-X-substituted-4,5-dimethoxyphenylisopropylamines, replacement of the ortho-H with any group

increases activity, but only the -OCH3 analogue has any substantial

potency.

Analysis of Conformational Properties

The hyperthermia results indicated that potent activity was generally

associated with analogues bearing an ortho methoxy group. Since ortho

substituents could be expected to influence the arylethylamine conformation, we have attempted to explain the inactivity of the ortho -H,

-SCH3, -CH3 and -Br analogues in conformational terms. Historically,

thel psychotomimetic potency of the ring-substituted phenethylamines,

phenylisopropylamines and tryptamines has been rationalized in terms

of the ability of the ethylamine side chain to assume conformations

which mimic certain aspects of the LSD structure. In the model proposed by Snyder et al. (1975), the crucial phenethylamine conformations are those which mimic the B and C ring of LSD. The “B Ring

Mimic” was envisioned to arise from a gauche conformation (T1=180°,

T2=0°) in which the side chain was planar with respect to the aromatic

ring and the amine was folded back toward an ortho-H substituent.

The C Ring of LSD was thought to be mimicked by a folded gauche conformation (T1=0°, T2=40°) in which the arylethylamine protons were

involved in an intramolecular interaction with the electron density

centered on the oxygen atom of an ortho methoxy substituent. Kang

and Green (1970) have suggested that the LSD structure could be

mimicked by an extended trans conformation (T1=150°, T2=170°) in which

the phenethylamine portions both LSD and the ring substituted

phenethylamines and phenylisopropylamines were directly superimposed.

Baker et al. (1973) have analyzed the X-ray structure of LSD and

2,4,5-trimethoxyphenylisopropylamine (HCl salt) and have proposed a

mimic conformation (T1=40° , T2=240°) in which the amine portions of

the phenethylamines were superimposed with the N(6) of LSD and an ortho

methoxy group was paired with the 9,10 double bond of LSD. Recently

Nichols et al. (1978a) have proposed a mimic conformation which was

similar to a trans conformation proposed by Kang and Green (1970), but

the phenyl ring oriented differently with respect to the indole

ring of LSD (T1=150°, T2=-120°). Nichols superimposed the N(6) and

C(2) portions of the LSD structure on the amine and ortho methoxy

groups of the ring substituted phenethyl and phenylisopropylamines,

Since these conformational theories were based on consideration of

analogues bearing either hydrogen or methoxy substituents at the

ortho position, it was of interest to reexamine the applicability

of these conformational predictions for the novel series of 2-Xsubstituted-4,5-dimethoxyphenylisopropylamines studied by us. Thus,

a PCILO conformational analysis of these analogues has been carried

out and is summarized in table II.

There have been many theoretical studies of phenethylamine conformation

using a variety of methods (Martin et al. 1975; Weintraub and Hopfinger

1973; Hall et al. 1975 and Pullman and Pullman 1975). These theoretical calculations have generally indicated that the side chain of these

variously substituted phenethylamines was conformationally quite

203
unrestricted and that the trans conformation was slightly less favorable than the gauche. The location of the global and local minima for

these compounds was similar to those obtained by us using PCILO.

Our PCILO calculations predict that in the gas phase or in an inert

solvent, the folded gauche conformation (T1=90°, T2=600

) was about

1 kcal/mole more stable than the extended trans conformation (T1=90°,

T2=180°). In the neutral phenethylamine,this can be rationalized by

N-H...π attractions which preferentially favor the folded forms. In

the protonated phenethylammonium, these attractions result in stabilizationofthe gauche foms relative to the trans conformations. The

introduction of electron donating groups such as a methoxy groups in

the para position further increases the electron donating capacity of

the aromatic ring and lowers the energry of the gauche confomers.

Comparison of the unsubstituted phenethylammonium with compounds

substituted in the ortho position by a -OCH3, -OH, -SCH3 groups

revealed that in these compounds, one of the gauche conformations

(T1=120°, T2=90°) was stabilized via N-H...OCH3, N-H...OH, N-H...SCH3

intramolecular attractions. In the orthomethoxyphenethylammonium

molecule, PCILO found the global minimum to be the gauche conformation

in which the ammonium hydrogen points to the oxygen electrons and

predicted this conformation to be 6.5 kcal/mole more stable than the

trans orientation. The intramolecular stabilization was considerably

 weaker in the orthothiomethyl case where the gauche conformation is

energetically only 1.7 kcal/mole more favorable the trans. In

the ortho -CH3 and -Br substituted phenethylammoniums, gauche

conformation (T1=90°, T2=-60°) in which the amine is folded toward

the aromatic ring on the side opposite to the ortho substituent was

found to be the global minimum and slightly more stable than the local

trans minimum.

Ab initio calculation of the trans and gauche PCILO minima for phenethylammonium showed the gauche to be preferred over the trans form by

0.24 kcal/mole, in excellent agreement with the PCILO energies (0.30

kcal/mole). A similar comparison of the energies calculated by the

ab initio and PCILO methods at the trans and gauche minima of orthomethoxyphenethylammonium found the gauche conformation to be more stable than the trans by 16.5 kcal/mole and 6.5 kcal/mole respectively.

The relevance of gas phase conformations in biological systems is

indirect at best and calculations at all levels which have considered

solvent effects have shown the trans conformations to be preferentially

stabilized relative to the gauche (Weintraub and Hopfinger 1973;

Pullman 1974). Determination of the T2 rotamer populations of phenethylamine (Ison et al. 1973), dopamine (Bustard and Egan 1971) and

numerous poly-substituted phenylisopropylamines (Bailey et al. 1971)

via NMR have shown almost equal populations of trans and gauche conformations in solution. X-ray structures have been reported for the

salts of phenethylamine (Tsoucaris 1961), dopamine (Bergin and

Carlstrom 1968), mescaline (Ernst and Cagle 1973), amphetamine (Bergin

and Carlstrom 1971) and 2,4,5-trimethoxyphenylisopropylamine (Baker

et al. 1973). In each crystal structure, an extended trans conformation (T1=70-89°, T2=171-175°) was found except for the 2,4,5-trimethoxy compound (T1=67°, T2=50°).

204
A number of workers have proposed phenethylamine conformations which

allow it to mimic LSD at a hypothetical hallucinogenic receptor. We

have used our PGILO calculations to evaluate the energy of these

conformations and have attempted to rationalize the conformational

effects of the ortho substituents in terms of these mimic structures.

If the phenethylamine binds to the receptor 103 times weaker than LSD

and has the same total intrinsic attraction at its LSD mimic conformation, one would expect that a reasonable upper bound for the conformational energy would be about 4 kcal/mole relative to the global

minimum (Δ G=-2.3RT log(10)3). If LSD has a greater intrinsic affinity

for the receptor than the LSD mimic conformation of the analog, then

the upper bound of the conformational energy must be correspondingly

less to compensate for this.

Since our calculations employed fixed internal bond lengths and angles,

the energy of conformations in which there are significant steric

effects (T1 between -40° and 40° or between 140° and 220°) is probably

an overestimate of the true energy for these conformations. Table II

lists the energies for the two LSD mimic conformations proposed by

Snyder and the mimics proposed by Kang and Green (1970) , Baker et al.

(1973) and Nichols (1978).

It is clear that the Snyder conformations are impossibly high in

energy but that the other three are energetically quite reasonable.

However, comparison of the energies of the latter conformational

mimics of the ortho-H and -OCH3 analogues predicts that the

ortho-OCH3 phenethylammonium should bind correspondingly weaker than

phenethylammonium, a prediction not supported by the biological

activities. Further, no explanation for the low potency of the

ortho substituted -SCH3, -CH3, and -Br analogues can be clearly

defined in terms of LSD mimic conformations.

Electron Donating Properties

The apparent inability of conformational arguments to rationalize the

variations in hyperthermic potency led us to a detailed investigation

of the electronic structure of compounds (1) - (14). A very simple

minded glance at the rearranged isomers might suggest that the

electron donating properties of the aromatic rings would be very

similar. However, CNDO/2 calculations as well as recent photoelectron

spectroscopy measurements (Domelsmith and Houk 1978) have indicated to

the contrary (see table I). On a qualitative level, these electronic

differences are apparent from the resonance structures. For example,

the dimethoxy benzenes, shown below, are accompanied by the number of

resonance structures which place excess charge on the carbon atoms of

the aromatic ring.

205
Table II. PCILO Conformational Analysis of a Series of Substituted Phenethylamines
The trends seen in the π densities are also reflected by the CNDO/2

calculated Mulliken population densities which account for both the

σ and π electrons.

Calculation of the HOMO energies for compounds (1)- (11) (excluding

the Br isomers which are suspect) has been made and a satisfactory

correlation (n=7, r=098) can be shown between these orbital energies

and the ionization potentials determined by photoelectron spectroscopy

in agreement with Koopmans Theorem. While the orbital energy differences between the various rearranged isomers were small, the CNDO/2

results indicated that the ionization potentials were ordered as

follows: 4-X<5-X<2-X. However, the regression of these HOMO energies

with the hyperthermic potencies (log H.P.) was quite impoverished

(r=0.65) and barely significant at the 95% confidence level. The

good correlations reported by others for the methoxy substituted

phenylisopropylamines were not to be found among the rearranged

isomers studied by us.

While quantitative electronic models seemed outside our grasp, a

qualitative picture of drug-receptor inter-reaction did emerge:

Two pharmacophoric sites are postulated in the drug binding, the first

involving the charged ammonium group and the second resulting from

an electronic association between the receptor and the aryl-moiety.

It is clear that good binding results from electron rich

aromatic systems which have both large negative electrostatic potentials at the van der Waals radius (=1.7Å) and also favorable electrostatic potential gradients parallel to the aromatic plane as have been

described by Weinstein at al. (1976) for the 4,5,6, and 7-OH tryptamines. In general, addition of electron donating substituents such

as a methoxy group was found to augment the π electron density of the

aromatic ring and presumably results in favorable electronic associations. The inplane electrostatic gradients were, however, quite sensitive to the position of the methoxy substituents. For example, in the

207
positional dimethoxy benzenes discussed previously, the resonance

arguments predicted low π density at C4 and high density at C1 for

the 2,4-dimethoxy substituted benzene, but little gradient was found

in the 2,5- or 3,4-dimethoxy substituted benzenes. (The Mulliken

populations are also consistent with this reasoning.) These gradients

predict that analogues which are electron rich at the 3,4, and 5

positions and relatively electron poor at the 1,2, and 6 positions

might be most potent at our diagrammatic receptor. While the

unfavorable electrostatic gradient of the 2,4-dimethoxy orientation

might be invoked to account for the low potency of the 5-X-substituted2,4-dimethoxyphenisopropylamines, apparently other arguments must be

advanced to separate the very potent 4-X-substituted-2,5-dimethoxyphenylisopropylamines from the weakly potent 2-X-substituted-4,5-

dimethoxyphenylisopropylamines.

Distribution Properties

If the molecular partition coefficient is assumed to be the linear

sun of the component π substituent values, then the Log P’s of the

mono-, di-, tri-, and tetramethoxy substituted phenylisopropylamines

would be almost equivalent (±0.10). Were this actually the case, one

would expect a rather poor correlation between the small differences

in the distribution characteristics and the rather large variations in

the biological activity of this class of phenylisopropylamines.

Further, the various positional isomers would be isolipophilic and

hence would be incorrectly predicted to be equiactive. These expectations have not been supported by experiment.

Barfknecht et al. (1975) have measured the partition coefficients of

various methoxy substituted phenylisopropylamines and have shown

conclusively that nonadditativity of π values was more the rule than

the exception for these compounds. Some Precedent for these findings

could be taken from the work of Leo et al. (1971) in which the π value

of the central methoxy group in 1,2,3-trimethoxybenzene was derived to

be -0.56 and much closer to the π value of an aliphatic methoxy group

(-0.47) than an aromatic methoxy group (-0.02). It was postulated

that unfavorable steric repulsions forced the central methoxy group

out of the plane making the hybridization of the oxygen more "sp3"

and hence resembling an aliphatic methoxy group.

Because of the nonadditive nature of these substituents, we have

analyzed the π values of the various positional mono- , di-, and trimethoxy configurations in a number of model compounds and have summarized these results in table III. As can be seen, the positional

monomethoxy isomers had almost equivalent π values (π =0.03 - 0.09)

and were reasonably close to the traditional value of -0.02. Similar

results were obtained for the 2,3-, 2,4-, and 2,5-dimethoxy orientations. However, in the case of the 3,4-dimethoxy grouping, a very

large neighboring group interaction substantially lowered the π substituent value. In the trimethoxy arrangements, the 2,4,6- pattern

appears to have a normal π value, whereas the 2,4,5-trimethoxy π value

derived from Nichols et al. (1978) measurement resembles the 3,4-

dimethoxy π value in that it is considerably more hydrophilic than

expected. The 2,3,4- and 3,4,5-trimethoxy π values are very similar

( π=0.20) and somewhat less lipophilic than expected.

208
Table III. Model System Study of the Positional Monomethoxy π Substituent Values, and

the Positional Di- and Trimethoxy π Multisubstituent Values

209
These unusual results have prompted us to explore neighboring group

effects in numerous other ortho oxygen substituted model compounds.

The substituent π values along with the CNDO/2 calculated conformational minima of these groupings have been summarized in table IV.

As can be seen from the table, the minimum energy conformation of

both 2,3- and 3,4-dimethoxytoluene and also 3-methoxy-4-thiomethyltoluene was predicted by CNDO/2 to be nonplanar; however, in the

latter two substitution patterns, a low-lying planar conformation

was also found. In 3-methoxy-4-hydroxytoluene, a nonplanar orientation

similar to the conformational minima of 3,4-dimethoxytoluene was computed to be slightly more stable than the planar form which differs

from the dimethoxy case in that the hydrogen of the phenol is directed

toward a methoxy group whose methyl substituent is pointed away. A

planar global minima was found in 3,4-dihyroxytoluene whereas nonplanar

conformations were favored in both 2,3,4- and 3,4,5-trimethoxytoluene.

The variations in the lipid solubility of these groupings appear to be

related to the solvation of the lone pair electrons of the methoxy

groups by water. The hydrophilicity of the 3,4-dimethoxy grouping can

be explained in one of two ways: 1) lone pair-lone pair repulsions in

adjacent methoxy groups favor nonplanar conformations which are more

strongly solvated than the planar structures, or 2) the planar orientation which exists as a low-lying local minima results in the buildup

of electron density between the methoxy substituents, and this region

provides a good site of solvation. It is difficult to rule out

one of these explanations. Since both the 2,3- and 3,4-dimethoxytoluenes exist in nonplanar forms and yet only the latter has an

abnormal π value, one might conclude that a specific solvation site

is implicated. However, the differences between the π values in these

two cases could also be explained by steric inhibition of solvation in

the 2,3-dimethoxy compounds by the ortho substituent (such as an

ethylamine side chain). Conversely, the methylenedioxy grouping,

which is more lipipophilic than the component substituents, is both

planar and the specific solvation site is blocked by the methylene

moiety. Similarly, 3-methoxy-4-hydroxy pattern is more lipophilic

than expected due to intermolecular hydrogen bonding in the low-lying

planar conformational minima.

In any case, it is clear that our initial assumption that the

“rearranged” positional isomers were isolipophilic was in error. The

adjacent methoxy group interaction makes the 4,5-dimethoxy-2-X-substituted analogues much more hydrophilic than the nearly isolipophilic

2,4-dimethoxy-5-Xand 2,5-dimethoxy-4-X-substituted compounds.

Regression analysis showed a rather poor correlation between Log H.P.

and HOMO energies; addition of the Log P resulted in no significant

improvement. Scrutiny of the log-log plot showed a fairly straight

line for the 2,5-dimethoxy-4-X-substituted compounds, but the H.P. of

the other positional isomers was not correlated with Log P.

Metabolism

A potentially important consequence of the electronic and distribution

properties of the “rearranged” isomers concerns their susceptibility

to biotransformation in vivo. Investigation of the metabolic fate of

DOM (5) (Zweig and Castagnoli 1977) had established the conversion of

210
Table IV. Model System Study of the π Substituent Values and the CNDO/2 Calculated Conformational Structure

of Various ortho-Oxygen Substituted Groups
this compound into the bis-O-demethylated metabolite (15), a close

analogue of the selective noradrenergic toxin 6-hydroxydopamine (1 6) .

The chemical behavior of the hydroquinone (15) and 6-hydroxydopamine

(16) is analogous in that both readily undergo spontaneous oxidation

leading to electrophilic intermediates (Zweig and Castagnoli 1974)

which in the case of 6-hydroxydopamine are likely to be responsible

for the observed destruction of noradrenergic terminals (Malnifors

and Thoenen 1971).

In this regard, the exceptional hyperthermic potency of phenylisopropylamines (5), (8), (11), and (13) (100, 54, 405, and 12 SRU respectively), all of which are 2,5-dimethoxy substituted, compared to the

low hyperthermic potency of the positional isomers (4 ), (6), (7) and

(12) (1, 0,5, and 3 respectively), none of which are 2,5-dimethoxy

substituted and are incapable of metabolic conversion to a parahydroquinone, focused attention on the special structural features

associated with the para-oxygen substitution pattern.

At the present time, no attempts have been made to determine if these

substituent effects relate, in general, to the metabolic profiles of

this class of compounds. Nonetheless, the potential association of

psychotomimetic activity with the metabolic formation of 6-hydroxydopamine like intermediates from the potent para-oxygen substituted

compounds studied here is intriguing. In vivo metabolic oxidation of

psychotomimetic amines to electrophilic species capable of covalent

interactions with brain enymes responsible for the control of central

amines could lead to biochemical lesions and account for the toxic

CNS effects of these analogues.

Summary and Future Directions

The excellent correlation between rabbit hyperthermia and human psychotomimetic potencies for a variety of psychotomimetic agents strongly

asserts the utility of this animal model in the pharmacological evaluation of this class of compounds. Consideration of the conformational,

electronic, metabolic, and distribution properties of the "rearranged"

isomers has led us to the conclusion that the psychotomimetic potencies

cannot be related satisfactorily to overall molecular properties, such

as Log P or the HOMO energies, but instead appears to be dependent upon

the regiospecific properties such as in plane electrostatic gradients,

local group lipophilicity, or metabolic conversion to reactive intermediates. While trends can be found in both the HOMO energies and

Log P which do reflect the variations in psychotomimetic potency, the

numerical formulation of the SAR using these variables results in

212
unimpressive regressions which have only marginal predictive capabilities and contribute little to our understanding of these novel compounds. Indeed, further analysis into the regiospecific properties

of the “rearranged” isomers and integration of these factors into a

QuaSAR equation constitute the direction of our researches. Towards

these ends, we have extended the analogue set by including both the

OCH2CH3 (1 7), (1 8), (19) and OH (2 0), (2 1), (2 2)substituted phenylisopropylamines, In the OCH2CH3 analogues, the overall electronic

and distribution properties of these positional isomers are very nearly

equivalent and may provide insights into the potential relationships

between the inplane electrostatic gradient and psychotomimetic potency.

The -OH substituted phenylisopropylamines provide an interesting example of positional isomers which are probably much more lipophilic than

previously anticipated due to the intramolecular hydrogen bonding of

the adjacent OH...OCH3 groups in compounds (2 0) and (2 1), and similarly

between the OH...NH2 moieties in the 2-OH analogue (22). In these

examples, the involvement of regiolipophilicity and metabolic effects

will be investigated.

Clearly, the “rearranged” phenylisopropylamines represent a unique.

set of analogues whose SAR are quite different from the methoxy

substituted phenlisopropylamines or 2,5-dimethoxy-4-alkylphenylisopropylamines studied previously. Further research into these analogues

may well reveal the critical factors related to the molecular expression of the complex phenomenon of psychotomimesis.

“In the infancy of a science generalizations are rarely true beyond

narrow and too often undefined limits. Always the question, How?

punctures the bubble of theory, and the answer is to be sought in

analysis and ever more analysis.”

K. S. Lashley, 1933

ACKNOWLEDMENTS

We are grateful to the Academic Senate (PAK) and the Earl Anthony

Fund (GMA) of the University of California, San Francisco and

MH-21219 (NC) for support of this research. PAK is grateful to the

NIH for a Career Development Award (GM-70718) and GMA III has been

supported by NIH Training Grant GM-00728. We would like to thank

Harrell Weinstein for the use of his electrostatic potential program

and Don Berkowitz and Gilda Loew for their programming assistance.

We gratefully acknowledge the use of the UCSF Computer Graphics

Laboratory (NIH-RR-1081) in this study.

213
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AUTHORS

George M. Anderson III

Graduate Student

Department of Pharmaceutical Chemistry

School of Pharmacy

University of California

San Francisco, California 94143

Neal Castagnoli, Jr., Ph.D.

Professor of Chemistry and Pharmaceutical Chemistry

Department of Pharmaceutical Chemistry,

School of Pharmacy

University of California

San Francisco, California 94143

Peter A. Kollman, Ph.D.

Associate Professor of Chemistry and Pharmaceutical Chemistry

Department of Pharmaceutical Chemistry

School of Pharmacy

University of California

San Francisco, California 94143

217
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Assessment of Quantum

Mechanical Techniques for Use in

Structure Activity Relationship

Development, and Application to

Analgesics and Other Drugs

B. Vernon Cheney, D.J. Duchamp, and Ralph E.

Christoffersen

I. INTRODUCTION

In the past fifteen years, the combination of advances in theoretical

methodology and computing capabilities has created a major new, avenue

of reseacrh. This research, Which can be described as the application

of theoretical chemical techniques to problerms of biological interest,

is now moving rapidly toward quantifying concents and providing significant insight in areas where only very qualitative discussions have

been possible in the past. For example, in the period between

and 1975, no less than 154 studies using quantum mechanical techniques

in the area of drug design appeased in the literature (Christoffersen

1976 a ). These studies encompass at least 27 different areas, including 25 studies on drugs having analgesic and/or hallucinogenic

action.1 During the last two years, both the interest and activity

in these areas have increased substantially, as this and the other

papers in this symposium will indicate.

Since most of the papers that follow this one will deal with the

use of one (or several) specific technique(s) to develop quantitative

structure-activity relationships on narcotic analgesics, antagonists,

and hallucinogens, this paper will describe several of the techniques

that are currently available or being developed, and the general

“state of the art." Included will be a discussion of the general

capabilities and deficiencies of the techniques, as well as an indication of the nature and extent of studies that are necessary if

they are to be used profitably within a program of drug design. In

addition, application of ab initio techniques to a variety of narcotic agonists and antagonists will be described.

II. METHODOLOGY ASSESSMENT

In order to place into proper context the vast majority of studies

already in the literature and most of those which are expected in

the near future, it is useful first to consider some of the major

ingredients that are needed if a reasonable description of drugs and

drug-receptor interactions is to be obtained using theoretical

methods. In addition, it is important to describe at least some of

the available theoretical techniques, so that their advantages and

218
limitations when applied to problems in drug design can be taken

into account.

First, it should be recognized that, for nearly all theoretical

studies, only one or at most several of the important factors

dealing with drug activity are considered. In particular, most

studies deal either with characterization of the isolated (or

solvated) drug, or with of model of the drug-receptor interaction.

While these aspects are indeed very important components of overall

drug activity, it most also be remembered that a proper understanding of overall activity will include many factors, e.g.,

absorption, excretion, catabolism, binding to plasma protein,

penetration of the blood-brain barrier, affinity for the receptor,

intrinsic activity, and perhaps others. Hence, if only isolated

drugs or model drug-receptor interections are considered, an

adequate understanding of overall drug activity can be expected only

if other factors play a relatively constant role. In addition,

if only isolated (or solvated) drug molecules are, considered, then

only a description of the initial stages of the drug-receptor

interaction can be expected. These initial stages will be

important considerations only if the transition state(s) resemble

the reactants. However, in spite of the complexity of the problem

and the many effects that need consideration in principle, remarkable progress is now occurring in providing insight into drug action

mechanisms, or at least, to provide very valuable information for

use in the direction of synthetic efforts.

In characterizing the available theoretical methods for correlating

overall drug action with molecular characteristics of drugs, (called

“quantitative structure-activity relationships”-QSAR studies), it is

of interest to note that there are essentially only two or three approaches in use currently. The first of these, exemplified by the

studies of Hansch (Hansch 1969) and several of the papers at this

symposium, employs the use of calculated or measured molecular properties of drugs, and attempts to find statistically valid linear (or

non-linear) relationships between activity and molecular properties

related to the thermodynamic free energy. The second approach, which

is similar in intent but differs in the methodology used, is generally

referred to as pattern recognition (Kowalski, Bender 1974). In this

approach much larger data bases are used in general, frequently including simply basic molecular information such as the nature of the

atoms, bond lengths, bond angles, etc. Mathematical searching and

sorting techniques quite different from linear (or non-linear) free

energy correlation techniques are then used to construct correlations

with biological data.

However, in both of these approaches there is an important similarity.

Since the resulting correlation(s) and/or equation(s) are derived on a

statistical basis, it cannot be expected that they will necessarily

give insight into the actual mechanism of action. Instead, they

should be viewed as relationships that may be quite useful in directing synthetic efforts toward development of increased activity in a

given series (“lead optimization”), but will not ordinarily be capable

of creating that is chemically unrelated to the series used

to develop the initial structure-activity relationship. To do the

219
latter ("lead generation") typically requires that information conconcerning the receptor as well as the drug be included in the study.

Since few receptors have to date been isolated, purified and characterized, studies such as these have been quite limited in number

to date (Scheiner, Kleier, Lipscomb 1975). However, it is expected that

theoretical studies of drug-receptor interactions will be of considerable importance and expanded significantly in the future.

In generating molecular properties for use in QSAR studies, one of

the techniques most commonly employed is molecular quantum mechanics.

Unfortunately, not only one technique is implied by this terminology,

but a whole variety of approaches that differ both in level of

accuracy attainable in principle and in the particular manner in

which the method has been implemenented.

In terms of the accuracy attainable in principle, the available

techniques can be classified as:

1. Hartree-Fock (SCF-MO) techniques

2. Correlation energy techniques

3. Empirical techniques

In the first case, the accuracy is limited to that of the molecular

orbital model, where the detailed correlation between electrons cannot be described (Schaeffer 1972). This deficiency is corrected in

the second case where, at least in principle, all non-relativistic

interactions can be described accurately. The third approach eschews

the first two entirely, and uses available experimental data to

develop a set of empirical potentials that accurately describe molecular properties of test systems. Then, as long as the drugs (or drugreceptor models) of interest resemble the test system in terms of the

kinds of interactions expected, an adequate description of the energetics of these systems can be obtained.

In the large majority of studies on drugs to date, the Hartree-Fock

model has been used, at various levels of approximation. This includes

several semi-empirical approaches, including Extended Huckel Theory

(EHT) (Lohr,Lipscomb 1963; Hoffman 1963; Hoffman 1964), Interacted Extended Huckel Theory (IEHT) (Rein, Win, Clarke 1966; Carroll, Armstrong,

McGlynn 1966; Zerner, Gouterman 1966), various methods based on neglect

of diatomic differential overlap (CNDO/2), INDO, PRDDO, and MINDO)

(Pople 1970; Halgren, Lipscomb 1973; Dewar, Haselbach 1970), as well as

ab initio approaches using a variety of basis sets(Christoffersen 1972).

Each of these methods has its own set of idiosyncracies (Pullman 1976),

and a potential user of any of these must become familiar with the

characteristics of the method before application to drug system, if

serious pitfalls are to be avoided.

In the second class of techniques, only two methods are in common

usage, the PCILO technique (Diner, Malriu, Jordan, et al 1969) and ab

initio formulations (Hackmeyer, Whitten 1971). The first of these is a

semi-empirical procedure that is designed primarily for conformational

studies of isolated molecules. It is based on a configuration interaction approach which allows, at least in principle, incorporation of

correlation effects into the parameters that are chosen. In the case

of ab initio approaches, only a few applications of configuration interaction techniques to systems of biological interest have occurred

220
to date(Hackmeyer, Whitten 1971), but represent an important trend for

the future that will allow, at least in principle, the proper description of reaction surfaces and spectra as well as the ground state.

In the third class of techniques, a variety of potentials has been

created (Scheraga 1968; Balasubramanian, Chidambaram, Ramachandran 1970;

Engler, Andose 1973) each designed to describe a particular class of

molecules. This class of techniques provides a potentially powerful

methodology for examination of relatively large numbers of conformational degrees of freedom, and hence is of substantial interest

to problems involving large molecular systems. However, there

remains a great need for development of a consistent set of

potentials that includes both intra- and intermolecular interactions

over a broad range of molecules.

In addition to the three basic classes of methods mentioned above,

other developments related to these should be noted, that add substantially to the techniques available for theoretical studies.

Perhaps the most useful and promising of these involves the use of

isopotential maps and interaction potentials(Weinstein 1975a,b) that.

are derived from wavefunctions obtained using Hartree-Fock and/or

correlation energy techniques to describe drug-receptor interactions.

Even if one or more methods can be identified as being suitable for

application to a given problem, significant difficulties remain in

the usual manner in which studies are carried out. As an example,

consider the methadone molecule, shown in Figure 1. Considering

conformational questions only, there are at least 11 dihedral angles

that should be varied simultaneously to find minima. If bond angle

and bond distance variations are also allowed, the dimension of the

non-linear parameter space that must be searched is beyond the size

that can be accommodated with contemporary computer systems. Even

if the energy evaluation at each point in this space is very rapid

(e.g., using empirical potentials), the techniques available for

optimizing non-linear parameters begin to fail for systems containing

>20 independent variables. Hence, serious simplifying assumptions

are typically required in order to devise a workable problem.

Fortunately, chemical experience and experimental data are frequently

available to aid in introducing realistic simplifying assumptions.

Other examples where currently used methodology is frequently inadequate include the proper use of statistical mechanical concepts

when interpreting conformational maps and the inclusion of solvent

effects. The first of these is relatively easily remedied(Washel,

Levitt 1976; Warshal 1977), but the second adds yet another serious

dimension to the problem. Work in the solvent effect area is currently quite active(McCreery, Christoffersen, Hall 1976; Beveveridge, Kelly,

Radna 1974; Beveridge, Schnuelle 1975; Pullman,Pullman 1975; Kollman,

Kuntz 1976), and several approaches are becoming both computationally

feasible and chemically realistic. However, proper consideration of,

e.g., solvent displacement at a model receptor site, is a very difficult problem to treat properly using current techniques and technology.

221
Hence, special care should be exercised in theoretical studies to

choose a suitable method, to include as many aspects as available

technology and resources allow, and systemtically study a relatively

large data base to avoid artifacts.

As difficult as such a procedure may appear, one current technological

development of great potential for alleviating several of the major

areas of difficulty is now available, and needs to be exploited.

In particular, the introduction during the last several years of

minicomputer systems that have both high speed and high internal

precision at relatively low cost is an event of particular importance.

Even using "off-the-shelf" hardware and software, minicomputer systems

that have comparable precision and are only 10-15 times slower than

near-state-of-the-art large scale computer systems (e.g., CDC 7600s)

can be purchased. Considering the access that is reasonably available to a dedicated system such as this, it is easy to show that such

a minicomputer system is far more cost effective for many problems

than large scale systems that are available, and that the total amount

of work that can be accomplished (e.g., in number of molecules studied, number of degrees of freedom considered etc.) is greatly enlarged. If the possibility of specialized hardware design and ease

of implementation on minicomputers is also considered, the costeffectiveness of a minicomputer system is further enhanced. Hence,

considering the likely advances and decrease in cost of minicomputer

technology in the near future, it is clear that substantial advances

in the extent of studies possible and general availability of

theoretical studies to the chemical and biological community are

now possible.

III. APPLICATION TO ANALGESICS AND RELATED DRUGS

A. Geometric Studies.

As noted as early as 1949(Shaumann 1949), there are several structural

features shared by nearly all strong analgesics(the "morphine rule"),

including: 1) a quaternary carbon atom, 2) an aromatic ring linked

to the quaternary carbon atom, 3) a tertiary amino group separated

from the quaternary carbon by two saturated carbon atoms, and 4) a

phenolic hydroxyl group situated meta to the quaternary carbon atom

if the tertiary nitrogen atom is part of a fused piperidine system.

In addition, replacement of the methyl substituent attached to the

quaternary nitrogen with an allyl or other substituents frequently

introduces a degree of antagonist activity to the molecule. Hence,

it is of interest first to see if these characteristics can be

rationalized on a molecular basis using electronic and geometric

features resulting from quantum mechanical and other theoretical

studies.

In order to begin such investigations, six classes of compounds that

exhibit various degrees of analgesic agonism and/or antagonism have

been examined. The 16 specific molecules iincluded in the study are

depicted in Figure 1, and include 1) the morphines, 2) oxymorphones,

3) morphinans, 4) benzomorphans, 5) 1,1-diphenylaminoalkanes, and

6) 4-phenylpiperidines.

222
In order to establish reasonable starting points for study of these

molecules, geometric structure data was sought from available x-ray

crystallographic studies. For morphine (Gylbert 1973; Mackay, Hodgkin

1971), naloxone(Karle 1974; Sime, Forehand, Sime 1975), cyclazocine

(Karle, Gilardi, Fratini et al 1969), methadone(Hanson,Ahmed 1958; Burgi,

Dunitz, Shefter 1973; Bye 1974), propoxyphene(Bye 1973), and meperidine

(Van Koningsveld 1970), x-ray studies have been reported earlier. In

the current studies, four new, high precision, single crystal x-ray

Studies have been carried out. The molecules studied include morphine,

nalorphine, fentanyl, and meperidine (Duchamp, Chidester, Olsen 1977).

Since one of the questions of interest pertains to the degree to

which these various molecules resemble each other, it was decided

that, before attempting electronic structural studies, it would be

important first to ascertain whether these molecules could be placed

in similar conformations without excessive energetic cost. To do

this, the techniques of molecular mechanics(Scheraga 1968; Balasubramanian, Chidambaram, Ramachandran 1970;Engler, Andose,Schlever 1973)

were employed but using a set of potential functions developed

specifically to be applied to large molecular systems such as the

analgesics. The form of the potential that was used is given by

The parameters for these potentials were chosen by studying experimental data for small molecules, and tested by predicting geometric

223
parameters for large molecules in the crystal. The level of accuracy

expected in predicted geometric parameters is -0.01Å for bond distances,  ̃1o

 for bond angles,  ̃3° for torsion angles, and -0.05Å for nonbonded distances. Details of the parameter development will be given

elsewhere(Oie,Duchamp,Christoffersen, to be published).

One of the first questions investigated was: How flexible is morphine

itself? While the molecule is frequently thought to be quite rigid

because of the presence of five rings, there is still some flexibility.

To illustrate this flexibility, rotation about the C12-C13 bond was

investigated, minimizing all other geometric parameters for each

value of the C4-C12-C13-C14 torsion angle. The results of this Study

are summarized in Figure 2. The first point of interest that is

evident in the figure is that there is a considerable amount of

flexibility in morphine about the C12-C13 bond, in that less than

3 kcal/mole is required for a variation of ±15° from the minimum.

This is an important flexibility, since it greatly changes the

distance of the basic nitrogen from the plane of the aromatic ring,

a feature which is inferred to be important in the drug-receptor

interactions(Hende,Nelson 1967). This flexibility is also exhibited

in other molecules, whose observed C12-C13 angles are also indicated

in Figure 2. In particular, it is seen that, in addition to the minimum at  ̃141° found here for the free, protonated morphine molecule,

a value of 135.7° is found in the morphine hydrochloride crystal

(A)(Gylbert 1973;Mackay,Hodgkin 1971), 134.1° is found in free base

morphine (B)(Duchamp,Chidester,OlSen 1977), 132.5° is found in the

nalorphine hydrochloride crystal (C-current study), and  ̃125° is

found for the O-CH3 derivative of etorphine (D)(Hende,Nelson 1967).

Thus, one of the first conclusions of interest is that morphine itself

has some flexibility, and when compared to other molecules, its

geometry must also be varied and should not be assumed to be rigid.

Next let us consider the fentanyl molecules, which has considerable

conformational flexibility, starting from the crystallographic data

(Duchamp,Chidester,Olsen 1977). In order to investigate whether

fentanyl and morphine can achieve similar conformations at reasonable

energetic cost, four points were matched, including the quaternary

nitrogen in both molecules, plus C3, C1, and C12 in morphine with the

para and two meta positions of the phenyl of the phenthyl moiety in

fentanyl, respectively. The technique used in this matching has

been described elsewhere (Duchamp 1977). By allowing the coordinates

of all other atoms to vary, a geometry for each molecule which

maximized the overlap of the four atom just mentioned was obtained.

The result for this case is that, for an energy cost of 2.6 kcal/mole

in morphine and 5.2 kcal/mole in fentanyl(compared to the minimum

energy for each of the molecules taken as isolated, free molecules),

an overlay of the two molecules can be obtained in which the three

atoms chosen from the phenyl groups are positioned within 0.1,

0.2, and 0.2Å of each other, and the nitrogen nuclei are within 0.5Å

of each other. It should be noted that the fentanyl geometry

found in the match to morphine is Substantially different than the

geometry found in the crystal. Of course, if higher energies were

allowed, even closer fits could be obtained. The conclusion that is

reached is that, for energies well within the range that might be

224
expected to be present in a drug-receptor interaction, both fentanyl

and morphine can be matched quite well.

For the case of meperidine,which was also studied crystallographically

in the current investigations, it was found that meperidine hydrochloride has 2 molecules in the asymmetric unit, which differ

primarily in the orientation of the ethyl group. The other point of

interest is that, in both the current studies and the earlier study

of meperidine hydrobromide by Van Konigsveld(Van Koningsveld 1970).

the phenyl group is in an equatorial position relative to the cyclohexyl ring system.

Earlier discussions of meperidine activity have involved suggestions

that an axial phenyl geometry might be the active form(Beckett,Casey

1965). As a free molecule, the equatorial phenyl form is found to be

favored by 0.8 kcal/mole over the axial phenyl form using the potential

functions described earlier. Both the axial and equatorial forms of

meperdine were matched to morphine, using the 4-point model described

earlier. The result is that both the axial and equatorial geometries

can be made to fit morphine, although the fit for the axial form is

somewhat better. In particular, for essentially a perfect match of the

phenyl moieties, a 0.38Å difference in nitrogen position results at

an energetic cost of -3.6 kcal/mole for the axial form of meperidine,

and requires 2.1 kcal/mole for morphine. For the equatorial form

of meperidine, 6.9 kcal/mole is required, and 2.7 kcal/mole required

for morphine, in order to achieve a nitrogen match within 0.4Å It

should be noted that, in considering the axial phenyl case, (which

involves a piperidine ring flip), the N-CH3 moiety is kept in an

equatorial form by inverting the configuration of the proton and

methyl group at the nitrogen. Thus, either axial or equatorial phenyl

meperidine can be matched to morphine without excessive energetic

cost. This result should be combined with the observation that,

in the equatorial case, the protron attached to the quaternary

nitrogen is no longer oriented in the same position as the corresponding proton in morphine after matching (while the corresponding

proton orientation in the axial case is the same). Thus, it appears

that the axial form of meperidine is favored, at least when

comparison to morphine is made, even though the crystal geometry

and lowest energy form is the equatorial phenyl form.

Now let us consider geometric features of antagonists, using

nalorphine as an example. One of the geometric features of particular

interest relates to the position of the allyl group, or of similar

moieties (e.g., the cyclopropyl group in cyclazocine). In the crystal,

it is found that the allyl group conformation in the current study

of nalorphine is different than that found in naloxone(Karle 1974;

Sime,Forehand,Sime 1975), but similar to the conformation found for

the cyclopmpyl moiety in cyclazocine(Karle, Gilardi, Fratini et al

1969).

To examine the extent to which the allyl (or cyclopropyl) group

orientation is variable, a scan of the two relevant torsion angles

for nalorphine are presented in Figure 3, and the results for

(τ1=C16-N-C17-C18 and τ2=N-C17-C18-C19) was carried out. The results

cyclazocine are presented in Figure 4. These particular calculations

225
were carried out using rigid rotations, varying only the two dihedral

angles The minima that are found are described in Table 1.

It is seen that the conformational energy surfaces for the two

molecules look quite similar, with four minima less than 1.0 kcal/

mole above the global minimum found for each molecule for approximately

the same ( τ1 τ2) values. In addition, interconversion among the four

low-lying minima in each molecule should be relatively easily

accomplished, since a barrier of only  ̃3 kcal/mole is encountered

between adjacent minima. The only difference between these two

molecules from the point of view of (τ 1,τ 2) flexibility is the presence

of 2 additional local minima at 3-5kcal/mole above the global

minimum, while cyclazocine has only 1 such additional minimum.

Hence, and nalorphine and cyclazocine look quite similar, at least with

respect to the possible orientations of the allyl or cyclopropyl

groups.

In addition, nalorphine and naloxone were matched using the technique

described earlier. Using the global minimum found for (τ 1 τ2) in

nalorphine as a starting point, a good match of the two molecules

can be obtained. In particular, the aromatic rings and quaternary

nitrogen can be placed within 0.2Å and the allyl groups within 0.6Å,

at an energetic cost of only 1.8 kcal/mole for nalorphine and 1.2

kcal/mole for naloxone. Similar results can be expected for the 3

other energetically favorable conformations. Hence, it is seen that

nalorphine and naloxone can assume quite similar geometries (e.g.,

for a drug-receptor interaction), even though their crystal structures

are different.

B. Electronic Structural Studies

We have now seen that, for several of the important classes of

analgesic agonists and antagonists, quite similar geometric environments for atoms believed to be important to the drug-receptor interaction can be attained at only modest energetic cost. It is thus of

interest to see whether electronic structural features can be found

which will distinguish e.g., agonists from antagonists, even though

geometric features may be similar.

To examine this possibility, ab initio SCF calculations using the

molecular fragment basis(Christoffersen 1972) have been carried out

on each of the 16 molecules illustrated in Figure 1. The geometries

chosen reflect several considerations. For morphine, meperidine, and

fentanyl, the geometries used were those found in the matching process

described in the previous section. For nalorphine, naloxone, and cyclazocine, methadone,and propoxyphene, the x-ray structure was used, while

in oxymorphone and naltrexone the basic geometric data from the naloxone

crystal structure was used with appropriate substitutions at the

quaternary nitrogen. Similarly, for metazocine, N-allyl normetazocine

and pentazocine, the cyclazocine crystal strtucture was used with

appropriate modifications at the quaternary nitrogen. Levorphanol

and levallorphan were built from the cyclazocine structure, using

molecular mechanics to orient the added substituents.

226
The results of these calculations are reported in part in Table 2,

where the molecular orbital (MO) energies for the five highest energy

occupied MOs and five lowest energy unoccupied MOs are given. In

addition, to indicate the shape and location of MOs of interest,

electron density maps for several MOs that are representative are

given in Figures 5-10. In each calculation, the protonated form of

the molecule was used.

IV. DISCUSSION

With the reasonably extensive geometric and electronic structural

data described above, obtained in a consistent fashion using the

same techniques, it is of interest to examine it further from at

least two different perspectives. First, since these techniques are,

relatively speaking, in their infancy, it is of interest from a

methodology point of view to compare the results obtained here with

other theoretical studies. Next, it is of interest to examine

whether the electronic and geometric structural features that are

identified as being of importance in these studies can be related

to the observed pharmacolcgical differences in the molecules, and add

to the understanding of these pharmacological differences based

upon the molecular properties of the drugs.

Turning first to geometric features, the minima found here for the

N-C18 and C18-C19 dihedral angle variations in nalorphine are consistent with those found by Loew and Berkowitz(Loew,Jester 1975) and

by Kaufman and Kerman(Kaufman,Kerman 1977) using the PCILO technique,

including both the location of the minima and the approximate energetic barrier separating them. This similarity of results using different methodologies is of interest, both from the point of view of

the adequacy of the techniques to describe these geometric features in

analgesics, and in the likelihood that the results represent the

actural physical situation (for the isolated molecule).

In the case of meperidine, the finding that the lowest energy form

is the equatorial phenyl form is consistent with PCILO studies(Loew,

Jester 1975;Beckett,Casey 1965). However, in contrast to earlier

results in which only two dihedral angles were varied(Loew,Jester 1975;

Loew,Jester,Berkowitz et al. 1975), the ability to relax all bond distances, bond angles and dihedral angles using the molecular mechanics

technique indicates that a good fit to the morphine structure can be

achieved at reasonable energetic cost, for both the axial and

equatorial forms of meperidine. This is consistent with the observation that significant potencies are observed for both phenylequatorial and phenyl-axial compounds(Portoghe,Mikhail 1968) and indicates that additional investigation is needed before identification

of only one form (whether axial or equatorial) as being solely responsible for meperidine activity is appropriate.

For the case of fentanyl, the results obtained here are consistent

with the features proposed by Feinberg(Feinberg,Creese,Snyder 1976)

to be important factors in determining its potency. In particular,

one of the reasons why the "F" ring plays such an important role in

fentanyl and similar molecules is that it can be closely matched with

the "A" ring of morphine without excessive energetic requirements.

However, it would then appear that the "A" ring region (and not the

227
"F" ring) of fentanyl and similar molecules is the "special feature"

that confers unusually high potency in the model proposed by Feinberg

et. al(Feinberg,Cresse,Snyder 1976).

While additional geometric features may play important roles in determining the potency and/or efficacy of these molecules(Loew,

Berkowritz,Newth 1976;Loew,Berkowitz 1978), it is clear from the few

examples presented here in abbreviated form that, even though the

molecules included here represent a rather broad range of chemical

structures of analygesics, they can be matched to morphine with only

modest energetic requirements. Hence, further examination of

electronic structural features may be appropriate in order to discern

reasons for potency differences.

First, comparison of the HOMO in morphine and naloxone or nalorphine

(Figs. 5 and 6) indicates that the general shape of the MO is nearly

identical in the two molecules, even though one is agonist and the

others are partial or pure antagonists. In addition, the MO energies are

nearly the same, as has also been noted earlier(Kaufman ,Kerman,Koski

1974;Popkie,Koskie,Kaufman 1976). This suggests that, if the analgesic receptor does indeed exist in two allosterically related conformations(Feinberg,Creese,Snyder 1976), the binding site for the

phenol moiety of either agonists and antagonists is unaffected by the

receptor being in either "agonist" or "antagonist" form, if the receptor acts as an electron acceptor.

On the other hand, comparison of the LUMO shape in morphine and

naloxone reveals important differences. In particular, the LUMO in

morphine is located in the phenyl region of ring "A", but in

naloxone the LUMO is associated with the allyl group attached to

the quaternary nitrogen. This is an important distinction, for it

allows a direct rationalization of the introduction of antagonist

behavior when N-CH3 in morphine is replaced by N-allyl to form

nalorphine. Specifically, if the receptor serves as au electron

donor in the drug-receptor complex, then introduction of the allyl

group produces a low-lying unfilled MO in the region of the quaternary

nitrogen that is well suited to the proposed drug-receptor interaction. In other words, one of the features that appears to be

important for introduction of antagonist behavior is the presence

of a low-lying unfilled MO in the region of the quaternary nitrogen.

Not only the allyl moiety can introduce "hole" density in the region

of the quaternary nitrogen to introduce antagonist characteristics.

For example, nlrexone and cyclazocine have a methyl cyclopropyl

moiety in place of the allyl moiety. In this case, the "hole"

density is still placed in the proper region of space, as indicated

in Figure 11. In this figure it is seen that the general shape of

the MO is quite similar to that of the corresponding allyl unoccupied MO, in spite of the chemical differences, and the introduction of antagonist characteristics to naltrexone and cyclazocine

is not surprising.

These observations immediately allow a qualitative rationalization of the behavior of many of the molecules in Figure 1, including morphine vs nalorphine, oxymorphone vs naloxone (or

223
naltrexone), levorphanol vs levallorphan, and metazocine vs Nallylnormetazocine (or pentazocine or cyclazocine).

It is of interest to note that the above observations are in contrast

to conclusions reached in earlier studies involving morphine and

nalorphine(Loew,Berkowitz 1975), in which it was concluded that "the

electronic properties of the fused ring skeleton including specifically the cationic region around the nitrogen are relatively unaffected

by varying N-substitueuts." The primary reason that the effect was not

seen in earlier studies is associated with the lack of ability of

INDO to describe the natrue of unoccupied MOs properly. This is not

surprising, since the parameterization of INDO was done to provide a

balanced description of the filled MOs only.

Also, the current suggestion that nalorphine and similar antagonists

act as electron acceptors (not donors) is in contrast to the suggestion

that the receptor "interacts with the π-electrons of the N-allyl"

group(Feinberg,Creese,Snyder 1976). Instead, the receptor electrons

are proposed to interact with the N-allyl "hole density", in the form

of the LUMO in nalorphine.

Finally, it should be remembered that the observation that the

location and energy of the LUMO may be an important factor in

determining antagonist behavior does not imply that it is the sole

factor of importance. Other electronic and geometric features may

also need to be taken into account, and the overall activity of a

molecule at the analgesic receptor will be determined by the detailed

balance of the various effects.

Clearly, much additional analysis and quantitative study is needed.

However, it is already clear that both geometric and electronic

structural features of the drugs play an important role in determining analgesic potency, and that currently available theoretical

techniques axe capable of probing these features in detail.

ACKNOWLEDGMENTS

The authors would like to express their appreciation to the University

of Kansas for partial support of the computing time required for

this work. In addition, many helpful discussions with E. C. Olson,

F. Kagan, R. Lahti and others at the Upjohn Co. are gratefully

acknowledged.

229
Table 1. Energetic Minima Found in Allyl (or Cyclopropyl) Rotations

a. Energies measured relative to the global minima for the molecule.
Table 2. Compilation of Selected Molecular Orbital Energies of Analgesicsa
Figure 1. Molecules Studied.

232
Figure 2. Study of Morphine Flexibility About the C12-C13 Bond.

A=morphine hydrochloride; B=free base morphine;

C=nalorphine hydrochloride; D=o-methyl etorphine.
Figure 3. Isoenergy contours for nalorphine. Contours are are drawn for 1.0, 2.0, 5.0 and

10.0 kcal/mole above the global minimum at (180, 120) - labelled "A" in the figure.
Figure 4. Isoenergy contours for cyclazocine. Contours are drawn for 1.0, 2.0, 5.0, and

10.0 kcal/mole above the global minimum at (175, 165) - labelled "A" in the figure.Text
Figure 5. Electron Density Contour Maps for the HOMO-1, HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Morphine.
Figure 6. Electron Density Controur Maps for the HOMO-1,HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Naloxone.
Figure 7. Electron Density Contour Maps for the HOMO-1, HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Levorphanol.
Figure 8. Electron Density Contour Maps for the HOMO-1, HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Pentazocine.
Figure 9. Electron Density Contour Maps for the HOMO-1, HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Fentanyl.
Figure 10. Electron Density Contour Maps for the HOMO-1, HOMO,

LUMO, and LUMO+1 Molecular Orbitals in Meperidine.
Figure 11. Density Contour Plot for MO#77 in Cyclazocine. The third

carbon atom of the cyclopropyl moiety is connected to

C18 and C19, and in a plane perpendicular to the plane

of the paper.
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Molec Pharmacol, 11:492-500, 1975;

Umeyama, H. Molecular-orbital study on solvolysis of asperin

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AUTHORS

B. Vernon Cheney, Ph.D.

D. J. Duchamp, Ph.D.

The Upjohn Company

Kalamazoo, Michigan 49001

R. E. Christoffersen, Ph.D.

The University of Kansas

Chemistry Deparment

Lawrence, Kansas 66045

249
‘QuaSAR’ Research Monograph 22

G. Gannett, M. Trsic, and R. Willette, eds

National Institute on Drug Abuse, 1978

Recent Physicochemical and

Quantum Chemical Studies on

Drugs of Abuse and Relevant

Biomolecules

Joyce J. Kaufman

I. INTRODUCTION

The objective of quantitative structure activity relation (QSAR)

studies is to establish a reliable quantitative relationship

between the structure of drug molecules and their pharmacological

activity. It is necessary in these considerations to distinguish

between the extremes of intrinsic activity (the activity of a

drug at its locus of action) (Ariens and Simonis 1964) as opposed

to whole animal or man in vivo potency which is influenced by

transport, metabolism and intrinsic activity. One of the most

striking examples of such a distinction and one which is illustrative of general phenomena in the field of central nervous

system(CNS) drugs is exemplified in the pioneering work of Herz

and coworkers (Kutter et al. 1970). In these studies they compared the analgesic potency in a series of narcotics by intravenous and by direct intraventricular injection. The intrinsic

activity of the narcotics as exemplified by direct intraventricular injection appeared to be primarily governed by the structure

and polarity of the narcotic molecule. The whole animal potency

of narcotics observed from intravenous injection was greatly

influenced by the lipophilicity as well as being dependent on

structure and polarity. [The in vitro inhibition by a narcotic of

the induced release of acetylcholine from the isolated guinea pig

ileum (Kosterlitz, Lord and Watt 1973) more closely reflects intrinsic activity while binding studies on the endogenous opiate

receptor reflect affinity (Pert and Snyder 1973).]

Thus quantitative structure activity (potency) relations are

governed by a combination of several factors:

i) Physicochemical properties of the drug, primarily its

lipophilicity

ii) Topological, topographical and substructure analysis

(and when appropriate. those of the relevant biomolecules)

iii) Electronic structure of the drug (and when appropriate

those of the relevant biomolecules)

250
iv) Systems analyses and control theory of how drugs interveneinto the pathways of normal biomolecules and the

various influences these drugs exert on the normal biomolecules, on their synthesizing and metabolizing

enzymes and processes, on their release from and reuptake back into the presynapse and their effect on the

postsynaptic receptors.

In our group we have been calculating or measuring the various

factors that contribute to QSAR. Our studies encompass the

gamut (Kaufman and Koski 1975a 1975b; Kaufman 1977a) from ab

initio quantum chemical calculations on large drug molecules

(Popkie and Kaufman 1976b, Popkie, Kaufman and Koski 1976) and

improved techniques for such calculations for large drug and

biomolecules (Popkie and Kaufman 1975, 1976a , 1977a, 1977b, 1977c;

Kaufman 1977b) to theoretical approaches such as systems analyses,

control theory and catastrophe theory (Kaufman and Koski 1975a,

1975b; Kaufman, Koski and Peat 1975), physicochemical studies

(Kaufman, Semo and Koski 1975) to experimental pharmacology,

teratology studies and interactions with physicians in the clinic

(Kaufman et al. 1975; Benson, Kaufman and Koski 1976; Kaufman

et al. 1975/76; Kaufman, Koski and Benson 1977).

In this presentation we consider QSAR aspects of drug action by

moans of experimentally measured pKa's, oil-water partition and

drug distribution coefficients as a function of pH for some hallucinogenic drugs of abuse, some additional narcotics as well as

some relevant endogenous biomolecules. Examples will be given of

ab initio quality wave functions for large drug and biomolecules

calculated by incorporating new desirable techniques. In addition

quantum chemical calculations have been carried out in an endeavor

to establish the utility of electrostatic molecular potential

contour maps in the study of drug action.

II. PHYSICOCHEMICAL PROPERTIES

The physicochemical properties such as pKa's, partition coefficients, distribution coefficients, and pH dependences were determined using the same procedures previously outlined in detail

(Kaufman,Semo and Koski 1975) so these will not be presented

here. The results for some hallucinogenic drugs of abuse, sane

barbiturates as well as for some relevant endogenous biomolecules

are included in Table I and the results for etorphine are contained

in Table II. Aknowledge of these parameters is of interest in

QSAR studies since, in general, such investigations follow the

approach of Hansch and coworkers (Hansch et al. 1973) which involves the use of linear free energy (LFER) relations

where c is the concentration in the biophase and P is the

partition coefficient,

251
However, a number of years ago we realized that, while the partition coefficient P (which we call Do/w)is an appropriate index for

rationalizing the pharmacology of a neutral molecule, the partition coeffiecent as defined by convention is not in general the

appropriate index for rationalizing the pharmacology of drugs

which can be protonated or deprotonated. The appropriate lipophilicity index for drugs which can be protonated or deprotonated

is the apparent partition coefficient Papp,(which we call PpH in

our tables to indicate its pH dependence) (which reflects the drug

distribution coefficient).

For a base:

We thus set up a microelectrometric titration method and a related

mathematical. deconvolution procecure (Kaufman, Semo, and Koski

1975) which permit

i) Deconvolution of overlapping pKa's not possible to resolve experimentally alone;

ii) Determination of both the partition coefficient and the

apparent partition coefficient from the shift in the

measured pKa's obtained by running the titration in a

mixed lipid-aqueous solution.

Often when there are two pKa's in a molecule, one deprotonating

an amine acid salt from a charged to a neutral species and the

other deprotonating a phenolic OH from a neutral to a charged

anion, it has been difficult to decide which measured pKa corresponds to which process. By the method we use this ambiguity never

arises. The shift in the measured pka (figure 1) in mixed oilwater is always toward lower pH in going from a protonated

amine(a charged species) to the more lipophilic free base (a

neutral species) and toward higher pH going from the more lipophilic neutral species (such as phenol or acid) to an anion

(a charged species).

252
FIGURE 1

Comparison of the aqueous titration and 10% oil-water titration of nalorphine, HCl.

Dashed line is the aqueous titration, solid line is the octanol-water titration.

The crosses mark the pKa values and the pKa's represent the shifts.

©1975, American Chemical Society3
Our very first experimental results on the narcotics and narcotic

antagotists showed several general phenomena crucial to their QSAR

and potencies.

i) Strong pH dependencies within the range of attainable

human physiologic pH's;

ii) Marked temperature dependencies which were widely

differing percentages (from 21 percent to 300 percent)

even for related compounds;

iii) Lipophilicities (apprrent partition coefficients) which

could differ by several hundred percent for such closely

related congeners that the molecules differed by only

one C and one H atom in a total of about 50 atoms.

We have established these results on narcotics and narcotic

antagonists to clinical practice and clinical observations

confirmed our findings.

We have extended such determinations of the pH dependence of

lipophilicities to a variety of drugs and biomolecules and to

compounds potentially teratogenic (Kaufman, Koski and Benson 1977).

We report here results on some CNS agents and biomolecules which

have relevance to the area of drug abuse:

A. Dissociative anesthetics: phenyclidine, ketamine and

some ketamine metabolites

B. Barbiturates

C. Normal neurotransmitters

D. Narcotics

Some comments on the results and their significance are in order

at this point

The dissociative anesthetics cause hallucinations in adults and

thus are not customarily employed in adult anesthesia although

they can be used clinically for children. These dissociative

anesthetics are among the latest and perhaps most violent drugs

of abuse. From our results (Table I) it will be noted that the

lipophilicities of the dissociative anesthetics have a similar pH

dependence as do the narcotics and narcotic antagonists previously

reported. Since the pKa listed corresponds to deprotonation of an

acid salt of an amine, a rise in pH leads to more neutral free base

and thus to a higher drug distribution coefficient which in turn

influences those pharmacologic actions that are dependent on the

drug concentration in the lipid. On the other hand, the metabolites

listed in Table I exhibit a mild dependence on pH compared to ketamine. They are considerably less lipophilic than ketamine and thus

will be excreted more rapidly.

In the case of barbiturates the pKa which is relevant to the pharmacological effects of the drugs is that of a deprotonation of the

254
TABLE 1

pK'a, Partition Coefficient and Drug Distribution Coefficient as a Function of pH (Temp = 37°C)

Drug Distribution Coefficient
Table I (continued0

pK'a, Partition Coefficient and Drug Distribution Coefficient as a Function of pH (Temp = 37°C)

Drug Distribution Coefficient
neutral phenolic compound to form the anion. Consequently.

as the pH goes up into the range of physiologically attainable pH

the drug distribution coefficient is expected to decrease. This

is supported by the data in Table I for pentobarbital, hexabarbital and thiopental.

Table I also includes data for the neurotransmitters dopamine and

norepinephrine. It will be noted that there are 2 pKa's for each

molecule (dopamine: 8.706 and 10.25; norepinephrine: 8.423 and

9.363). The data indicates that the physiologically significant

pKa refers to deprotonation of the phenolic group since the lipophilicity rises with decreasing pH.

In our earlier research (Kaufman, Semo and Koski 1975) we measured

the pH and temperature dependence of the lipophilicities of a

number of narcotics and narcotic antagonists. The lipophilicities

of narcotics and narcotic antagonists were found to be extremely

sensitive to pH within the range of attainable human physiologic

pH. For an acidotic patient, only about 60 percent as much of a

narcotic or narcotic antagonist would penetrate the blood brain

barrier as it would at a physiologically normal pH. On the other

hand. in a patient whose pH is alkaline about 60 percent more of

a narcotic or narcotic antagonist would penetrate the blood brain

barrier than at normal pH. This has significant implications in

the clinical usage of such drugs as well asinthe drug abuse area

(Benson, Kaufman and Koski 1976; Kaufman, et al. 1975/76;

Kaufman, Koski and Benson 1977).

Etorphine is about 500 times more potent than morphine when given

intravenously but only about 34 times more "potent" when given

intraventricularly (Kutter et al. 1970). The high intravenous

potency is due in large part to the lipophilicity of etorphine

(Table II) which is of the order of 1000 times greater than that

of morphine under physiological conditions. Etorphine would be

expected to have a rapid onset of action due to its high lipophilicity and preliminary clinical trials confirm this (Jasinski,

Griffith and Carr; Lewis1). The high intravenous potency of

etorphine makes a knowledge of the pH dependence of the lipophilicity an important property, especially in avoiding accidental overdose. The lipophilicity of etorphine is 46% greater at pH 7.7

than at the physiologic norm of 7.4. Consequently, care must be

taken in the clinical use of etorphine especially during surgery

when this narcotic might be used concomitantly with a general

anesthetic under which the patient might hyperventilate and have

a rise in blood pH to 7.7.

Since there was interest from the veterinarians in the lipophilicity

of etorphine at the higher body temperatures of the larger animals

(20°, 37°, 38°, 40°C) as well as of pH.

we also carried out the determinations as a function of temperature

257
TABLE II

pK'a, Partition Coefficient and Drug Distribution Coefficient as a Etorphine as a Function

pH and Temperaturea
III. ELECTRONIC STRUCTURE

A. LCAO-MO-SCF,MC-SCF and CI

We have long had an interest in quantum chemical calculations of

the electronic structure of large drugs and biomolecules (Giordano

et al. 1967; Giordano et al. 1968; Kaufman and Kerman 1972; Kaufman 1973a, 1973b; Preston and Kaufman 1973; Preston et al. 1973;

Kaufman and Kerman 1974a, 1974b, 1974c; Kaufman, Koski and Kerman

1974; Kaufman and Koski 1975a, 1975b; Saethre et al. 1975; Kaufman

and Kerman 1976; Kaufman and Kerman 1977; Popkie and Kaufman 1976b;

Popkie, Koski and Kaufman 1976;. Petrongolo, Preston and Kaufman,

in press 1977; Petrongolo et al. submitted 1978). In our first paper

dealing with pharmacological research in 1964 we performed extended

Huckel calculations on the pyridine aldoxime antagonists to the

organophosphorus intoxicants to determine both their conformational

energy profiles and their charge distributions (Giordano et al.

1967; Giordano et al. 1968). In our earlier studies on the neuroleptics and the narcotics (Kaufman and Kerman 1972; Kaufman 1973a ,

1973b; Kaufman and Kerman 1974a, 1974b, 1974c; Saethre et al.

1975; Kaufman and Kerman 1976) we used the CNDO and INDO methods

(Pople and Beveridge 1970). Probably the most reliable use of such

CNDO or INDO calculations seems to be in comparison of calculated

charges on the same atom in closely related molecular environments

(Saethre et al. 1975). We have used the semirigorous PCILO method

(Diner et al. 1969) (perturbative configuration interaction based

on localized orbitals) for conformational analysis (Kaufman and

Kerman 1977). We also carried out completely ab initio calculations for narcotics (Popkie, Koski and Kaufman 1976) (morphine

and nalorphine) and for neuroleptics (Popkie and Kaufman 1976b )

(chlorpromazine and promazine).

However, more recently our major attention has been devoted to the

development and implementation of procedures for calculating

nonempirical ab initio quality wave functions for the valence

electrons of a molecule (Popkie and Kaufman 1975, 1976a , 1977a,

1977b, 1977c, Kaufman 1977b, 1978a, 1978b) by incorporating into

our own new fast ab initio computer programs (Popkie, 1974; 1975;

1976) two desirable options: ab initio effective core model

potentials (MODPOT) (Bonifacic and Huzinaga 1974) for the inner

shell electrons which allow one to treat only the valence electrons

explicitly and a nonempirical charge conserving integral prescreening approximation [which we have named VRDO - variable

retention of diatomic differential overlap (Popkie and Kaufman

1975)] especially effective for spatially extended molecules.

The charge conserving integral prescreening approximation cuts

down significantly on the number of two electron integrals which

have to be calculated, stored or processed in the calculation of

a large molecule. If too many of the millions of two electron

integrals are thrown away by size alone, then the SCF calculation

will not converge. This is because there is a delicate balance

between one and two electron integrals and while there are

relatively few one electron integrals, many of them are large.

Thus an effective procedure to discard integrals must still

259
retain the delicate balance between one- and two-electron

intergrals. Our nonempirical VRDDO approximation (Popkie and

Kaufman 1975) (variable retention of diatomic differential

overlap) was inspired in part by a suggestion from solid state

physics by Wilhite and Euwema (Wilhite and Euwema 1974; Euwema

and Green 1975). They had proposed several approximations. These

the point where they might become competitive with less rigorous

speed up considerably minimum basis set SCF calculations even to

techniques. These approximations reduce the number of integrals

that have to be determined in an ab initio LCAO-MO-SCF calculation on a large molecule. Their first approximation consists of

neglecting all one- and two-electron integrals that involve basis

function products φi(1)φj(1) whose pseudo-overlap

is less than some tolerance τ1 The pseudio-function φi*(1) is

represented by a single normalized spherically averaged Gaussian

function whose exponent is equal to that of the most diffuse primitive in the contracted Gaussian function φi(1). A second threshhold τ2 controls the accuracy of the two-electron repulsion

integrals (integrals are accurate to n decimal places); those

integrals whose absolute magnitude is less than τ2 are neglected.

Alternatively, if the pseudo overlap of any pair of orbitals in

the two electron integrals is less than τ3 the integrals are

neglected. The results (Popkie and Kaufman 1975,1976a, 1977a,

1977b;1977c; Kaufman 1977b, 1978a, 1978b)show both these approximations to be numerically accurate. The computer CPU time

required to generate the two-electron integral list for a large

molecule is substantially reduced as τ2 τ3 are increased. The

introduction of the VRDDO approximation with τ 1 = 10-2 and τ 2 = 10-4

a.u. (1 a.u. = 27.21 ev) hardly affects at all the accuracy2 of

the computed properties. The valence orbital energies and gross

atomic populations are essentially accurate to 3 decimal places

(in a.u.). The error in the total energy increases slightly on

going from the smaller to the larger molecules. The maximum error

found in the total overlap populations is 0.008 and in the dipole

moment is 0.009 a.u.. In the alternative procedure the same

accuracy is found if τ1 = 10-2 and τ3 = 10-2.

Recently Bonifacic and Huzinaga have given a new formulation of

the model potential (MODP0T) method of the core projection type

for carrying out LCAO-MO-SCF calculation considering only the

valence electrons (Bonifacic and Huzinaga 1974, 1975a , 1975b,

1976, 1977; McWilliams and Huzinaga, 1975). The effect of the

core electrons is taken into account through the use of a

modified model potential (MODPOT) Hamiltotian:

260
In equations (1) and (2), nv is the number of valence electrons, nh

is the number of hydrogen atoms and nc is the number of atoms with

core electrons. is the number of core electrons in the k-th core.

are inner shell atomic orbitals for the k-th

core. The parameters A1k, A2k, 

α

1k, 

α

2k and B

k

 have been tabulated

by Bonifacic and Huzinaga (Bonafacic and Huzinaga 1975a ) for the atoms

Li to Ne (for the particular atomic basis sets they chose). The A1k,

A2k,

α

1k and α2k parameters are virtually independent of basis set.

The Bk parameters must be optimized from atomic calculations for each

different choice of Atomic basis set.

We have implemented the coding for the ab initio effective core

model potential for inner s, p (Popkie 1974; 1975) and d (Popkie

1975) orbitals with s, p and d orbitals. We extended the derivation to inner f orbitals in our f orbital package (Popkie 1976).

261
We verified the accuracy of the MODPOT method for a variety of

molecules by carrying out the completely ab initio reference

calculations with two-electron integrals accurate to 6 decimal

places. Then the MODPOT calculations were carried out with

valence shell two-electron integrals accurate to 6 decimal places

and the MODPOT modified one-electron integrals. The same reference

valence atomic basis set was used for both calculations. Comparison

of the MODPOT results to the completely ab initio ones (including

inner shell electrons) showed the valence orbital energies

and gross atomic populations are accurate to better than 2 decimal

places (the average error is 0.005 a.u.). The maximum error in the

orbital energies for the largest molecule studied to date is only

0.011 a.u. and in the gross atomic populations is only 0.010 a.u..

It should be emphasized that the accuracy obtained with the MODPOT

approximation is more than acceptable if one wants to reproduce ab

initio results for large molecules. Both these options (MODPOT

and VRDO) cut down significantly on the time necessary for quantum

chemical calculations on large molecules yet retain the chemical

accuracy.

Since both the MODPOT and VRDDO techniques are independent, they

can be used together. The combined MODP0T/VRDDO method is just as

accurate compared to the completely ab initio calculations as is

the MODPOT method itself. The relative accuracy of the MODPOT/

VRDDO method along a potential energy surface, such as for molecular conformational changes or interactions between two species,

(0.0001 - 0.0002 a.u.) is even greater than its absolute accuracy.

The results of our testing of the MODPOT, VRDDO and MODPOT/VRDDO

methods on a variety of molecules including heteroaromatics and

substituted aromatics indicated that these methods give excellent

agreement with the completely ab initio results using the same

valence atomic basis set. Part of the excellent agreement

arises from our careful prior optimization of the atomic model

potential parameters in both the isolated atom and in the small

prototype molecules containing these atoms.

Moreover we have previously devoted considerable attention to

optimization of small completely ab initio atomic basis sets to

match as closely as possible the results of larger completely

ab initio atomic basis sets and to examining the influence of

basis set size and composition on calculated orbital energies and

charge distributions.

We have been carrying out nonempirical MODPOT/VRDDO calculations on

a variety of drugs, endogenous biomolecules, and other compounds

suspected of being teratogens and carcinogens. As an example, the

timings for some prototype endogenous biomolecules (Kaufman 1977b;

1978a, 1978b) using a carefully optimized MODPOT atomic basis

set corresponding to a 6-3G full basis set, indicate the speed

of this method.

262
MOLECULE NO. ATOMS NO. BASIS FNS. TIME (Minutes)

C,N,O, H Integrals SCF

Neurotransmitters

Norepinephrine 12 11 5 9 16.9 8.4

6-Hydroxydopamine 12 11 59 17.2 8.3

Histamine 8 9 41 6.8 4.1

Serotonin (5-HT) 13 1 3 6 5 22.9 10.9

CDC 6600 computer, FIN, OPT = 0. (The integrals run even 50

percent faster under OPT = 2 on a CDC 6600 computer which

supports a full OPT = 2 compiler).

CDC 6000 computer, FIN, OPT = 2.

More recently we have been carrying out MODPOT/VRDDO calculations

on even larger molecules (teratogens and carcinogens and their

metabolites) and the increase in computer time with the number of

basis functions (n) appears to rise by much less than by the

theoretical n4 for the completely ab initio calculations retaining

all integrals. The timings by the MODPOT/VRDDO method (as well as

by completely ab initio calculations) are influenced also by the

size and shape of the molecules. In particular, there is very

little increase in integral or SCF time for the almost planar

polyclic aromatic hydrocarbon methyl cholathrene (100 basis

functions) over that for the closely related benzopyrene (92 basis

functions).

While many of the phenomena of interest for large drug and biomolecules involve properties related to the ground states of these

molecules, there are certain biological and chemicophysical phenomena which involve excited states. Thus several potential energy

surfaces of different spin or symmetry may be involved. To calculate appropriately molecular interactions one needs configuration

interaction (CI) (Shavitt 1976) or multiconfiguration SCF (MC-SCF)

(Hinze 1973) potential energy surfaces. To interpret measured UV

and visible spectra it is necessary to calculate excitation energies, which can only be carried out properly by CI or MC-SCF calculations.

Thus we meshed the MODPOT, VRDDO and MODPOT/VRDDO methods into the

MC-SCF and CI programs (Shavitt and Pipano). We tested the combined MODPOT/CI program and the results are accurate to 0.005 ev

compared to a completely ab initio calculation including the inner

shells and the MODPOT/CI results are accurate to 0.01 ev compared

to the experimental values. The most time and space consuming

part of a CI calculation is the transformation of integrals from

integrals over atomic basis orbitals to integrals over molecular

263
orbitals. A procedure has been implemented which allows folding

into a new effective CI Hamiltonian the influence of the molecular

orbitals from which no excitations are allowed (Raffenetti 1973-

74; Raffenetti 1974-76). This permits us to transform only over

molecular orbitals from and to which excitations are allowed.

Thus, with the combination of MODPOT, VRDDO and MODPOT/VRDDO SCF,

MC-SCF and CI procedures, rapid and reliable calculations on large

drug and biomolecules are more than feasible at present.

B. Electrostatic Molecular Potential Contour Maps

In this phase of the study we present the calculations of electrostatic molecular potential contour maps and illustrate their application to drug action.

The electrostatic potential V(r), arising from molecule A is

completely defined at every point of the space if one knows the

charge distribution (electronic and nuclear) of the molecule

(Scrocco and Tomasi. 1973).

In general it is preferable to use ab initio [or now ab initio

effective core model Potential (Kaufman 1978a, 1978b; Pullman 1977,

Weinstein and Osman 1977; Osman and Weinstein 1977)] wave functions

employing a well balanced basis set from which to calculate electrostatic molecular potential contour maps. Frequently it is desirable

if the molecules are quite large to use small atomic basis sets and

the question arises whether maps generated from such basis sets

using completely ab initio wave functions match the results of

those using larger atomic basis sets. We have calculated the

electrostatic potential map for pyrrole (Petrongolo, Preston and

Kaufman, in press 1977) using both the results of our previous

large basis set ab initio calculations (Preston and Kaufman 1973)

and those using a minimal basis set which had been carefully optimized to reproduce closely the properties calculated from the wave

functions with a large basis set. The results are illustrated in

figure 2 and they show that the large basis set (LBS) and the

matching minimal basis set (MBS) wave functions generate maps

virtually identical in shape and in numerical values. It will be

noted, on the other hand, the electrostatic molecular potential

contour maps generated from deorthgonalized CNDO wave functions as

suggested by Pullman (Giessner-Prettre and Pullman 1968) do not

appear similar in shape let alone in numerical values (figure 2)

confirming the results of others (Giessner-Prettre and Pullman

1972; Petrongolo and Tomasi 1975).

It would appear reasonable that one of the theoretical requisites

for a drug that acts as a direct agonist to a normal neurotransmitter is that the electrostatic molecular Potential contour map

which is generated around the pharmacophorically significant parts

of the drug molecule must resemble very closely in shape and

numerical magnitude the contour that is generated around the

corresponding pharmacophorically significant parts of the normal

264
FIGURE 2

Pyrrole - molecular plane

Electrostatic molecular

potential contour map

LBS - large basis set

MBS - minimal basis set

CNDO - CNDO deorthogonalized

© 1978, J.J. Kaufman
neurotransmitter itself. In these applications one must be careful to distinguish between direct and indirect agonists. In the

former case the direct agonist binds to the postsynaptic receptor

site and so perfectly mimics the true endogenous normal neurotransmitter that the receptor cannot distinguish the agonist from

the neurotransmitter. In the case of the indirect agonist the

concentration of the normal neurotransmitter is raised by an

indirect means at the postsynaptic receptor either by decreasing

the metabolism, increasing its synthesis, increasing its release

from the presynapse or interferring with its reuptake back into

the presynapse. It is clear that in the latter case potential

contour maps would not be applicable.

An interesting example of this concept is shown in figure 3

where we have generated (Petrongolo, et al. Submitted 1978) the

electrostatic molecular potential contour map around the pharmacophorically significant N atoms of morphine (an N-methyl narcotic

agonist) and nalorphine (an N-allyl narcotic agonist-antagonist)

from our ab initio calculations on morphine and nalorphine (Popkie,

Koski and Kaufman 1976). Using the criterion defined above, namely

that two related drugs which extert a common pharmacological effect

must generate similar or identical maps around their relevant

pharmacologically significant pharmacophores, it is clear in

figure 3a that the molecular potential contour maps generated

around the nitrogen in the lone pair direction are nearly identical

in shape as well as in numerical value. This suggests that it is

in this direction from the N atom that both morphine and nalorphine

exert their common narcotic agonist effect. On the other hand,

maps generated around the substituent groups on the N atoms in

morphine and nalorphine from a different perspective (figure 3b)

are completely different. In particular there is a region around

the isolated double bond in the allyl group of nalorphine which is

attractive to an electrophilic reagent in contrast to the completely repulsive region around the methyl group in morphine. This

corresponds also to a low lying unoccupied C = C antibonding

orbital in all of the narcotic antagonists. This low lying unoccupied orbital contributes to the paramagnetic polarizability

type term which causes the 15N NMR shift of nalorphine to be

different than that of morphine (Kaufman 1977a ).

Moreover, to investigate in a thorough and comprehensive manner

the agreement or disagreement of electrostatic molecular potential

maps generated from CNDO or INDO wave functions (either orthogonalized or deorthogondlized), to determine the influence of the

various approximations when CNDO or INDO wave functions are used

and to assess the influence of the GTO expansion to STO'S, in

collaboration with Petrongolo (Petrongolo et al. Submitted for

publication) we generated the maps using both approximations III

and IV(Giessner-Prettre and Pullman 1972) and STO-3G and STO-6G

expansions. Striking changes are obtained deorthogonalizing both

the CNDO and INDO eigenvectors. Different expansions, STO-3G or

STO-6G, give the same quantitative results. The significant

conclusion is that if the minimum around the N atom is compared

266
FIGURE 3a

Morphine and Nalorphine - N lone pair direction

Electrostatic molecular potential contour map

©1978, J. J. Kaufman; printed by permission

267
FIGURE 3b

Morphine and Nalorphine - N substituent direction Electrostatic molecular potential contour map

©1978, J. J. Kaufman; printed by permission
with the minimum around the O atoms, the semehipirical relative

ordering is greatly changed with respect to the ab initio one.

Near the oxygen atoms of the OH groups the semiempirical potential

holes are as deep as near the N atom or even deeper. This is in

contrast with the trend found in present and previous (Scrocco

and Tomasi 1973; Petrongolo and Tomasi 1975) ab initio calculations.

Also, ab initio and semiempirical calculations, give even different

qualitative results in the π region of the benzene ring in morphine and nalorphine since the ab initio attractive region due to

the π-electron distribution of the benzene ring becomes repulsive.

In the π-region of the allyl group, only the semempirical results

using the approximation IV and the STO-6G expansion are in agreemerit with the ab initio ones. From these and other results (Scrocco

and Tomasi 1973; Petrongolo and Tomasi 1975; Petrongolo, Preston

and Kaufman - in press 1977; Petrongolo et al, - submitted for

publication) the conclusion remains that the CNDO and INDO methods

can only be used with caution to get V (r) and that it seems difficult

to derive any general rules.

IV. SYSTEMS ANALYSIS, CONTROL THEORY AND CATASTROPHE THEORY

We formulated (Kaufman, Kerman and Koski 1974; Kaufman and Koski

1975a, 1975b; Kaufman, Koski and Peat 1975; Kaufman 1976; Kaufman,

Koski and Peat 1977) a system analysis and control theory and

catastrophe theory (Thom 1972; Thom 1975) framework in order better

to clarify the dynamic balance of the influence of endogenous

diseases or exogenous CNS drugs and for combined interactions of

several CNS drugs with diverse modes of action (Kaufman 1976).

Using this we have been able to delineate the conditions for normal,

abnormal, and "catastrophic" dynamic balance of neurotransmitters.

We have also been able to program this for an interactive computer

system that ties into a patient clinical test and drug record keeping system and allows computer generated diagnostics and prescriptions.

Moreover, drugs which cause physiological addiction appear to be

categorized by causing a particular type of neurotransmitter

imbalance while drugs which may be addictive psychologically but

not physiologically present a different neurotransmitter profile.

FOOTNOTES

1

J. Lewis. Private communication, 1975.

2

Where accurate implies accuracy with respect to the completely

ab initio calculations with the same atomic basis set retaining

all integrals 10-6 a.u. or greater.

269
3

Figure 1 is reprinted with permission, from Kaufman, J. J., Semo,

N. M., and Koski, W. S. Microelectrometric titration measmement

of the pKa's, partition and drug distribution coefficients of

narcotics and narcotic antagonists and their pH and temperature

dependence. J Med Chem 18(7), 1975, p. 649. © American Chemical

Society, 1975:

ACKNOWLEDGMENTS

The research on the carcinogens, their metabolites and DNA constituents is supported by NCI under Contract N01-CP-75929. We are

indebted to Dr.Kenneth Chu.

The research on the neurotransmitters, the barbiturates

and a variety of suspected teratogens and the computer graphics is

supported by NINCDS under Contract N01-NS-5-2320. We appreciate

the interest of Dr. Eknst Freese, Chief, Laboratory of Molecular

Biology, NINCDS.

The development of the experimental procedure for the microelectrometric determination of the lipopphilicites and their pH and

temperature dependence studies were supported by NIDA. We thank

Dr. Monique Braude and Dr. Robert E. Willette, NIDA.

The measurements on the ketamine metabolites were supported in

part by a grant-in-aid from Parke Davis Company with the late

Dr. Duncan McCarthy.

We should also like to thank the following for their gracious gifts

of some of the compounds used in this research.

Phenyclidine - Dr. E. E. Trainor, Phillips Roxanne, Inc.

Ketamine (and its metabolites) - Dr. D. A. McCarthy, Parke Davis

& Co.

Etorphine - Dr. R. E. Willette, National Institute on Drug Abuse

270
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Giordano, W., Hamann, J.R., Harkins, J.J. and Kaufman, J.J. Quantum

mechanically derived electronic distributions in the conformers of

2-Pam. Ariens, E.J. ed. Physico-Chemical Aspects of Drug Action.

Vol. 7. New York: Pergamon Press, 1968. pp. 327-354

271
Hansch C., Leo, A., Unger, S., Kim, K.H., Nikaitani, D. and Lien,

E. J. "Aromatic" substituent constants for structure-activity

correlations. J Med Chem 16:1207-1216, 1973.

Hinze, J. MC-SCF. I. The multi-configuration self-consistent-field

method. J Chem Phys, 59:6423-6432, 1973.

Jasinski, D.R., Griffith, J.G. and Carr, C.B. Etorphine in man.

I. Subjective effects ands suppression of morphine abstinence.

Clin Pharm Ther, 17:267-272, 1975

Kaufman, J.J. Quantum chemical and theoretical techniques for the

understanding of action of psychoactive drugs. Proceedings of the

Symposia at the VIII Congress of the Collegium Internationale

Neuro-Psychopharmacologicum on Psychopharmacology, Sexual Disorders

and Drug Abuse. North-Holland Publishing Co. Amsterdam-London,

Avicenum, Czechoslovak Medical Press, Prague, 1973. pp.31-42. (a)

Kaufman, J.J. Quantum chemical and theoretical techniques for the

understanding of action of psychoactive drugs and narcotic agents.

Int. Conference on Computers in Chemical Research and Education,

Ljubljana, Yougoslavia, July 12-17, 1973. Comput. Chem. Res. Educ.

Proc., 3:5/1-5/20, 1973. (b)

Kaufam, J.J. Molecular neurotransmitter disorders - theoretical

and chemicophysical approach to their understanding and treatrment.

Int J Quantum Chem, QBS3:187-215; 1976.

Kaufman, J.J. Theoretical approaches to pharmacology. Int J

Quantum Chem, QBS4:375-412, 1977. (a)

Kaufman, J.J. Ab initio and approximately rigorous calculations

on small, medium and large molecules. Int J Quantum Chem, S11: 

1005-1015, 1977. (b)

Kaufman, .J.J. Theoretical insights to pharmacology. Invited

lecture presented at: Int. Symp. on Biomolecular Structure, Conformation, Function and Evolution, Madras, India, January 1978. (a)

Kaufman, J.J. Nonempirical MODPOT/VRDDO calculations on drugs,

carcinogens and biomolecules. Invited lecture presented at the

Int. Symp. on Quantum Biology and Quantum pharmacology, Florida,

March 1978. (b)

Kaufman, J.J. and Kerman, E. Quantum chemical calculations on antipsychotic drugs and narcotic agents. Int J Quantum Chem, 6S:319-

335, 1972.

272
Kaufman, J.J. and Kerman, E. Quantum chemical and other theoretical techniques for the understanding of the psychoactive action

of the phenothiazines. Third Int. Conference on Phenothiazines and

Related Drugs, Rockville, Md., June 25-28, 1973. Forrest, I.S.

Carr, C.J. and Usdin, eds. Phenothiazines and Structurally Related

Drugs. Vol. 9 New York: Raven press, 1974, pp. 55-75

Kaufman, J.J. and Kerman, E. Quantum chemical and theoretical

techniques for the understanding of action of drugs which affect

the central nervous system: antipsychotics, narcotics and narcotic

antagonists. Int. Symp. on Chemical and Biochemical Reactivity,

Jerusalem, Israel, April 9-13, 1973. Jerusalem Symposia on Quantum

Chemistry and Biochemistry. Vol. VI. Jerusalem, Israel, 1974.

Kaufman, J.J. and Kerman, E. The strutcture of psychotropic drugs

(including theoretical prediction of a new class of effective

neuroleptics). Int J Quantum Chem, QBS1:259-289, 1974. (c)

Kaufman, J.J. and Kerman, E. Quantum chemical calculations on

thioridazine. Int J Quantum Chem, 10:559-567, 1976.

Kaufman, J.J. and Kerman, E. Conformational profile of nalorphine

by PCILO calculations; Int J Quantum Chem, 11:181-184, 1977.

Kaufman, J.J., Kerman, E. and Koski, W.S. Quantum chemical, other

theoretical and physicochemical studies on narcotics and narcotic

antagonists to understand their mechanism of action. Int J Quantum

Chem, QBS1:289-313, 1974.

Kaufman, J.J. and Koski, W.S. Physicochemical, quantum chemical

and other theoretical techniques for the understanding of the

mechanism of action of CNS agents: psychoactive drugs, narcotics

and narcotic antagonists and anesthetics. Ariens, E.J. ed.

Drug Design. Vol. V. New York: Academic Press, 1975, pp. 251-

Kaufman, J.J. and Koski, W.S. Physicochemical, quantum chemical

and other theoretical studies of the mechanism of action of CNS

agents: anesthetics, narcotics and narcotic antagonists and psychotropic drugs. Int J Quantum Chem, QBS2:35-57, 1975. (b)

Kaufman, J.J., Koski, W.S. and Benson, D.W. Temperature and pH

sensitivity of the partition coefficient as related to the bloodbrain barrier to drugs. Exp Eye Res, 25:201-203, suppl., 1977.

Kaufman, J.J., Koski, W.S., Benson, D.W. and Semo, N.M. Measurement of the pKa's, partition and drug distribution coefficients of

narcotics and narcotic antagonists, their pH and temperature

dependence and their significance in clinical practice. Drug

Dependence Meeting, Washington, D.C., May 1975. Problems on Drug

Dependence, National Academy of Sciences, 1975. pp. 433-452.

273
Kaufman, J.J., Koski, W.S., Benson, D.W. and Semo, N.M. Narcotic

and narcotic antagonists pKa's and partition coefficients and

their significance in clinical practice. Drug and Alcohol Dependence, 1:103-114, 1975/76.

Kaufman, J.J., Koski, W.S. and Peat, D. A systems and control

theory approach to dynamic neurotransmitter balance in narcotic

addiction and narcotic antagonism. Life Sciences, 17:83-84, 1975.

Kaufman, J.J., Koski, W.S., and Peat, D. A systems and control

theoretic approach to dynamic neurotransmitter balance: normal

abnormal and "catastrophic". Int J Quantum Chem, 11:485-504,

1977.

Kaufman, J.J., and Manian, A.A. Topological conformational

similarities among antipsychotic drugs, narcotics and biogenic

amines. Int J Quantum Chem, 6S:375-381, 1972.

Kaufman, J.J., Semo, N.M. and Koski, W.S. Microelectrometric

titration measurement of the pKa's, partition and drug distribution coefficients of narcotics and narcotic antagonists and

their pH and temperature dependence. J Med Chem, 18:647-655,

1975.

Kosterlitz, H.W., Lord, J.A.H. and Watt, A.J. Morphine receptor

in the myenteric plexus of the guinea pig ileum. In: Kosterlitz,

H.W., Collier, H.O.J. and Villarreal, J.E. eds. Agonist and

Antagonist Actions of Narcotic Analgesic Drugs. Baltimore, Md.;

University Park Press, 1973, p.45-61.

Kutter, E., Herz, A., Teschemacher, J.H. and Hess, R. Structureactivity correlations of morphine-like analgetics based on

efficiencies following intravenous and intraventricular application.

J. Med Chem, 13:801-805, 1970.

Malrieu, J.P., Huron, B. and Rancurel, P. Iterative perturbation

calculations of ground and excited state energies from multiconfigurational zeroth-order wave functions. J Chem Phys, 58:5745-

5759, 1973.

McWilliams, D. and Huzinaga, S. Gaussian basis sets suitable for

accurate valence-shell calculations using the model potential

method. J Chem Phys, 63:4678-4684, 1975.

Osman, R. and Weinstein, H. Near and ab initio methods for calculations of large molecules - comparison of pseudopotential and ab

initio results. Chem Phys Letts 49:69-74, 1977.

Pert, C.B. and Snyder, S.H. Properties of opiate-receptor binding

in the rat brain. Proc Nat Acad Sci USA 70:2243-2247, 1973.

274
Petrongolo, C., Popkie, H.E., Preston, H.J.T. and Kaufman, J.J.

Ab initio LCAO-MO-SCF calculation of the electrostatic molecular

potential of morphine and nalorphine and comparison with semiempirical results. Submitted for publication, J Am Chem Soc.

Petrongolo, C., Preston, H.J.T. and Kaufman, J.J. Ab initio

LCAO-MO-SCF calculation of the electrostatic molecular potential

of chlorpromazine and promazine. In press Int J Quantum Chem. 1977.

Petrongolo, C. and Tomasi, J. The use of electrostatic molecular

potential in quantum pharmacology. I. Ab initio results. Int J

Quantum Chem, QBS2:181-190, 1975.

Popkie, H.E. MOLASYS: (Molecular Orbitals for Large Systems):

Computer programs for molecular orbital calculations on large

systems. The Johns Hopkins University, 1974.

Popkie, H.E. GIPSY (Gaussian Integral Program System): A system

of computer program for the evaluation of one- and two-electron

integrals involving s-, p-, d-type contracted Cartesian Gaussian

functions. The Johns Hopkins University, 1975.

Popkie, H.E. GIPSY (Gaussian Integral Program System): A system

of computer programs for the evaluation of one- and two-electron

integrals involving s-, p-, d- and f-type contracted Cartesian

Gaussian functions. The Johns Hopkins University, 1976.

Popkie, H.E. and Kaufman, J.J. Test of charge conserving integral approximations for a variable retention of diatomic differential overlap (VRDDO) procedure for semi-ab-initio molecular

orbital calculations on large molecules. Int J Quantum Chem,

QBS2:279-288, 1975.

Popkie, H.E. and Kaufman, J.J. Molecular calculations with the

VRDDO, MODPOT and MODPOT/VRDDO procedures. I. HF, F2, HCl, C12,

formamide, pyrrole, pyridine and nitrobenzene. Int J Quantum Chem,

10:47-57, 1976. (a)

Popkie, H.E. and Kaufman, J.J. Ab initio LCAO-MO-SCF calculations

of chlorpromazine and promazine. Int J Quantum Chem, 10:569-580

1976. (b)

Popkie, H.E. and Kaufman, J.J. Molecular calculations with the

MODPOT, VRDDO and MODPOT/VRDDO procedures. II. Cyclopentadiene,

benzene, diazoles, diazine and benzonitrile. J Chem Phys, 66:

4827-4831, 1977. (a)

275
Popkie, H.E. and Kaufman, J.J. Molecular calculations with the

MODPOT, VRDDO, and MODPOT/VRDDO procedures. III. MODPOT/SCF + CI

calculations to determine electron affinities of alkali metal atoms.

Chem Phys Letts, 47:55-58, 1977 (b)

Popkie, H.E. and Kaufman, J.J. Molecular calculations with the

MODPOT, VRDDO and MODPOT/VRDDO procedures. V. Ab initio and MODPOT

LCAO-MO-SCF calculations on the chlorofluoromethanes. Int J Quantum

Chem, S11:433-443, 1977. (c)

Popkie, H.E. and Kaufman, J.J. Molecular calculations with the

MODPOT, VRDDO and MODPOT/VRDDO procedures. IV. Boron hydrides and

carboranes. Int. Symp. on Quantum Chemistry and Solid State Physics, Sanibel Island, Fla., Jan. 1977. In press: Int J Quantum

Chem.

Popkie, H.E. Koski, W.S. and Kaufman, J.J. Ab initio LCAO-MO-SCF

calculations on morphine and nalorphine and comparison with their

measured photoelectron spectra. J Am Chem Soc, 98:1342-1345, 1976.

Pople, J.A. and Beveridge, D.A. Approximate Molecular Orbital

Theory. New York: McGraw-Hill, 1970

Preston, H.J.T. and Kaufman, J.J. Ab initio SCF calculations on

pyrrole and pyrazole. Int J Quantum Chem, 78:207-215, 1973.

Preston, H.J.T., Kerman, E., Kaufman, J.J. and Cusachs, L. C.

Comparison for pyrrole and pyrazole of orbital energies and

population analyses from ab initio SCF, CNDO/2, INDO extended

Huckel and ARCANA calculations. Int J Quantum Chem, 7S:249-260,

1973.

Pullman, A., Gresh, N., Daudey, J.P. and Moskowitz, J.W. The use

of pseudopotentials for the study of cation-ligand and intermolecular interactions. Int J Quantum Chem, S11:501-507, 1977.

Raffenetti, R.C. The Johns Hopkins University, 1973-74.

Raffenetti, R.C. ICASE 1974-76.

Saethre, L.J., Carlson, T.A., Kaufman, J.J. and Koski, W.S.

Nitrogen electron densities in narcotics and narcotic antagonists

by X-ray photoelectron spectroscopy and comparison with quantum

chemical calculations. Mol Pharm, 11:492-500, 1975.

Scrocco, E. and Tomasi, J. Electrostatic molecular potential as

a tool for the interpretation of molecular properties, Topics In

Current Chemistry. Vol. 42, Heidelberg: Springer Verlag, 1973,

276
Shavitt, I. The method of configuration interaction. In: Modern

Theoretical Chemistry. Vol. II, Electronic Structure Ab Initio

Methods, ed. Schaefer, H.F. New York: 'Plenum press, 1976.

Shavitt, I. and Pipano, A. The basic CI programs described below

are those written originally by I. Shavitt and A. Pipano and continued by A. Pipano while he was a postdoctoral in our group at

The Johns Hopkins University. We have subsequently written a

number of additional features to those CI programs.

Thom, R. Stabilité Structurelle et Morphogenese, Essai d'une

Theorie Generalle des Modeles, W.A. Benjamin, Reading, Mass., 1972.

Thom,R. Structural Stability and Morphogenesis; An Outline of a

General Theory of Models, trans. form French by Fowler, D.H.,

W.A. Benjamin, Inc., Reading, Mass., 1975.

Weinstein, H. and Osman, R. Models for molecular mechanisms in

drug receptor interactions. Serotonin and 5-hydroxyindole with

imidazolium cation. Int J Quantum Chem, QB4:253-267, 1977.

Wilhite, D.L. and Euwema, R.N. Charge-conserving integral approximations for ab initio quantum chemistry. J Chem Phys, 61:375-

382, 1974.

AUTHOR

Joyce J. Kaufman, Ph.D.

Department of Anesthesiology

The Johns Hopkins University School of Medicine

Baltimore, Maryland 21205

277
‘QuaSAR’ Research Monograph 22

G.Barnett, M. Trsic, and R. Willette, eds,

National Institute on Drug Abuse, 1978

Structure-Activity Studies of

Narcotic Agonists and Antagonists

from Quantum Chemical

Calculations

Gitda H. Loew, Donald S. Berkowitz, and

Stanley K. Burt

In this paper, the application of quantum chemical methods in elucidating electronic and structural requirements for opiate narcotic

analgesic agonism and antagonism will be discussed. In particular

the relevance of this method to two specific problems of opiate

activity will be described.

One major effort centers on characterizing molecular features which

determine the extent of agonism and antagonism in a given opiate.

This problem is of clinical importance since there is growing evidence that a clinically useful analgesic with little or no addiction

liability will be a mixed agonist/antagonist with just the "right"

mixture of both activities. It is also particularly suited to

quantum chemical studies, since the ratio of agonism to antagonism

in a given compound should be a receptor-related event. To help

understand how chemical modifications in a series of opiate analogues

alter their agonist/antagonist potency ratios, calculations have been

done on N-substituent variations in a series of rigid opiates with

and without an axial C14-OH group and for a series of oripavine

derivatives with varying C7 substituents. In addition, interaction

of these different agonist/antagonist with a model for an anionic

receptor site have been studied.

Another focus of attention has been on the diversity of chemical

structures which have narcotic analgesic activity. Conformational

studies have been made for a number of different types of flexible

opiates including methadone, meperidine and enkephalins. Since

all of these classes are thought to act at the same receptor as rigid

morphine like opiates, low energy conformers for each type of opiate

were characterized and compared with each other and with rigid

opiates. Studies of this type should help understand how these more

flexible opiates interact at the receptor site. They should also

indicate to what extent results of structure activity studies for

one class of opiates should carry over to another class.

278
I. STRUCTURAL FEATURE OF RIGID OPIATES THAT MODULATE

AGONIST/ANTAGONIST ACTIVITY

It is now generally acknowledged that compounds possessing potent

analgesic activity yet having some degree of antagonist properties

are good candidates for clinically useful anagesics with low

addition potential. N-substituents of fused ring opiates (la-f)

play a central role in determining the relative analgesic agonist/

antagonist potencies of these molecules. However, other structural

modifications such as an axial C14-OH group in the morphone (6 keto

7,8 dihydro morphine) series and ong chain (R1,R2) tertiary

carbinol substituents on C7 in the thebaine (R5=CH3) and in the

oripavine series (R5=H) mitigate this ratio for a given N-substituent.

To help understand how these chemical modifications in a series of

opiate analogues alter their agonist/antagonist potency,

semiempirical quantum chemical calculations have been done for

N-substituent variations in a series of rigid opiates with

(oxymorphone) and without (morphine) an axial C14-OH group and for

a series of oripavine derivatives with varying substituents on C7.

In addition, preliminary studies of the interaction of different

agonist/antagonist with models for anionic receptor site have been

made. The goal of these studies was to identify structural

properties which might explain the observed variation in relative

agonist/antagonist potencies.

In early work (Loew, Berkowtiz, et al 1975) the effect of polar

groups on the relative agonist potency of morphine derivatives

was explored by mapping of electrostatic potentials of low energy

conformers of morphine, codeine, heroin, 6 monoacetyl and 2 amino

morphine, in the presence of a model anionic receptor site (fig. 1

morphine). This work established the relevance of a dynamic

interaction pharmacophore (Weinstein, et al 1973) for opiates.

Only by initial interaction of the quaterinized amine group with

an anionic receptor site do the remainder of the polar groups

279
Figure 1. Calculated electrostatic potential pattern of cis

morphine in the plane of the benzene ring

©1975, D. Reidel Publishing Co., Dordrecht-Holland1

(e.g., 3-OH, furan oxygen) develop significant negative potential

to effectively interact with cationic receptor sites. These

electron rich regions are greatly diminished in neutral species

and in the isolated opiate cation.

In another study (Loew, Weinstein, Berkowitz 1976) to help understand the origin of the enhanced agonist activity of a C14-OH group

in pure agonists we have investigated the ionization and partition

behavior of the related opiates hydromorphone and oxymorphone

including the effect of solvent on them. The C14-OH group was shown

to enhance the proton affinity of the nitrogen in the presence

or absence of solvent, and to increase the pKa in agreement with

experiment (Jolliffe, Ahmad 1971; Kaufman, Semo, Koski 1975) and

contrary to the usual inductive effect of an OH group. By studies

of hydration energies, we were also able to conclude that both the

apparent and intrinsic partition coefficients of the C14-OH oxy

compounds should be lower than the C14-H analogue. If our

predictions are correct, the intraven ricular potency of oxymorphone

280
relative to hydromorphone should be greater than the apparent two

fold differences obtained from standard ED50 measurements (Mellet,

Woods 1963).

In contrast to agonist potency, relative agonist/antagonist potency

in a given compound is a receptor related event. In a series of

related compounds, variations in this ratio would most likely be

due to differences in binding and interaction at the receptor site.

Thus correlations of structural features of the isolated molecule

with this clinically important behavior should be particularly

successful since the influence of such factors as transport and

metabolism are kept to a minimum. To establish such correlations

all of the conformational calculations reported here. for

N-substituents with and without C14-OH and for C7 substituents

were done on the isolated protonated form of the Opiate though; to

be the active form (Casy 1973). Solvent effect on conformation

are not expected to be substantial at the lipophilic receptor site.

The possibility of induced conformational change at the receptor

site is included in all our studies by considering many low energy

conformers rather than a single global minimum as receptor site

candidates.

semiempirical

All calculations were performed using the

all valence electron program called PCILO (Perturbative

Configuration Interactive. using Localized Orbitals) developed in the

Laboratory of Professor Bernard Pullman (Diner; et al 1969)

specifically for energy conformational studies of large molecules.

It has been used extensively to study a variety of pharmacological

and biological molecules ( Christoffersen 1976) and has proven its

reliability by consistent agreement with x-ray structures and results

of more sophisticated calculations.

A. EFFECT OF N-SUBSTITUENT AND AXIAL C14-OH GROUP ON

AGONIST/ANTAGONIST POTENCY

1. Origin of Mixed Agonism/Antagonism in Nalorphine.

In our first study of the effect of N-Substituent on agonist/antagonist potency (Loew, Berkowitz 1975), nalorphine and N-phenethyl

morphine were chosen as prototype compounds. From the results of

those studies, we proposed that the dual agonist antagonist behavior

of nalorphine was due to the existence of two low energy “types”

of equatorial N-allyl conformations at the receptor (fig. 2).

Differences in activity between agonist and antagonist could not be

attributed to electronic effects (Loew, Berkowitz 1975; Kaufman,

Kerman 1974; Popkie, Koski, Kaufman 1976). Such differences in

drug conformation, as well as in receptor conformation, could account

for the differences between agonist and antagonist binding associated with the observed Na+ effect (Pert, Snyder 1974).

2. Origin of Mixed Agonism/Antagonism in Oxymorphone Series.

In addition to our hypothesis for nalorphine, several other investigators have speculated on the roles of N-substituents and the axial

281
Figure 2. Two types of low energy allyl group conformers in

nalorphine: (a) low energy, noninterfering (agonist) conformer;

(b) minimum energy, protruding (antagonist) conformer

©1975, the American Chemical Society2

C14-OH group in conferring agonist or antagonist activity on the

him-like fused ring structure. It has been suggested

(Smythies 1976) that axial C14-OH groups might act by direct steric

interference with certain equatorial N-substituent conformations.

In another widely publicized hypothesis (Feinberg, Cresse, Snyder

1976), it has been suggested that agonist and antagonist activities

are associated with axial and equatorial N-substituents, respectively, and the efffect Of the C14-OH group enhancing antagonism is

explained by assuming its presence is responsible for a shift from

an axial to equatorial N-substituent conformation. Although there

is evidence for rapid interconversion between axial and equatorial

N-substituent orientation of unhindered piperidines in solution

(Jones, Katritsky, et al 1970), nitrogen inversion is unverified

in the highly rigid fused ring opiates. Further, in all crystal

structures of neutral and protonated 3,4,5 and 6 fused-ring opiates

reported to date N-substitents have been found to be equatorial.

To further explore these hypothesis and the role of the C14-OH group

and various N-substituents in mediating agonism and antagonism, a

systematic quantum chemical study has been made of six oxymorphone

derivatives (2a-f)

This series is a good example of the complex role the N-substituent

plays in controlling pharmacological behavior ranging from potent

agonism (2a and 2f) to mixed agonism-antagonism (2c, 2e) to pure

282
potent antagonism (2b, 2d) (Kosterlitz, Waterfield 1975; Blumberg,

Dayton 1974; Pert, Snyder 1974; Kosterlitz, Lord, Watt 1972; Deneau,

Villareal, Seevers 1966). While there are three structural differences between the morphine and oxymorphone parent compounds, it

is the C14-OH group that appears to most drastically alter the

morphine-like pharmacological profile for varying N-substituents

(Cordon, Priciro, et al 1974; Kutter, Herz., et al 1970;

Knoll 1975). In mixed agonist-antagonist, the C14-OH drastically

diminishes agonism while increasing antagonism.

The geometry of the oxymorphone fused ring structure was taken from

a recent crystal structure of naloxone (Karle 1974) and is comparable

to other recent oxymorphone derivative structures (Sime, Dobler, Sime

1976). With this basic ring structure, energy conformation calculations including geometry relaxation were made for both axial and

equatorial N-substituents. Energy differences between optimized

axial and equatorial and barriers to inversion conformers were

obtained for each compound (2a-f) in their protonated and neutral

form as well as for similar forms of morphine and nalorphine.

Possible interaction of the C14-OH group with these substituents

was also considered.

The results summarized in table I do not support the hypothesis that

axial N-substituents are required for agonist activity while equatorial substituents confer antagonist activity. No correlation

exists between agonist/antagonist potency variation and calculated

axial-equatorial energy differences in either the base or protonated

form of the opiates studied. For all compounds studied, the equatorial conformer is the lowest energy form. This result is consistent with crystal structure data as are the optimized values of

equilibrium bond angles about the nitrogen. Morphine and nalorphine

have comparable axial-equatorial N-substituent energy differences,

as do oxymorphone and naloxone. Some effect of the C14-OH can be

seen by larger energy differences in the oxymorphone series over

those in morphine and nalorphine. However, axial conformations are

equally accessible in naloxone and oxymorphone indicating an equally

hindering effect of the C14-OH group on agonists and antagonists.

In general there is no systematic increase in axial conformational

energies as agonist potency decreases.

The inversion barriers calculated for the neutral molecules are

insensitive to N-substitution and to the presence or absence of

C14-OH and hence show no correlation to agonist/antagonist potencies.

The inversion barriers correspond to the calculated differences in

energy between the lowest energy (equatorial) and planar structures.

Calculated barrier heights for ammonia and aniline were 12 and 7

kcal/mole respectively, compared to known values of 6 and 2. Thus

while the PCILO method overestimates these quantities, their relative

values are reasonably reliable.

Comparison of the calculated inversion energies (table I) with those

calculated for ammonia and aniline indicates that the inversion process can occur in these fused ring opiates at about the same rate as

in ammonia and that the axial conformer is accessible, particularly

283
TABLE I: ANIAL-EQUATORIAL ENERGY DIFFERENCES IN THE PROTONATED

AND NEUTRAL SPECIES AND NEUTRAL MOLECULE INVERSION ENERGIES
in the protonated form of the isolated molecule. There is no

evidence, however, that this inversion or the presence of axial

conformers is related to drug action at the receptor. No direct

interaction of the C14-OH group with low energy axial or equatorial

conformations of the N-substituents occurs and hence cannot explain

the effect of Cl4 substitution on pharmacological activity.

The rotational behavior of the equatorial N-allyl group in naloxone,

with the C14-OH in its minimum energy conformation, is identical

within computational accuracy to that calculated for the allyl group

in nalorphine. The allyl group in naloxone retains the two types

of low energy conformers found in nalorphine which we previously

associated with its dual agonist and antagonist behavior (Loew,

Berkowitz 1975). In low energy regions of axial nalorphine and

naloxone the same statements can be made. Thus, the total abolition

of agonist activity in naloxone cannot be explained by differing

accessible allyl conformations from those in nalorphine nor by direct

interaction of the axial C14-OH with low energy N-allyl conformers.

As mentioned above we have suggested that agonist/antagonist behavior

could be regulated by different low energy equatorial N-substituent

conformers (Loew, Berkowitz 1975). We have also recently reported

calculations (Berkowitz, Loew 1976) which suggest that the C14 OH

group and various N-substituents cannot interact directly but might

interact indirectly through a common anionic receptor site such as

R-SO4 or R1R2PO4 Both of these hypotheses together can be used to

help understand the variation of agonist/antagonist potency in the

series of oxymorphone derivates studied.

Drug receptor complex formation could occur by simultaneous interaction of the C14-OH, cation nitrogen, and certain N-substituents

with a model antonic receptor site (sulfate or phosphate). The

ternary complex formed is stable only for a small range of

N-substituent conformations. These could correspond to effective

antagonist-receptor interaction and account for the relatively strict

structural requirements for pure antagonism. The role of the C14-OH

group in diminishing agonism then would be to cause preferential

selection of only one type of low energy conformer for receptor

complex formation from among the several hypothesized “agonist” and

“antagonist” N-substituent conformations.

With this suggested hypothesis, allyl and methylcyclopropyl

N-substituents would be prototypes for optimum formation of such

ternary complexes since they have a comparatively high degree of

felxibility and π character. Nalmexone and nalbuphone are less

flexible and have less π character and thus would form weaker

ternary complexes reducing the effect of the C14-OH group (i.e.,

creating a less pure antagonist).

If this hypothesis is correct, any substitution that detracts from

the strict geometric requirements found for (C14-OH)- (anion)- (N-allyl)

antagonist complexing should enhance the agonist/antagonist potency

ratio. Two such alterations, the Cl4-OCH3 derivative of naloxone and

285
the C15-OH analogue, should restore agonism. Removal of the C14-OH

group together to make N-allyl hydromorphone would also produce a

agonist/antagonist. The ability of the axial C14-OH group alone to

reduce intrinsic agonism has been recently demonstrated by the

decreased agonism of naltrexone relative to its hydromorphone

analogues in the guinea pig ileum. Also, one would predict that

substitution on cyclopropyl ring carbons would alter the locations

of conformational local minimas and reduce the pure agonism of

naltrexone by reducing overlap with the local minima of the allyl

group.

The lack of antagonist activity of methyl and phenethyl N-substituents could then be explained by their lack of interaction with

the receptor anionic site. The N-phenethyl substituent could,

however, interact with a more distant binding site enhancing its

binding and agonist patency. Further characterization of the

interaction of different N-substituents with a model anionic site

is now in progress.

B. EFFECT OF C7 SUBSTITUENTS ON AGONIST/ANTAGONIST IN ORIPAVINES

The oripavine and thebaine series of opiates synthesized by Bentley,

Lewis and co-workers (Lewis, Bentley, Cowan 1971) in the search for

clinically useful narcotic agonists and antagonists are structurally

similar to morphine and dihydromorphine but contain a C6-C14 etheno

bridge and C7 substituents. Among other reasons, the series became

a focus of attention because some of the compounds had unexpectedly

high agonist [>1000 x morphine (M)] and antagonist [>100 x nalorphine

(N)] potencies, and because the relative agonist/antagonist

potencies of N-substituted compounds were dramatically sensitive

to substitution at C7 and because some of the compounds showed

unique pharmacological profiles.

To provide additional insight into causes of sane of these

variations in activity, we have made a conformational study of

the series of C7 oripavines (3a-k)

Calculations were performed cm compounds 3a-k to determine the most

energetically feasible conformations of the C7 substituents. In

286
these studies attention was given to possible hydrogen bonding

interaction between the C6-OCH3 and the C7-OH group. Although the

calculations reported here were done on compounds in the oripavine

series (C3OH) the results are equally applicable to compounds in

the bridged thebaine series (C3-OCH3) since the C3 and C7

positions are separated enough to preclude conformational interaction.

The pharmacology of this and related series has been reviewed

(Lewis, Bentley, Cowan 1971) and only illustrative points will be

mentioned here. The compound most similar to M, differing only by

a C14-C16 ethyl bridge and a methoxy group on C6 is 30 x M

in the rat tail pressure test (Lewis Redhead, Smith 1973) and only

six times as potent as C6-OCH3 morphine (Soine, Willette 1966). Thus

the greatly increased agonist activity of etorphine (31) [1,100 x M

RTP] (Lewis, Redhead 1970) must be strongly linked to the additional

substituents present at the C7 position. The relative agonist/

antagonist potencies of the well-studied N-methyl cyclopropyl and

N-allyl derivatives of (3) are also highly sensitive to C7

substitution. For example, in the N-methyl cyclopropyl derivatives

of 3d and 3i lengthening R2 from a CH3 group to CH2CH2CH3 (i)

changes the activity from strong antagonism (50 x N RTP1) to potent

agonism (1,000 x MRTP1). In the N-allyl derivatives, the same

variation in R2 causes a similar change in activity from moderate

antagonism (2 x N RTP1) to strong agonism (60 x M RTR1).

The sensitivity of agonist/antagonist potency ratio to C7 substitution is most likely related to binding and interaction of the C7

substituent at the receptor site. To explain the effect of C7 substituents on the observed activity of oripavines, several hypotheses

have been presented. Based on the Beckett and Casy receptor model

(Beckett, Casy 1954) it has been proposed (Lewis, Bentley, Cowan

1971) that the C7 substituents interact with a lipophilic receptor

site since polar C7 substituents reduce agonist activity (Bentley,

Lewis, Smith 1972) while large lipophilic substituents substantially

enhance it (Lewis, Bentley, Cowan 1971). This idea has been extended

to suggest that in phenethyl etorphine, (R1=CH3, R2=CH2CH2φ ) the

phenethyl moiety, by complementarity, describes a receptor site which

stabilizes a conformation of the receptor associated with agonism

(Feinberg, Creese, Snyder 1976). Much has been made of the "F" ring

binding but without results of extensive binding studies the

importance of aromaticity is unclear since at least five other compounds with R2 saturated have been synthesized (Bentley, Hardy

1976) with higher in vivo potencies than the phenethyl derivatives.

It is hoped that conformational studies of the C7 substituents 3a-k

would provide some insight into how they modulate agonist/antagonist

activity.

Torsion angles for substituents at all positions which would not

interact with the C7 substituent or with each other were determined

by independent variation. For the C6-OCH3 group whose conformation

could be coupled to the C7 substituent conformation, rotations were

performed in etorphine with the C7 substituent in hydrogen bonding

and nonbonding conformations. In all cases only one conformer was

287
the only local minima and was held fixed in all subsequent calculations. The most extensive calculations were performed for the

lowest energy intramolecularly hydrogen bonded structures (τ3 = 300°,

τ 1 = 60°, τ10 = 60°) where energies were determined for C19 alkyl

group(s) using all combinations of rotation angles for each optical

isomer. Local minima were determined in this way for hydrogen bonded

forms. These, as well as those determined using models and less

complete rotational variations, were then optimized to determine

local minima accurate to a few degrees and energies accurate

(within the computational method) to a few tenths of a kcal/mole.

The results of conformational studies (table II) indicate that

intrarmolecular hydrogen bonding to the C6-OCH3 in oripavines does not

appear to play a dominant role in determining the conformation of

C19 carbinol substituents on C7 if R1 are R2 hydrogen or methyl

groups (3a-d). This result is in keeping with the observation of

several conformers of these C7 substituents in the NMR spectra

(Fulmor, et al 1967). With these small groups, the C7 substituents

have substantial conformational freedom in binding to the receptor.

Thus it is not surprising that their apparent potencies are similar,

increasing somewhat with lipophilicity of the alcohol group (17, 15,

37 and 63 x M RTP for 3a-d, respectively), and that the relative

Table II

Energy Differences Between H Bonded and Non H Bonded*

Conformers of C7 Substituents of Oripavine

Compound Δ E(HBond-NoHBond)

3 a

3 b

-0.4 kcal/mol

+0.3

3c +0.3

3 d +0.1

3 e +0.9

3f +1.9

3 g

3 h

+3.4

+1.4

3i +2.3

*H bond between C60CH3 + C7 OH groups.

agonist/antagonist activities conferred by different N-substituents

are consistent with structure activity relationships found in other

fused-ring opiates.

In compounds with tertiary carbinol substituents of the type

C1 9 CH(CH2 ) ηCH3OH with η >1 (table II) hydrogen bonding is favored

in both diastereoisomers. This constraint fixes the C7 substituent

in one of two distinct spatial regions which are different for the

(R)- and (S)-diastereoisomers (fig. 3). It is suggested that this

difference in orientation determines the extent to which the long

288
Figure 3. Lowest energy conformers of C7 R- and S-substituent

diastereoisomers for etorphine showing by complementarity the lipophilic site for accommodation of the most potent isomer

alkyl group of the C19-carbinol can be accommodated at the postulated

lipophilic receptor could account for the difference in observed

agonist potencies in the (R) and (S) isomers of N-CH3 compounds. For

example, in compounds 3e, f, h and i the R-diatereoisomers are much

more potent than either the (S) diastereoisomers or compounds (3a-d)

with smaller alkyl groups. The calculated low energy conformers of

the more potent (R) isomers place the longer alkyl chain in one

spatial region. This region should, by complementarity, define optimum binding to a lipophilic receptor site. We further suggest that

it is the interaction of these conformationally restricted groups

with this site that directs the overall orientation of these compounds at the receptor and imposes a change in N-substituent binding

relative to morphine like compounds. Specifically, in keeping with

our hypothesis, the change in orientation imposed on the N-substituent

by binding of the C19 n-CH2 CH3 group in a rigid conformation could

interfere with the antagonist "type" of N-substituent binding mode

while enhancing the agonist "type" (fig. 4). The anomalous structureactivity profiles of the N-allyl and N-methyl cyclopropyl derivatives

of etorphine (3i) could then be due to this altered overall drug

receptor interaction. If this hypothesis is correct, replacement of

the C6OCH3 or the C19-OH group by a H or CH3 substituent should

enhance or restore antagonist potency to N-allyl and N-cyclopropylmethyl derivatives of oripavines and thebaines with long chain, C7

carbinol substituents.

289
Figure 4. Perspective drawing of

two superimposed etorphine molecules

shown perpendicular to phenyl ring

plane. One molecule is rotated slightly

about the phenyl ring axis (dashed line)

to show the directing effect of the C7

substituent. Note particularly the

altered N-methly orientation.

290
In compounds 3j and 3k, related to C19 (R) and (S) diatereoisomers of

buprenorphine, the bulky t-butyl substituents must be accommodated

at nearly identical positions in the two diastereoisomers thus allowing an intramolecular C6-O---H-O-C19 hydrogen bond in the (S) isomer

(3k) and not in the other. The pharmacology of the (S) isomer of

buprenorphine has been extensively studied (Cowan, Lewis, MacFarlane

1977; Cowan, Doxey, Harry 1977) but there appears to be no published

data on the (R) diastereoisomer or any such pair of C19 t-butyl

isomers. In contrast to the C19 straight chain deriivated compounds

(such as 3h and 3i ) we predict that intrinsic activity in the t-butyl

compounds 3j and 3k should have a rather low sensitivity to C19 optical

isomerism as the t-butyl group occupies similar positions in th

isomers.

SUMMARY AND PRACTICAL IMPLICATIONS

These studies have given us some insight into important opiate

structural features which modulate agonist/antagonist potencies.

Specifically, in the morphine series we have suggested that

restriction of the conformational freedom of the N-substituent to.

two types of low energy equatorial conformers causes the molecule to

adopt binding modes favoring either agonism or antagonism.

Our results refute any connection between agonist/antagonist potency

ratios and the extent of axial/equatorial N-substituent conformers.

The role of C14-OH group in modulating agonism and antagonism of

N-substituent in oxymorphone is not by direct interaction but

possibly thorugh their simultaneous binding to the anionic receptor

site to form a stable tertiary complex. In oripavines, the

agonist/antagonist profile of N-substituents is altered relative to

morphine by the directing effect of the C7-substituents that

hydrogen bond to the C6-OCH3 group. These C7-substituents are

relatively rigid in both diatereoisomers with long alkyl chains on

C19. This rigidity could also account for the differences in

potency between the (R)- and (S)- isomers. On the basis of these

hypotheses a number of predictions of altered agonist/antagonist

with specific chemical modifications have been made. For example,

in the morphine series we have suggested that further restrictions

of the conformational freedom of the N-substituent could cause the

molecule to adopt binding modes favoring either agonism or

antagonism. Placing of substituents at appropriate locations on the

alkyl side chains should create such conformational restrictions.

Colleagues at SRI International have thus synthesized a series of

morphine compounds:

and tested them for agonism and antagonism with promising results

(DeGraw, Lawson, Crase 1978). Other compounds in this series are

also being investigated.

291
In the oripavine series we have suggested that the replacement of

the C6-OCH3 by a nonhydrogen substituent would restore antagonism

to N-allyl and N-methylcyclopropyl derivatives. In the oxymorphone

series we predict that replacement of C14-OH by C14-CH3 or C14-OCH3

would restore agonism to naloxone and naltrexone as would

substitutions on the cyclopropyl ring of naltrexone. These

prediction, if true, would further verify our hypothesis and could

lead to synthesis of other clinically useful analgesics.

II. CONFORMATIONAL STUDIES OF FLEXIBLE OPIATES AND THEIR RESEMBLANCE

TO RIGID OPIATES

A. Methadone and 4-Phenyl Piperidines

Rigid opiates (la-d) have in common the three fused rings (A, B, D)

of the 6,7-benzomorphan nucleus. The salient common features of such

opiates which appear to be necessary for analgesic activity are: a

tertiary amine group which is part of a piperidine ring; an aromatic

ring which is constrained to be axially connected to the piperidine

ring at the central carbon atom (C13) para to the nitrogen, and

polar oxygen groups, particularly an OH group at the C3 position

of the phenyl ring.

In the 4-phenyl piperidine class of opiates, the phenyl ring can be

axial or equatorial to the piperidine ring with free rotation about

the bond between them. Meperidine (fig. 5)

Figure 5. Minimum energy conformer of meperidine with piperidine

ring superimposed on that of morphine and calculated net atomic

charges.

©1975, the American Chemical Society4

292
and its reverse ester desmethyl prodine (fig. 6) are common examples

Figure 6. Desmethyl (or (+) a)

Figure 9. Minimum energy conprodine with piperidine ring former for (-) a, (-)ß prodine

superimposed on that of morphine with piperidine ring superand calculated net atomic chaarges imposed on that of morphine

©1975, the American Chemical Society4

of such opiates. In addition to the relative orientation of the

two rings, another defining conformational feature in these

compounds is the relative orientation of the ester chain.

Meperidine is 1/10 as potent as morphine when administered subcutaneously in mice and tested by a modified hot plate method

(Portoghese, Larson 1973). However, intraventricular data determined

by a tooth pulp test in rabbits indicate that it is only 1/60 as

potent as morphine when transport and distribution factors are

eliminated (Kutter, Herz, Teschemacher 1970). Evidence for

meperidine-type opiates acting at the morphine receptor is given by

the fact that cross tolerance is observed and both are antagonized

by nalorphine and naloxone (Aceto, McKean, Pearl 1969).

The reversal of the ester chain from meperidine to desmethyl

prodine enhances in vivo potency by a factor of 10, making desmethyl

apparently equal to morphine in potency (Portoghese, Larson 1973).

As seen in table III they span a potency range of 100 with (+)

isomers more potent than (-). While no intraventricular potency

data is available for these compounds, metabolic and brain

concentration studies (Portoghese, Abdel-Monen, Larson 1972)

indicate that differences in potency among them is due solely to

receptor events.

Despite the fact that crystal structures indicate that protonated

meperidine (Van Konigsvel 1970), (+)α (Ahmed, Barnes, Kartha 1960)

and (-) ß (Ahmed, Barnes 1963) prodines are all in a phenylequatorial conformer, there was a tendency to believe that because

of its greater similarity to morphine an axial conformer was

implicated at the receptor (Beckett, Casy 1954) and that the lower

its energy the more potent the drug (Porteghese 1966). The recent

synthesis of active 4-phenyl piperidine analogues constrained to

293
Table III. Salient Energy-Conformation Characteristics of Six Compounds Studied
be in a phenyl equatorial position has altered such hypotheses

(Portoghese, Mihail, Kupferber 1968). No conformational studies

of 4-phenyl piperidine compounds exist to further explore the

general question of similarities between morphine, meperidine,

and prodines in their low energy conformers.

Methadone as shown below (4) has a central carbon atom (C7) to

which are attached two phenyl rings, a COC2H5 carbonyl group and a

CH2CH(CH3) N(CH3) amine group This compound retains the feature

of a phenpropyl amine group in common with both morphine and

4-phenyl piperidines . The tertiary amine is acyclic. Moreover

methadone has 11 degrees of rotational freedom as shown above with

defining torsion atoms for each angle listed in table IV.

There is evidence that methadone also acts at the same receptor

site as morphine (Goodman, Gilman 1970; Smits, Takemori 1970).

Tests for analgesic activity in mice have shown an ED50 for

methadone approximately equal to morphine and five times stronger

than meperidine (Smits, Takemori 1970; Eddy, Halback, Braenden

1956). However, in intraventricular administration (Kutter, Herz,

Teschemacher 1970) the potency of methadone is 1/30 that of

morphine and only twice that of meperidine. These results are

consistent with its enhanced lipophilicity (Kutter, Herz, et al

1370) and a less efficacious mode of action at the receptor site

than morphine.

Speculation over the past 20 years on morphine-like conformers for

methadone has centered on intramolecular interaction, particularly

of the amine and carbonyl groups. In 1954, Gero proposed a

protonated confomer of methadone with a hydrogen bond between one

of the N-methyl groups and the oxygen presumably confining the

molecule in a rigid position and causing formation of

pseudorpiperidine ring (5).

Another structure for the protonated form has been proposed based

on intramolecular bonding between the nitrogen proton and the

295
Table IV. Minimum Energy Conformers of Cadidate Methadone Structures
oxygen leading to a seven membered “piperidine” ring with the

methyl group on the asymmetric carbon equatorial (6a) or axial

(6b) to the pseudopiperidine ring.

A methadone conformer with maximum atom for atom overlap with

morphine has also been proposed (Goldstein, Aronow, Kollman 1968).

However, no conclusive evidence supporting or refuting the existence

of any of the postulate conformers has been obtained (Henkel, Bell,

Portoghese 1974), and an x-ray crystallographic study of (+)

methadone hydrobromide showed no intramolecular hydrogen bonding

between the nitrogen proton and carbonyl oxygen (Hanson, Ahmed 1958)

or siginificant morphine overlap.

Thus, in spite of longstanding consideration of the problem, the

question of the most likely conformation of methadone at the

receptor site and its similarity to morphine remains open.

In this study, using the PCILO method, we have investigated the

energy conformational behavior of meperidine, desmethyl, α and ß

prodines, and methadone. The objectives of the study are: to

identify low energy conformers of each compound as candidates for

interaction at the receptor site; to correlate these conformers,

together with calculated net atomic charges, with observed analgesic

potencies; to compare these conformers with morphine; to understand

how and with what degree of overlap they can act at the morphine

receptor site.

For meperidine, desmethyl and ±α and ±ß prodine, a regularized

geometry based on the x-ray structure of the prodines (Kartha, Ahmed,

Barnes 1960: Ahmed, Barnes 1963) was used and nested rotations about

τ1(C3C4–C7C8 in figs. 5 and 6) and τ2 (C4C7–C13C15 in fig. 5 and

C4C7–O13C14 in fig. 6) were perfomed for phenyl axial and phenyl

equatorial positions. The ester chain was kept in the extended,

staggered conformation found in the crystal structure. Rotations of

the methyl group on C8 (C12) in the prodines were performed as needed

to identify minima. Table III summarizes the salient energyconformation behavior for the six compounds studied. Figures 5 and

6 show meperidine and desmethyl prodine in their minimum energy

conformers with calcualted net atomic charges. For comparison,

similar results for morphine obtained from a previous calculation

(Loew, Berkowitz, Weinstein 1974) are given in figure 7.

For methadone, with 11 degrees of rotational freedom, low energy

conformers could not feasibly be identified by systematic nested

rotations about each axis. Instead, a plausibility approach was

taken to find a candidate low energy conformer.

297
Figure 7. Minimum energy conformer of morphine from x-ray structure

with calculated net atomic charges.

©1975, the American Chemical Society2

Eight protonated structures Mere considered: the x-ray structure;

an extended chain structure to calibrate the effect of the proposed

intramolecular hydrogen bonding structure; internal hydrogen

bonding (NCH 2 H—O C) between a carbonyl group and a hydrogen

of the N—CH3 group as proposed by Gero (Gero 1954); structures

6a and 6b with internal hydrogen bonding (N — H --- 0 C )

between the two carbonyl groups and the proton of the nitrogen as

previously suggested; and three structures with an increased

similarity of methadone to morphine. Table IV summarizes the

results obtained from energy optimization of all of these structures

except the last (conformer 8), which was constructed to maximize

similarity to morphine instead. Energies are all expressed relative

to the minimum energy conformer.

For each compound shown in Table III the calculated low energy

conformers are consistent with the x-ray data, but our results show

additional low energy local minima in τ1 and τ2. In each case the

energy of the best phenyl equatorial conformer was significantly

lower than the best phenyl-axial one. These results, combined with

the fact that significant potencies have been measured in forced

phenyl-equatorial 4-phenyl piperidine compounds (Portoghese, Mikhail,

Kupferberg 1968), dispel the idea that a phenyl-axial conformation

per se is more efficacious at the receptor.

Since complete superposition of a phenyl-equatorial form of these

compounds on morphine is impossible, it is evident that they occupy

only partially overlapping receptor sites, which also agrees with

observed differences in some of the physiological effects of

meperidine and morphine (Portoghese 1966). The question remains as

to the actual orientation of the phenylpiperidine compounds at the

298
receptor. We have considered two likely orientations of these

compounds at the opiate receptor.

In one orientation (I) (figures 5 and 6) the piperidine ring of

these compounds is directly superimposed on the piperidine ring

of morphine, used as the prototype rigid opiate, implying a fixed

cationic receptor for both types of compounds. The phenyl ring is

then displaced to a position identical with that proposed for the

phenyl substituent of 5-phenylbenzomorphan (GPA 1657) (Clarke,

Hill et al. 1974). This displacement is reasonable without a

m-OH group in the phenyl ring corresponding to the crucial 3-OH

group in morphine. However, the presence of a m-OH group could

direct the phenyl group of 4φ-piperidines to the same receptor

site as in morphine. In this orientation (II) (figure 8) the

Figure 8. Pharmacophore II. Minimum energy conformer of prodine

with phenyl ring superimposed on that of morphine.

©1975, the American Chemical Society4

phenyl rings of the two types of compounds are assuned to make the

same receptor contact, displacing the piperidine rings. An

optimum correlation between observed potencies and calculated

properties is obtained using the first orientation which is the

more appropriate one without a m-OH group.

While meperidine and desmethyl prodine have similar low energy

conformers, the reversal of the ester chain places the polar

O C—O groups in different relative positions, which could

account for their 10-fold difference in apparent potency. In

desmethyl prodine (fig. 6) the O C—O group more closely

mimics the charge distribution in morphine. The highly negative

carbonyl oxygen (O15) is very close to the negative furan oxygen of

299
morphine (fig. 7); the positive carbonyl carbon (C14) is near

the positive C4 of morphine; and the negative ester oxygen (O13)

is nearly superimposed on the negative C12 of morphine. By

contrast, the reversal of the ester group to meperidine (fig. 5)

seems less advantageous. The polar O C—O group is superimposed on the relatively neutral part of the phenyl ring in

morphine far from either the negative furan oxygen, the positive

C3, or the phenolic oxygen, therefore possibly. accounting for

its lower potency.

The reasons for (+)α, (+)ß, desmethyl prodine potency differences

do not appear to be conformational, as all three compounds have

accessible energy minima at similar values of τ1 and τ2 (fig. 6)

and have the same electron distribution even on C8. An explanation

has been offered (Portoghese, Gomma, Larson 1973), however, that

a hydrophobic pocket is present in the receptor which preferentially

fits the methyl group in the axial position. Recent data have

been obtained (Portoghese, Mihail, Kupferberg 1968; Casy 1968;

Casy, McErlane, Jones 1973) on the potencies of the 3-allyl and

3-propyl prodine derivatives confirming this idea.

The differences in potency between the (+) and (-) isomers of

prodine may, however, be due to conformational differences since

both τ1 and τ2 have different values for low energy conformer of

the two diastereosiomers (table III, figs. 6 and 9). This torsion

angle difference in τ1 for the (+)/(-) isomers confirms previous

speculation based on x-ray structures (Portoghese, Larson 1973).

However, since the proposed phenyl binding site is different from

morphine, it is difficult to assess how the relative phenyl

position (τ1) affects potency. Nevertheless, the nitrogen is

7Å in front of the phenly ring plane in the (-) isomers, and is

.7Å behind the plane in the (+) isomers, as in morphine.

Most convincing evidence for the enhanced potency of the (+) isomers

comes from the results of the variation of τ2. The minimum energy.

conformers of the least potent isomers (-) have τ2 = 60°, causing

the ester chain to protrude into the original phenyl receptor site

on the same side as the piperidine ridge atoms (fig. 9, page 293),

possibly hindering receptor interaction with the phenyl, and piperidine

rings. A value of τ2 = 300° in the most potent (+) isomers (fig.

6) places the ester chain in a nonhindering position away from the

receptor (on the opposite side as the piperidine ridge atoms) but

still close enough to the phenyl receptor site to favorably

interact with the receptor. It is interesting to note that the

least potent isomer (-) is “locked” into the unfavorable value of

τ2 = 60° whereas (-), though favored at τ2 = 60°, has an accessible

energy minima at 300° as well. Thus on the basis of τ2 = 60°, has an accessible

predicted order of potency would be (+)α = (+)ß > (-)ß2> (-)α, in

good agreement with potencies.

From the results summarized in table IV, it is clear that morphine

like conformers of methadone (conformers 6, 7, 8) are high energy

forms and that methadone cannot occupy the identical receptor sites

as morphine.

300
The minimum energy conformer was one based on the hydrogen-bonded

(N—H---O—C) structures proposed by Portoghese. It was 11.6

kcal/mole more stable than the extended chain. Figure 10 shows this

minimum energy conformer of protonated methadone in an orientation

with its nitrogen superimposed on that of morphine, implying a fixed

cationic receptor site. Also shown in this figure are the net atomic

charges calculated for methadone. In this orientation the central

carbon atom (C7) superimposes on its counterpart in morphine and

meperidine. In fact, the minimum energy conformer of methadone

greatly resembles the minimum energy conformer for meperidine shown

in fig. 10 in the same orientation. The ester chain in meperidine and

the carbonyl chain of methadone could occupy similar positions at the

Figure 10. Minimum energy conformer of protonated methadone with

net atomic charges showen with a nitrogen atom superimposed on

that of morphine.

©1976, the American Chemical Society5

receptor site reinforcing the possibility that the receptor can,

to some extent, accommodate a polar group in this vicinity. The

displaced phenyl groups are also in similar positions in the two

compounds. The two fold increase in methadone potency over

meperidine in intraventricular studies could be attributable to

the interaction of the second phenyl group with a new receptor

site. This φ group is in the vicinity of the tertiary alcohol

substituents on C7 in the potent etorphine type rigid opiates.

Comparing net atomic charges for meperidine and methadone, the

cationic head atoms (nitrogen and the atoms binding to it) have

similar charges, as do the C O groups, consistent with the

idea that these two types of analgesics could act in a similar

fashion at the opiate receptor.

The net charges on the cationic head atoms are in fact, similar in

all three compounds: meperidine, methadone and morphine. Thus

these molecules could have the same delocalized interaction with

301
a diffuse anionic receptor site as has been previously proposed

for morphine. However, surprisingly, in methadone the proton on

the nitrogen points down towards the phenyl ring position in

morphine, a consequence of hydrogen bonding to the C O group,

while for meperidine and prodine with a true piperidine ring, the

proton is in a more morphine like position. Thus it appears

that a morphine like position of the nitrogen proton is not required

for activity, at least in flexible opiates where the phenyl

binding site to the receptor is apparently displaced.

B. PEPTIDE OPIATES

Two endogenous pentapeptides with morphine-like activity have been

isolated from mammalian brain (Hughes, Smith, Morgan 1975;

Simantov, Snyder 1976). These pentapeptides have been characterized

as methionine-enkephalin and leucine-enkephalin, and have the amino

acid sequence Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu,

respectively. Both pentapeptides mimic the actions of morphine in

the guinea pig ileum (Hughes, Smith, Morgan 1975; Simantov, Snyder

1976) and the mouse vas deferens assays (Morgan, Smith, Waterfield

1976), exhibit cross tolerance to morphine (Waterfield, Hughes

Kosterlitz 1976), and inhibit the stereospecific receptor binding

of 3H-naloxone in rat brain homogenates (Simatov; Snyder 1976).

In addition, intracerebroventricular injections of enkephalin can

produce transient analgesia (Jacquet, Marks 1976; Feldberg, Smyth

1977) which can be blocked by the potent opiate antagonist naloxone

(Beluzzi, et al 1976). These findings, plus the fact that enkephalin

appears to be localized in synaptosomal fractions that are rich in

nerve terminals (Simantov, Snowman, Snyder 1976) provide evidence that

the enkephalins may be involved in analgesia or pain suppression.

Since the enkephalins and rigid opiates appear to compete for the

same receptors, it would seem that there must be chemical and

conformational similarity between these two different classes of

opiates. The most striking feature common to both the enkephalins

and the rigid opiates is the presence of a p-OH phenethylamine

(tyramine) moiety formed by the terminal amino group and the tyrosine

residue. It has been established by structure-activity studies that

for potent opiate activity the tyrosine residue is critical and, in

common with rigid opiates, the amine and p-OH groups must remain

intact (Chang et al. 1976).

A number of conformational comparisons with rigid opiates have been

made by model building and comparing overlaps of different assumed

critical regions of Met-enkephalin and rigid opiates such as

7-[1-phenyl-3-hydroxybutyl-3-]endothenotetrahydrothebaine (PET)

(Bradbury, Smith,Snell 1976; Horn, Rodgers 1976; Smith, Griffin

1978). In addition, extensive conformational analyses of Metenkephalin have been made by means of empirical energy calculations

(Isogai, Nemethy, Scheraga 1977; Momany 1977). Of the fifty-two

conformations reported (Isogai, Nemethy, Scheraga 1977), the lowest

energy structures, i.e., those with ΔE<2.5 Kcals/mole were found to

be G-PßII' type bends with glycine3 and phenylalanine4 taken as

302
the central residues. Many other conformers were in the relative

energy range of 5<Δ E<11 Kcal/mole. The lowest energy structure

was found to be stabilized by a hydrogen bond between the tyrosine

OH group and the glycine backbone C=O group. This low energy

structure is consistent with chemical shifts and coupling constants

in reported NMR studies of Met-enkephalin (Garbray-Janeguiberry,

Roques, Oberlin 1976; Jones, Gibbons, Garsky 1976).

While valuable in describing numerous low and medium energy

conformers of Met-enkephlain, none of the optimized conformers

previously reported (Isogai, Nemethy, Scheraga 1977) have significant spatial overlap with several functional groups in morphine

like opiates. On the other hand the conformers proposed by

overlap with rigid opiates are based on model building with no

estimate of their relative energies. Deviations from minimum

energy conformations of the isolated molecule could occur by an

induced fit at the receptor site allowing enhanced resemblance to

rigid opiates, but with sane energy required.

In order to determine the most likely conformers for interaction

at the receptor site, we have made systematic energy conformational

studies by both empirical and quantum mechanical methods. The

aim was to determine the energy of met-enkephalin conformers with

varying degrees of similarity to rigid opiates and select as most

likely those conformers with modest energy expenditure and

significant overlap with rigid opiates. An addititonal requirement

in our selection strategy was the accommodation of D-ala2 but not

L-ala2 in place of the Gly2 residue. This second criterion is

consistent with the observation of the essential retention of

activity when Gly2 is replaced by D-ala2 (Walker, Bernston,

Sandman 1977) but a large decrease of receptor affinity upon

replacement by L-ala2 (Pert, Pert, Chang 1976).

Starting with the fifty-two low and medium energy conformers

previously obtained(Isogaii, Nemethy, Scheraga 1977), successive

imposition of these criteria allowed the selection of a small

number of low energy conformers as most likely candidates for

receptor site interaction. While the empirical and quantum mechanical energy calculations did not give identical results, a number

of likely candidates common to both methods were obtained. Both

methods also showed a trend towards higher energies for conformers

with increasing resemblance to rigid opiates.

The conformational energy calculations reported here were performed

a CDC 7600 using the Empirical Conformational Energy Program for

Peptides (ECEPP) (Momany et al 1975) and the Perturbative Configuration Interaction using Localized Orbitals (PCILO) (Diner,

Malrieu et al. 1969) method of quantum chemistry. Energy minimizations for ECEPP were carried out by a Quasi-Netwon method (Gill,

Murray, Pitfield 1972) which employs a gradient search. The

convergence criterion was 0.01 kcal/mole. Local minima were also

found by parabolic fits to sequentially varied sets of torsion

angles. In the PCILO method, local minima were found only by

parabolic fits.

303
Table V.6

Energy of enkephalin conformers with maximum overlap

with the rigid opiate PETa

Conformer ΔE

h Δ E

i

Maximum PET overlap (w1=0)b

Relaxed PET overlap (w1=0)b

Modeled GG II (w1=180°)b

Modified GG II (no optimization)B

Opitmized GG II (ψ1,x1x2 fixed)c

Optimized GG II (x1x2 fixed)d

Totally optimized GG I'(x1x2=-110,-130)e

Totally optimized GG II'(x1x2=-168-100)f

-107

135

628

119

25.5

17.3

13.3

6.5

613

2 5

---g

---g

14.4

9.7

10.7

5.4

a

PET = 7-[1-phenyl-3-hydroxybutyl-3-]endoethenotetrahydrothebaine

b

These conformers were chosen by similarity to PET and were not

optimized.

c

Optimized GG II keeping ψ1, x1 and x2 at values which mimiced PET.

d

Optimized GG II letting ψ1 relax.

e

Totally optimized GG bend which gave closest tyramine overlap.

f

Lowest energy GG bend. It has no similarity to PET.

g

Energy not calculated.

h

ΔE in Kcal/mole calculated by ECEPP method.

i

ΔE in Kcal/mole calculated by PCILO method.

304
The faster ECEPP method was used to obtain totally optimized

conformations and energies for each conformer considered. The

PCILO method was also used to calculate the energies of the totally

optimized conformations obtained from ECEPP calculations with

local optimization for important structural features such as the

tyramine p-OH phenethylamine overlap with morphine.

As shown in table V, a conformer constructed to have maximum overlap

with PET, and not energy optimized, gave extremely high energies by

both the ECEPP and PCILO methods. These high energies are due to

the crowding of the terminal nitrogen and hydrogen atoms with the

Gly2 carbonyl carbon and oxygen atoms. Relaxation of the Tyr1 and

Gly2 backbone angles to a local minima, while maintaining w1 = 0°,

decreased the energy significantly. Step-wise relaxation of

another starting structure with w angles 180° yielded a series of

relatively high energy structures by both methods.

In another approach it was assumed that a minimum viable overlap

of Met-enkephalin with rigid opiates should involve the tyramine

(NH 2 - Cα -Cß-φ-OH) moiety of both opiates. Imposition of this constrain on the fifty-two previously optimized Met-enkephalin

conformers, resulted in twenty-two with relative energy 9-20

kcal/mole above the lowest energy conformer without the imposed

overlap. These relative energies calculated by the PCILO method

ranged from 1.0 to 8.5 kcal/mole, implying many more accessible

cmformers. The there condition that D-ala2 be accommodated more

readily than L-ala2 in place of Gly2 reduced the number of viable

conformers from twenty-two to twelve. These twelve conformers were

reoptimized with the constraint of accommodating both the Gly2 and

D-ala2 residues and the tyramine overlap with morphine.

The results of this procedure were that the minimum energy conformers

calculated by both the ECEPP and PCILO methods did not have any

overlap with rigid opiates beyond the imposed tyramine overlap.

On the other hand, the lowest energy conformer with signigicant

additional rigid opiate overlap was the same by both methods: a

G-G ßII' bend with a hydrogen bond between the carbonyl group of

tyrosine and the amine group of phenylalanine. This is a particularly viable candidate for interaction at the receptor site since it is

only 3 kcal/mole above the minimum energy form as calculated by

the PCILO method.

Figure 11 shows the overlap of this structure with the potent

agonist PET. Not only do the important phenethylamine moieties

overlap, but the phenylalanine side chain overlaps with the phenethyl

C19 substituent of PET and the methionine backbone C=O group and

side chain are in the region of the C6 methoxy group of PET. This

conformer is very similar to the one previously proposed by model

building (Bradbury, Smyth, shell 1976) and is confirmed by these

studies as a very likely candidate at the receptor site.

Further dramatic verification that this type of enkephalin

conformer can interact with the opiate receptor is provided by a

just published x-ray crystal structure determination of

305

Leu-enkephalin (Smith, Griffin 1978). The structure reported is a

G-G ß bend stabilized by hydrogen bonding between the tyrosine and

phenylalanine residues; This structure is different from the

minimum energy conformer obtained by previous calculations and

from structures deduced from NMR studies in solution. It. is

consistent with the lowest energy confomer we have chosen by a

combination of energy calculations and a selection rationale based

on resemblance to rigid opiates and accomodation of D-ala. This

approach to conformational studies thus appears to be a powerful

tool for determining important opiate peptide receptor

interactions. An implication from this study is that enkephalin

binds to the receptor by an induced fit initiated at the crucial

tyramine region, followed by binding of the C-terminal residues.

This mode is similar to a proposed “zipper” mechanism (Burgen,

Roberts, Feeney 1975) or the concept of a dynamic pharmacophore

(Weinstein 1975) which our previous work (Loew, Berkowitz,

Weinstein 1975) indicated was appropriate even for rigid opiates.

C. SUMMARY

Three classes of flexible opiates have been studied: 4-phenyl piperdines, methadone and enkephalins. Our results show that low energy

conformers of the 4-phenyl piperidines have equatorial phenyl rings

and cannot completely overlap with rigid opiates at the receptor. A

combination of calculated conformational and electronic properties

could account for observed potency differences in meperidine, desmethyl, α+, α-, ß+ and ß- prodines.

Our results also indicate that both meperidine and its reverse ester

bind to the receptor in a similar mode with the φ ring in approximmately the same position as the phenyl substituent in 5-phenyl benzomorphans.

Conformers of methadone which maximally resemble morphine have very

high relative energies. The lowest energy conformer has a partial

H-bond between the NH and O=C groups. In this conformation methadone

resembles meperidine more than morphine. The electronic structure

of all three types of opiates indicate a similar cationic charge

distribution around the amine nitrogen and imply that their binding

to an anionic receptor site could be similar.

The determination of peptide opiate conformations present a challenge

of a different order of magnitude than the most flexible exogenous

opiates. Because of the extremely large number of possible

conformations, search strategies based on energy optimized conformations alone are not adequate to select plausible receptor site

candidates. Other criteria such as consistency with known structure

activity data and similarities to rigid opiates must be used. With

this rationale, we have predicted and characterized a low energy

conformer of Met-enkephalin and D-ala2 Met-enkephalin as a likely

candidate at the receptor site. With a modest energy input (ΔE 3

kcal/mole) significant overlap of this conformer with the potent

opiate PET was obtained. The tyrosine and phenyalanine side chains

307
and the terminal amine and carboxyl groups play crucial role in

this overlap. It is hoped that this calculation will help

establish a template for peptide opiate receptor interactions.

III. POTENTIALLY USEFUL AREAS OF QUANTUM CHEMICAL RESEARCH IN

OPIATE NARCOTICS

The results reported here have established the reliability of

semiempirical quantum chemical methods for conformational studies

of both flexible and rigid opiates. Crystal structure conformers

are always obtained as low energy local or global minima. More

subtle conformations effects such as intramolecular hydrogen

bonding and conformational mixtures determined by NMR spectra have

also been verified.

Quantum chemical calculations supply additional information not

readily accessible to experimental techniques relevant to pharmacological behavior. These include: characterization of compounds

not yet synthesized; characterization of separate diastereoisomers;

and characterization of a large number of stable conformers within

a range of energies, many of which are beyond detection in the

isolated molecule but which could be viable candidates for induced

fit at a receptor site.

These studies can be made relatively efficiently and can serve as

a guide in many ways to more costly and time consuming synthesis

and testing. Thus one area of productive use of theory is

in predictive structural-activity efforts, ideally in collaboration

with medicinal chemists and pharmacologists. Such calculations

can also be used to compare different classes of opiates for region

of similarities and differences. Thus they can be of use in

delineating crucial regions of the opiate receptor common to many

classes of opiates and those unique to specific ones.

Future effort is thus envisioned in two related areas: predictive

medicinal chemistry and opiate-receptor theory. Specifically,

we plan to use the working hypothesis we have developed for rigid

opiates which relate their structure to their agonist/antagonist

potency ratios to continue to predickt potentially useful rigid

opiate analogues. We also plan to extend this effort to unusual

classes of flexible exogenous opiates such as ketobemidone type

4-phenyl piperidines and to peptide opiates. Our central goal

is to understand requirements for agonism/antagonism in all these

series of compounds and to compare these requirements among

members of different classes of opiates. If successful this effort

should lead to prediction of other potentially useful analgesics

and also help to understand the nature and degree of flexibility

of the opiate receptor.

308
FOOTNOTES

1. From Loew, G.H., Berkowitz, D.S., Weinstein, H., and Srebrenik, S.

Quantum chemical studies of morphine-like opiate narcotics: Effect of

polar group variations. In: Molecular and Quantum Pharmacology,

Bergmann, E., and Pullman, B., eds. Copyright 1975. Reprinted by permission of D. Reidel Publishing Company, Dordrecht-Holland.

2. Reprinted with permission, from Loew, G.H., and Berkowitz, D.S.

Quantum chemical studies of morphine-like opiate narcotic analgesics:

I. effect of N-substituent variations. J Med Chem, 18(7):656-662,

1975. Copyright by the American Chemical Society.

3. Reprinted with permission, from Loew, G.H., and Berkowitz, D.S.

Quantum chemical studies of N-substituent variation in the oxymorphone

series of opiate narcotics. J Med Chem, 21(1):104-106, 1978. Copyright

by the American Chemical Society

4. Reprinted with permission, from Loew, G.H., and Jester, J.R.

Quantum chemical studies of meperidine and prodine. J Med Chem,

18(11):1051-1056, 1975. Copyright by the American Chemical Society.

5. Reprinted with permission, from Loew, G.H., Berkowitz, D.S., and

Neuth, C. Quantum chemical studies of methadone. J Med Chem, 19(7):

863-869, 1976. Copyright by the American Chemical Society.

6. Reprinted from Loew, G.H., and Burt, S.K. An energy conformation

study of Metenkephalin and its D-ala2 analogue and their resemblance

to rigid opiates. Proc Natl Acad Sci USA, 75(11):7-11, 1978.

ACKNOWLEDGMENTS

The authors gratefully acknowledge support of this work from the

National Institute of Drug Abuse Grant No. DA-00880-01, -02. They

also wish to thank their colleagues Drs. R. M. Featherstone,

E. Leong Way, and Horace Loh of the Department of Pharmacology,

University of San Francisco, and Dr. William Forrest at Standord

for early support and helpful discussions. Helpful discussions

with Dr. Vincent Dole at Rockefeller University, Drs. J. DeGraw, J.

Lawson and H. Johnson at SRI International, and Dr. R. H. Willette

of NIDA are also gratefully acknowledged.

309
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AUTHORS

Gilda H. Loew, Ph.D.

Donald S. Berkowitz

Stanley K. Burt, Ph.D.

Department of Genetics

Stanford University Medical Center

Stanford, California 94305

316
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Wilette, eds.

National Institute on Drug Abuse, 1978

An Extended Isolated Molecule

Method and Its Applications to the

Design of New Drugs: General

Aspects

Alfredo M. Simas, Roy E. Bruns, and

Richard E. Brown

INTRODUCTION

The aim of the isolated molecule method is the study of chemical

reactions in which the attacking species is always the same (Greenwood and McWeeny 1966). As the isolated molecule method has been

successfully used in the study of chemical reactivities for various

types of chemical reactions, these reactivities are then considered

to depend only on the differences in the substrates. Also the

isolated molecule method is convenient for the study of biological

activities (BA) of drugs, because here only the substrates are

known. The receptor or any other interactive species which might

be involved are subject to speculation.

In a previous work (Brown and Simas 1978), many of the π indices

used in the isolated molecule method were justified for the more

advanced semiempirical methods where all of the valence electrons,

antisymmetry, and interelectronic repulsions were included. This

allows a more credible study of nonplanar and heteropolar systems.

The objective of this paper is to extend such an advanced isolated

molecule method to the study of Quantitative Structure Activity

Relationships (QSAR) of drugs.

Approaches to QSAR, such as the isolated molecule method, have

rarely been employed. Many of the studies which have been reported

in the literature have used only π electron approaches focussing

on the calculation of electronic charge distributions (Brownlee

and Taft 1968; Kang and Beveridge 1971; Kang and Cho 1973). Two

works, however, are of special importance. The first is a paper

by Wohl where the extended Hückel method was used to calculate

the indices for the π system of a family of benzothiadiazines

(Wohl 1971). Using a similar approach, which also yielded good

results, Peradejordi studied the tetracycline family of drugs

(Peradejordi 1971).

317
Many criticisms have appeared against this kind of approach including the meaningfulness of the indices employed, the exaggerated

importance given to the statistical analysis, the nonexistence of a

solid theoretical foundation to the BA phenomena, the necessity of

utilizing good experimental data for correlating the MO indices,

etc. However, the theoretical calculations possess two great advantages over the purely experimental correlations. First, these

calculations provide information about submolecular properties

such as charge distributions and polarizabilities in the atoms.

These properties are of obvious importance in understanding the

differing chemical aspects in a series of substrates. Second, and

probably more important, these calculations provide a reasonably

objective way to predict the properties of a drug before it has

been synthesized.

Here a conceptual set of hypotheses which can extend the applicability of the all valence isolated molecule method to the study

of BA is presented and discussed. In an extension of our previous

work (Brown and Simas 1978), a new rationalization of the superdelocalizabilities is also presented. In a subsequent paper, this

methodology, including these newly derived indices, will be applied

to the chloramphenicol family of drugs (Simas et al. 1978).

BASIC APPROXIMATIONS

The objective of the isolated molecule approach is to determine

the differences in the free energies of activation of the reactions associated with the various compounds of a homologous

series from the initial steps of the reactions. Suppose that a

set of compounds belonging to a homologous series are subjected

to one type of reaction. The rate constant for the ith compound,

ki, is given by

where K, k, h, T and R are the transmission coefficient, the

Boltzmann constant, Planck's constant, the absolute temperature and

the ideal gas constant (Glasstone, Laidler and Eyring 1941). The

molar free energy of activation, , can be expressed in terms

of the entropy and the energy of activation in the usual manner.

The free energy has the important property of being proportional

to the logarithm of the rate constant, and is the unique quantity

which varies largely from one reaction to another.

With a homologous series it is always possible to make the initial

free energies of the substrates equal, such as in figure 1. The

free energy profile can be, at least in principle, approximated by

a straight line. Its slope is then proportional to the free energy

of activation:

(2)

318
where the proportionality constant Δλ represents the change in the

reaction coordinate from λ0, the states of the reagents, to λ

the transition state. Further, let us consider that the free energy

profiles do not cross from the state of the reagents to the transition state, and that AA is constant through the series, which

implies that the mechanisms of the reactions are always the same

for the homologous series. In this way it is always possible to

find a λ' between λ0 and λ where the derivatives of the

free energy with respect to the reaction coordinate can be described

by a linear transformation

(3)

Equation 3 yields good estimates for the free energies of activation

of the reactions. In this equation, a and b are constant through

the series.

The above relation allows the study of processes which are kinetically controlled. In order to study equilibrium controlled processes, one can assume that there exists a relationship of the type

expressed in equation 3 involving ΔA

0

, the standard free energy of

reaction, instead of ΔA Reactions where the free energy curves

are approximately parallel past the transition state could be common

for a homologous series, and we assume this here. This is particularly true where the product resembles somewhat the transition

state. For both of these processes, a compensating effect appears.

The derivative of the free energy at the beginning of the reaction,

at λ 0, where one finds a minimum in the reaction profile, must be

zero. For this point, considering the relations between A, E and

S, the equation

(4)

is valid for both the equilibrium and kinetically controlled reactions. For this reason an index related to a first order perturbation does not always correlate well with the reactivities of the

compounds, whereas a second order One does (e.g., induced charge and

self atom polarizability). The change in energy associated with the

first order index was completely absorbed by the change in entropy

in the initial steps of the reaction. This implies another approximation; the second order changes in entropy will be very similar for

all reactions since the attacking species, solvent, etc., are always

the same.

Finally, with these hypotheses, equation 3 can be rewritten by substituting the derivative of E for the derivative of A to obtain

(5)

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As attempts will be made to find indices which are proportional to

the changes in energy at the beginning of the reactions, one must

also consider that each index will be related to the same displacement in the reaction coordinate. Generalizing equation 5, one has

(6)

where Xi is the value of index X for compound i.

Although some large assumptions have been made up to this point, it

is important to note that this isolated molecule method is consistent with the Hammett treatment, where the constant of reaction,

is constant for a homologous series and the substituent constant

σi accounts for the differences in the reactivity.

INDICES FOR THE ALL-VALENCE ISOLATED MOLECULE METHOD

As a first step in the perturbation procedure, consider only the

electrostatic interaction of the attacking species with the substrate. The electrostatic field and its potential have been derived

using the point charge approximation as discussed elsewhere (Brown

and Simas 1978). The coulombic interaction energy between the

electrostatic potential Vs of the substrate and the charge distribution YA of the attacking species is

(7)

(8)

where ra and Qa are the position and net charge of atom a of

substrate. For points distant from the substrate, the point

charge approximation is adequate.

An advantage of studying the electric field around the substrate is

that reasonably far from the molecule, this field can be considered

to be relatively constant in a small region of space. If the

attacking species has a permanent dipole moment, , its interaction with the electric field may be written as

(9)

(10)

In this way the electric field can be decomposed into its components

and the differing correlations of the components of with the reactivities provide information about the direction of attack.

320
Equations 7 through 10 illustrate how the potential, field components

and net atomic charges are proportional to the energy of interaction.

The gross environmental effects on the drug molecule can be considered, in a first approximation, as the interaction of an electric

field with the substrate. The interaction energy can then be

written as:

(11)

where is the dipole moment of the substrate and αS is its

polarizability tensor. The derivations and a discussion concerning

the applicabilities of these indices have been reported (Brown,

Simas and Bruns 1977; Brown and Simas 1978).

Now consider the attacking molcule as a charged or polar species

which can produce some perturbation to the substrate molecule. We

treat this as an empirical perturbation to only one atom where the

energies of the atomic orbitals on atom v are modified by the

amount δαμ. For advanced semiempirical methods such as the CNDO

method, it was previously discussed and derived that the modification to the electronic density at atom v and the energy of

interaction are given respectively by (Brown and Simas 1978),

(12)

(13)

The quantity Πvμ is the atom-atom polarizability, Πμμ is the selfatom polarizability and q μ is the electronic density at atom μ.

Obviously both qμ and Πμμ are proportional to the energy of

interaction. As MO indices, they have proven useful in parameterizing reactivities.

If the attacking species approaches the substrate more closely, it

must not be described simply as a distant point charge or polar

species, but as an atom or molecule possessing electronic energy

levels. Bond formation through exchange or charge transfer must be

considered. Suppose that we have two species, the attacking

reagent T and the substrate S, and that each satisfies the eigenvalue equations,

(14)

(15)

these zeroth order wave functions ψ

(0)A are the usual normalized

determinantal functions.

321
We have similar expressions for the excited state functions

and where an electron is promoted from the occupied MO i of

S or j of T to the unoccupied MO s and t respectively.

These excited state functions are defined as

(16)

The MO with bars denote those with ß spin, and the two singly

occupied MO are singlet coupled. The MO are expanded in terms

of the atomic orbitals

(17)

where NA is the number of atoms in the molecule, and Nμ is the

number of valence AO's for the μth atom. Such excited state

functions as described in equation 16 are approximate representations of the excited states of S and T with energies approximately εs-ε i and εt-εj respectively above that of their

ground state functions. Suppose that upon approach of the attacking

species, there is an interaction between atom σ of S and atom

τ of T and that every atomic orbital of atom σ can interact

with every atomic orbital of atom t with the same energy of interaction, δßστ. Then the empirical perturbation can be written as,

(18)

where σ, λ, τ and φ are atoms, δ is the Kronecker delta and

δ ßστ is the change in the one electron resonance integral due to

the interaction between σ and τ. Note that the resonance

integral was zero prior to the reaction. The and are one

of several atomic orbitals on the respective atoms.

Now the appropriate zeroth order functions for the total complex,

S-T. must be constructed which represent the eigenfunctions of the

total zeroth order Hamiltonian, These are the

antisymmetrized and normalized products of the wavefunctions for S

and T which we represent as: and

Multiple promotions do not contribute in first and

second order since the perturbation is a one electron operator. We

will also include the charge transfer functions where an electron

in MO i on S or MO j on T is transferred to MO t on T

or s on S respectively. The resulting singly occupied orbitals

are then singlet coupled in the same manner as in equation 16. We

322
represent these charge transfer functions as and

We include their effect in the same perturbational

scheme as the other zeroth order functions described above. In

all cases above, we use the difference of the initial and final

orbital energies to approximate the difference in the energies

of the zerothorder functions.

Since such an empirical perturbationis taken as nonzero only

between atoms on different molecules, the first order correction

is zero. That is, all integrals involve MO localized on either

S or T only.

The second order correction to the energy is given as:

(19)

(20)

The other zeroth order functions involving promotions localized on

only S or T do not contribute for the same reason; the perturbation is nonzero only between atoms σ and τ on the two

molecules. Upon substituting in the expression for the zeroth

order functions and the MO and then evaluating the integrals, we

obtain the simple result,

(21)

Here the energy levels, both the occupied and unoccupied, of S and

T are represented by s and t respectively, with ns and nt

being the number of electrons in the s and t levels.

Now it is of interest to derive an index which depends only on the

characteristics of the substrate, such as the one used in the

Hammett equation. Suppose that the attacking species possesses

just one level which interacts strongly with the substrate. If the

attack is electrophilic, the level t is empty and will interact

only with occupied levels of S. An electrophilic interaction

energy can be defined as

(22)

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The coefficient of the atom τ factors out because it has only one

normalized level. If the attack is nucleophilic, the analogous

interaction energy is

(23)

It is now possible to define the superdelocalizabilities which are

assumed to be proportional to the interaction energy between S

and T but independent of T as described in the equations as

follows

(24)

and

(25)

Here and are the electrophilic and nucleophilic superdelocalizabilities of atom σ in S. The quantities and

are considered to be constants of reaction in the same spirit

as found in the Hammett equation. The denominators of equations 22

and 23 prevents such a separation. However, assuming the Hammett

equation valid, the superdelocalizabilities can be defined as

(26)

and

(27)

It is obvious that such a separation is arbitrary. It is based on

the validity of experimental relations, such as the Hammett equations. Attempting to expand the denominators in equation 22 or 23

in terms of the ratios εt/ε i or εt

/ε a respectively, gives a

convincing argument only when the ratio is less than unity in

magnited. (It also indicates that perhaps higher order terms in

1/ε i and 1/ε a should be included in any correlations.) For

reactions where we are proposing weak bond formation, i.e., only a

partial transfer of charge, this is not uncommon since the energy

level εt may lie close to the zero level in either case. However,

for the general case, such an argument is pure speculation. These

new formulae are only slightly different from those used previously

(Brown and Simas 1978) to successfully parameterize the chemical

reactivities of several homologous series of compounds.

324
If the molecule being studied has a plane of symmetry which contains atoms forming unsaturated bonds, the total wave function of

the molecule satisfies the σ-Π separability conditions of Parr

(Parr 1963). It can then be written as an antisymmetrized

product of the antisymmetrized functions {Σ} and {Π} which

contain MO of only σ and Π symmetry respectively.

(28)

The total electronic energy can then be divided into a contribution

from the {Π} function and another from the {Σ} function. If

the plane of symmetry is conserved during thecourse of the reaction, the transition state electronic energy can also be divided

into σ and π contributions. Indices calculated using only

the {Π}. functions might more accurately reflect the trends in

reactivity. But these arguments are only valid for substitution

reactions in which the attacking species is chemically equivalent

to the leaving species and the plane of symmetry is conserved

during the reaction. However, if the principle established in the

Hammett equation is considered to be valid, the indices which can

describe this reaction must also describe others in which the

attacking species is not chemically equivalent to the leaving

species and vice-versa. All indices calculated using the MO of

only one symmetry are indicated with the appropriate superscript,

e.g., and .

One can also use frontier indices to estimate trends in reactivity.

The frontier electron treatment (Greenwood and McWeeny 1966) has

been very successful in describing satisfactorily not only the

aromatic hydrocarbon reactivities but also the stereoselectivity

of reactions and the mechanisms of transition metal catalysis. To

use a frontier treatment one can compute the indices previously

described using only the HOMO (highest occupied molecular orbital)

and the LUMO (lowest unoccupied molecular orbital). However, there

is a fundamental conceptual difficulty associated with frontier

treatments. Frontier indices, like molecular orbitals, are not

observables andthereareno fundamental reasons to assume that

relationships exist between them and observable quantities, such

as biological activities. As such, the interpretation of frontier

indices must involve a high degree of caution.

SOME SIMPLE APPLICATIONS

Two of these MO indices, the polarizabilities and superdelocalizabilities, have been extended from the simple Hückel theory to

the more advanced CNDO method and should be tested on simple

systems to verify their effectiveness in parameterizing chemical

reactivities. Since the CNDO method includes all the valence

electrons and is parameterized for heteropolar molecules, all of

these indices should be applicable to the study of reactivities of

both cyclic and exocyclic sites in either polar or nonpolar systems.

325
Four simple homologous series were studied to verify the effectiveness of these indices. These were the aromatic hydrocarbons, the

substituted benzene compounds, the substituted benzoic acids, and

the substituted phenyl amines. Since the details of these calculations can be found elsewhere (Brown and Simas 1978) we present

here only a short description of these results. The MO indices

described above gave acceptable correlations with the chemical

reactivities as measured by the appropriate Hamett-like parameter.

For example, correlations against the Hamett σr parameter for the

14 unique reactive sites in the first group which includes benzene,

biphenyl, napthalene, anthracene and phenanthrene gave results

very similar to those of the Hückel indices. Since the CNDO method

was parameterized for the more general case, this is an acceptable

result. For the other three series, the results were as good or

better. This is particularly significant in that these other series

are not only polar, but in the case of the benzoic acids and

phenyl amines, have the reactive site off of the aromatic ring.

For all of these cases, the best indices were the superdelocalizabilities which showed good correlations for both perturbationally

and electrostatically controlled reactions. It is also significant that the frontier indices gave poor results, which only confirms

the suspicion that one should exercise caution in the use of

frontier indices. Only with the aromtatic hydrocarbons did the πcomponents of the indices show good correlations with the reactivities, however the total indices gave comparable results. The selfatom polarizabilities were good parameters in addition to the

electrophilic polarizability. For the other three series, all of

which are polar, some potential field and group charge indices

produced correlations nearly as good as those of the electrophilic

superdelocalizabilities.

In general, correlation coefficients between 0.86 and 0.90 were

obtained with the best indices used in these studies. The number

of data points used in these regressions were 14, 25, 19 and 19

respectively for the aromatic hydrocarbons, the substituted

benzenes, the benzoic acids and the phenyl amines. These results

engender confidence in the application of these CNDO indices to

the study of more complicated problems such as QSAR of drugs.

AN EXTENSION OF THE ALL-VALENCE ISOLATED METHOD TO THE

STUDY OF BIOLOGICAL ACTIVITIES OF DRUGS

A model which can satisfactorily describe the biological activities of drugs, based on quantum theory, must have the following

important characteristics:

(1) As BA are observable quantities, the model must be based

also on observables, such as the dipole moment, electric fields

around the molecules, etc., and

(2) the parameters or the indices used must provide a consistent set of physico-chemical quantities, useful in the interpretation of the results.

326
As pointed out by Lin (Lin 1974), the drug molecule may follow a

very complicated path, passing through a great number of stages

including dissolution, absorption, distribution, penetration,

binding, metabolism and excretion in order to give a particular

response. It is clear that the success of the drug as a whole is

related to its behaviour in all these stages (if they can really

be distinguished from each other).

Now, consider the molecule in one general stage of the pharmacological process. The system may be divided into two subsystems;

the drug molecule and the biological environment which contains

all the biological complexes which surround the drug. The central

idea is that the success of the drug in each step will depend on

its recognition by the environment. This recognition will be

associated with a measuring process where the measuring apparatus

is the biological environment and what is being measured is the

drug molecule. In this way the drug molecule can be seen as a

collection of observables. During the steps, the various environments will recognize the drug with the degree of success depending

on the expectation values of the observables. The metabolism is

assumed to occur only after all the previous steps, including the

interaction with receptor, have been completed.

So, the set of the observable expectation values will determine

unequivocally the drug's biological activity. There are two

problems here: the lack of knowledge of the biological environmentand the large quantity of observables pertaining to the drug

molecule, many of which mimic each other. Therefore in the application of this method, it will be necessary to know the BA of some

compounds of the homologous series of drugs, and to have a guarantee

that the global mechanism of action is identical for all cogeners.

Only observables related in some way to the interaction energies

between the molecule and the environment will be considered. These

are the indices previously described.

There is one additional problem. For which geometrical conformation will the indices be calculated? To solve this we will use

the Kier hypothesis of "Remote Recognition of the Preferred Conformation" (RRPC) (Kier 1973, 1975). This hypothesis states that

there is always a significant amount of binding between the two

molecules when they approach each other from infinity, yet both

initially retain their preferred isolated conformations. Also,

this binding is almost always greater than kT (where T is

about 310°k). Moreover, their change in conformation only has

significance when they are very near each other.

First consider the drug molecule inside a cavity of infinite

radius whose surface reflects the biological environment. Letting

the system relax it will progress from λ0 (the initial state) to

λ R (the state which really occurs) with the corresponding changes

in the biological environment and the geometry of the substrate.

Supposing that for the interval including λ0 and λ', Kier's

hypothesis is valid and that the reaction profiles for every compound of the homologous series do not cross, then all the concepts

of the isolated molecule method can be applied.

327
To be consistent with the indices considered here, the BA must be

expressed as a quantity which can be related to the free energy

such as the logarithm of rate constants or equilibrium constants.

A step of the biological process, for the purposes of this method,

can be defined operationally as a stage which can be explained by

only one index or an expansion of indices which represent just one

perturbation. Suppose that the ith step can be explained by a set

Of indices Xiμ calculated in the manner pointed out previously

for each compound μ of the homologous series. Following the

isolated molecule method, a linear transformation of the index

will give a good estimate of the free energy of interaction of the

μth compound, ΔA i μ

(29)

We now define rμ as the BA of the μth compound, which is proportional to the free energy, ΔAμ , being considered from the

administration of the drug until the interaction with the receptor

and subsequent metabolism. ΔAμ can be written as a summation of

the free energies involved in each step i, ΔAiμ , plus the interaction free energy between the various steps, ΔAi ,j u

(30)

Many steps will possess the same free energy for all the homologous

compounds with the exception of a small number of them represented

by ΔAkμ which will differentiate the compounds. Neglecting the

free energies of interaction between the steps, one can write:

(31)

(32)

Applying equation 29 in equation 31 one has,

This is the basic equation of the extended isolated molecule method.

CHANGES OF MECHANISM THROUGH THE HOMOLOGOUS SERIES

Eventually, some subset of compounds of the homologous series can

have their BAs well explained by a small number of indices. If

the BA of the remaining compounds are not well explained by those

indices, this implies that the mechanism of BA has changed, or that

the steps which are rate determining for one subset are different

for the other subset. This implies the crossing of the reaction

328
profiles for these compounds. In many cases, this is caused by

steric effects of the substituents. In the domain of the extended

isolated molecule method, recognition of this change of mechanism

is possible. Also attempts to establish rules allowing the differentiation of those compounds belonging to this or that subset

can be made.

CONCLUSION

From the initial set of hypotheses established here, it is not

possible to deduce which of the more highly correlated indices is

related with one or more particular stages of drug action. As a

result all of these stages are taken into account. The more

highly correlated indices reflect the importance of strategic

positions in the molecule, or which of its global properties,

such as the dipole moment, are significant. Of course, some

indices will be more closely related to one stage than to others,

but in the domain of the extended isolated molecule method, it

is not possible to make this discrimination. The model can only

reflect the complete biological process. On the other hand, this

is the model’s great advantage, as the particular stages such as

solvent effects cannot be considered explicitly.

It is important to note that all the experimental parameters used

in free energy relationships (such as the Hansch analysis), are

compatible with equation 32 and can be employed together with the

theoretical indices. This outlook is very promising for the

method since a complete set of indices (a set which can span

every perturbation in a molecule) is difficult to obtain. However, looking at the mixed equations, one must be careful because

the physical models involved in the two sets of parameters are

different, although they can complement one another.

One additional point must be made. The free energy relationships

such as in the Hansch analysis, the Free-Wilson analysis, etc.,

explain in principle only those parts of the BA that are equilibrium controlled. The extended isolated molecule method can

treat not only these, but also the kinetically controlled processes.

In this sense it is, at least in principle, more powerful.

The extended isolated molecule method has been applied to the

chloramphenicol family of drugs to clarify the mechanism of the

drug potency and to predict new active drugs in this family. We

comment briefly on these results. The chloramphenicol drug as

illustrated here is an effective antibiotic, particularly for the

treatment of typhoid fever. The parent chloramphenicol has

X= p-NO2 and R = CHCl2. None of the 40 odd cogenors which have

been synthesized have proven to be more active. The propanediol

moiety, the phenyl group and the acylamino side chain are critical

to the drug potency. However, substitutions can be made for the

p-nitro group and the dichloromethyl group without destroying the

biological activity.

329
Statistically significant correlations have been obtained for the

X-series for X = p-NO2, CN, OCH3, Cl, H and NH2. The single variable regressions are given below where the biological activities

are fitted to equation 32.

R denotes the correlation coefficient and SNC the superdelocalizability of the benzyl carbon. and are the orthogonal

component of the electric field and the coulombic potential at a

point 4.5A below the center of the benzene ring. These parameter

do not strengthen the argument that the mechanism involves the

formation of a radical by the loss of the α-hydrogen atom. An

electrostatic mechanism agrees with these results. The potential

field below the benzyl carbon and α-hydrogen increases with the

drug potency for a positive charge which indicates that an attack

by a nucleophile them is indeed probable. The high correlation

of SNC agrees with this. Electron withdrawal by the X-group is

also important since Qx has a high correlation coefficient.

330
However, α-deutero-chloramphenicol experiments show a 20% drop in

activity. In all, these results indicate that the most probable

mechanism involves a loss of the α-hydrogen as a proton rather

than a radical. The importance of indicates that an accompanying interaction between the benzene ring and the receptor

is also likely. The details of this work as well as further

applications to the prediction of new drugs can be found elsewhere (Brown, Simas and Bruns 1977; Simas et al.1978).

These results give only a glimpse of the way in which this method

can be used. It can be seen that the isolated molecule method,

as translated to an all valence formalism and extended to the

study of the BA of drugs can be used in clarifying the mechanism

of drug action as well as in predicting the activities of new

drugs. This method represents a potentially useful combination of

experimental and theoretical techniques.

ACKNOWLEDGMENTS

Partial financial support from FINEP, the Financiadora de Estudios

e Projetos, and FAPESP, the Fundacão de Amparo à Pesquisa do

Estado de São Paulo, is gratefully acknowledged. One of the

authors (AMS) is indebtedto CNPq, the Conselho Nacional de

Pesquisas, for the grant of a graduate fellowship.

REFERENCES

Brown, R.E., Simas, A.M. and Bruns, R.E. An MO study of the

chloramphenicol family of drugs: a preliminary report. Int

Quantum Chem. Quantum Biology Symposium 4: 357-362, 1977

Brown, R.E. and Simas, A.M. On the applicability of CNDO indices

for the prediction of chemical reactivity. Submitted for publication, 1978.

Brownlee, R.T.C. and Taft, R.W. Concerning the σ

0

 scale of

substituent Π delocalization parameters. Theory and experiment.

J Am Chem Soc, 90: 6537-6539, 1968.

Dewar, M.J.S. The Molecular Orbital Theory of Organic Chemistry.

New York: McGraw Hill, 1969.

Glasstone, S., Laidler, K.J. and Eyring, H. The Theory of Rate

Processes. New York: McGraw Hill, 1941.

Greenwood, H.H. and McWeeny, R. Reactivity indices in conjugated

molecules: the present position. Adv Phys Org Chem. New York:

Academic Press, 1966, Vol 4, Chap 3.

Kang, S. and Beveridge, D.L. Molecular orbital aspects of substituent effects. Theoret Chim Acta, 22: 312-314, 1971.

331
Kang, S. and Cho, M.H. Molecular orbital aspects of substituent

effects II. Int J Quant Chem, Symp. 7: 3193-27, 1973.

Kier, L.B. The prediction of molecular conformation as a biologically significant property. Pure Appl Chem, 35: 509-520, 1973.

Kier, L.B. A stochastic model of the remote recognition of

preferred conformation in a drug receptor interaction. J Theoret

Biol, 49: 401-416, 1975.

Lin, T.K. Quantum statistical calculation for correlation of

biological activity and chemical structure I: drug receptor

interactions J Med Chem, 17: 151-154, 1974.

Parr,R.G. The Quantum Theory of Molecular Electronic Structure.

New York: W.A. Benjamin Inc., 1963.

Peradejordi, F. Aspects de la Chimie Quantique Contemporaine,

Paris: CNRS, 1971: pp 261.

Simas, A.M., Bruns, R.E., Dunn, W. and Brown, R.E. An extended

isolated molecule method and its applications to the design of

new drugs: the chloramphenicol family of drugs. Submitted for

publication, 1978.

Wohl, A.J. A molecular orbital approach to quantitative drug

design. In: Ariens, E.J., ed., Drug Design. New York: Academic

Press, 1971. chap. 4. pp. 382-404.

AUTHORS

Alfredo M. Simas*

Roy E. Bruns

Richard E. Brown**

Instituto de Quimica

Universidade Estadual de Campinas

Campinas, S.P., BRASIL

*Present Address: Department of Chemistry

Queen's University

Kingston, Ontario, CANADA

**Present Address: University Computing Services

State University of New York/Buffalo

Amherst, New York 14226

332
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic. and R. Willette, eds.

National lnstitute on Drug Abuse, 1978

Recognition and Activation

Mechanisms on the LSD/Serotonin

Receptor: The Molecular Basis of

Structure Activity Relationships

Harel Weinstein, Jack Peter Green, Roman

Osman, and W. Daniel Edwards

Most studies on the relation between the molecular structure of drugs

and their biological effects are aimed at deducing what structural

characteristics of the drug are recognized by the receptor and what

characteristics evoke the tissue response - all based on the assumption that the interaction between drugs and receptors can be described

in the same terms as those used for chemical interactions and reactions. Early work - and to a great extent, work beiug done today -

focussed almost exclusively on functional groups such as a hydroxyl

group, a phenyl ring, a tertiary mine, and on the geometrical and

stereometrical relationships among them. Implicit in such an approach

is the tacit assumption that the functional group necessarily reacts

directly with the receptor, and that its role is independent of its

effect on the physical and chemical properties of the entire molecule.

A pertinent example is the conclusion that because 5-hydroxytryptamine

(5-HT) is more potent than other tryptamine derivatives, the hydroxyl

group at the C5 position interacts directly with the receptor. In

fact the hydroxyl substitution markedly alters the electronic properties and thereby the reactivity pattern of the molecule so that, for

example, the magnetic circular dichroism spectrum of 5-HT in solution

is markedly different from that of tryptamine (Sprinkel et al. 1975)

and the self association of 5-HT in solution is very different from

that of 6-hydroxytryptamine (6-HT) (Sapper and Lohman 1976). It

became evident, even from very early theoretical structure-activity

studies that the differences in potency in the tryptamine series are

attributable to changes in the electronic structure of the entire

indole ring (Green and Kang 1970; Kang and Green 1970; Johnson and

Green 1974). This level of understanding of the molecular determinants for drug activity is closely related to the development of new

physicochemical and theoretical methods of analysis that provide the

proper tools for the study of molecular mechanisms.

The classical theoretical method from which a quantitative measure is

obtained in relating molecular properties to biological activity is

the "quantitative structure activity relationship" (QSAR) procedure

pioneered by Hansch (for reviews see Hansch 1971; Van Valkenberg

1972). The basic assumption underlying this approach is that the

pharmacological activity is a numerically quantifiable function

of several mutually independent parameters which are themselves

333
dependent on the molecular structure. For example, experimentally

measurable partition coefficients between model media (e.g.,

oil:water) are considered to be dependent on substituents on the

molecule, and they determine the amount of drug that reaches the

receptor site (the π coefficient). Other parameters considered to

affect the biological response are electronic affects of substituents (the σ coefficient from Hammett's models), steric affects (the

Es parameter introduced by Taft) and recently also quantum mechanical indices such as frontier electron densities (fi) and energies

of the highest occupied molecular orbital (EHOMO) and the lowest

unoccupied molecular orbitals (ELUMO). Assuming that the effects

of the parameters are independent of each other, one formulates the

dependence of the biological response (BR) on the parameters (Pi) as

Log (BR) where Ci is a coefficient.

With the parameters measured or calculated, and the use of multiple

regression analysis, it is possible to obtain the coefficients (Ci)

by fitting the biological data for a series of compounds to the (Pi)

values for each molecule. The resulting equation is then assumed to

predict the biological potency of any other drug for which the Pi

values are known. This quantitative correlation between molecular

properties and potency has proved useful in drug design and can, in

principle, indicate the molecular structural factors that are most

likely to affect biological activity (see review quoted above; also,

Hansch 1971; Redl et al. 1974).

The main shortcomings in this approach are related to the nature of

the parameters used, the assumption that they are mutually independent, and the inherent impossibility to yield Inferences on the

molecular mechanisms of interaction between the drug and a receptor.

Thus, au important question is the meaning of parameters such as the

Hammet sigma constant. These parameters reflect the effects of

structural changes at a particular reaction center in a model compound. But such parameters cannot indicate which sites in the

molecule are involved in the reaction. This problem is overcame to

a certain extent by the use of parameters obtained from quantum

mechanical calculations. For example, the quantum chemical concepts

of Fukui's frontier orbital theory (for a review see Fukui 1964)

provide a means of defining site-localized reactivity parameters by

the use of frontier densities (fi). These numbers represent the

electronic charge localized on a certain atom and originating from

a Mulliken population analysis involving only the Highest Occupied

or Lowest Unoccupied orbitals (HOMO or LUMO). Such parameters have

been used in QSAR studies on tryptamine and LSD (Johnson and Green

1974; Green et al. 1978), and with success in predicting biological

aetivities: the mescaline-like activity of 2-amino-7-hydroxyteralin

(Green, Dressler, and Khazan 1973) was predicted in a study (Kang

and Green 1970) that also led to the correct prediction that the

R-enantiomers of the hallucinogenic amphetamines were the active

forms (Kang, Johnson, and Green 1973).

However, reactivity criteria from point charge distributions generated by a Mulliken population analysis, such as frontier densities,

334
cannot provide a physically rigorous description of the fundamental

mechanisms that control the reactions of drugs with macromolecular

receptors. Such an understanding requires a description of the

structural features of the molecules , their electronic distributions

and the energies required to modify and perturb them during interactions. From these basic elements one can obtain a more complete

characterization of the molecular reactivity of the drugs and of

the modes of interaction, the electronic consequences and the energetic characteristics of drug-receptor complexes. In principle and

in fact, most of the information required for such a dynamic representation of biological interactions can be acquired by a judicious

use of quantum mechanical and physicochemical methods.

The success of quantum mechanics in describing the physical and

chemical properties of organic compounds and their reactions is

evident in every facet of chemical research and is very well documented. The main advantage of theoretical studies with quantum

mechanical methods are: the ability to predict processes and mechanisms of interaction with the use of model systems that cannot readily

be obtained experimentally but are amenable to experimentation once

the most probable mechanism is established (for a very recent assessment of this successful role in a specific example see Maugh 1976);

and the ability to analyze, interpret and rationalize all experimental observations on molecular processes in a unified language

based on the primary principles of physics. During the past decade,

quantum chemical methods have been increasingly applied to drugs and

related compounds (Kier 1971; Bergmann and Pullman 1974), and these

studies have recently been reviewed by us and others (Green, Johnson

and Kang 1974; Christoffersen 1976).

Until very recently, most theoretical studies on drug molecules have

concentrated on the calculation of conformation in an attempt to

predict the most probable geometry of the drug at the receptor and

the relation between conformation and activity. With very few exceptions, notably the calculation of aeetylcholine by a classicalquantum chemical method (Gelin and Karpilus 1975), the total energy

of the molecules is calculated for a small number of variable parameters and most bond lengths and bond angles fired at their initial

values. Unfortunately. freezing most of the molecule in its initial

geometry may influence the outcome of the calculation: an ab-initio

calculation of acetylcholine (ACh) using the crystal geometry of

AChCl showed a gauche form to-be 3.2 kcal/mole more stable than the

trans form, whereas with a standard geometry the trans form was 3.4

kcal/mole more stable than the gauche (Port and Pullman 1973).

Analogously, PCILO calculations based on crystal geometries of AChCl

and AChBr produced different preferred conformers (Pullman and

Courriere 1972). However, the energy differences are generally small

and do not invalidate conclusions as to which conformations are energetically permissible and which improbable: the single conformation with the lowest calculated energy is not necessarily the conformer at the receptor, as the many possible interactions of the

drug with its environment may stabilize other allowed conformations

that are only a few kcal less stable than the optimal one. Moreover,

a certain degree of flexibility may be a requirement for agonist

335
activity (Weinstein et al. 1975; Richards et al. 1975). In our

studies we have regarded the conformational data obtained from quantum mechanical calculations as a starting point rather than an end.

By indicating which conformations should be considered for further

work, they simplify the problem of defining the reactive regions of

the molecule and the sites of likely intermolecular interaction.

Whatever ambiguities evolve from theoretical studies on conformation

can often be eliminated or at least reduced by studying a series of

similar compounds and by the judicious use of experimental date from

x-ray and NMR studies as well as pharmacological data from studies of

rigid analogs.

Good theoretical calculations on molecular structure provide more

than conformational date: the electronic structure in each conformation of the molecule can be obtained from the corresponding wave

function and the chemical behavior of the drugs can be studied from

the changes in this electronic structure upon interaction with model

reagents such as models for receptor sites. On this basis, the

wealth of methods and formulations developed in quantum chemistry,

which were shown to provide reliable predictions and understanding

of chemical reactivity, can be directly used to study drug receptor

interactions (Weinstein 1975). Parameters related to the reactivity

of biologically active molecules may be sought from the molecular

wave functions and from the calculated properties, providing insight

into the likely molecular mechanisms of drug receptor interactions

and to the biological implications of such interactions. As with

experimental studies, theoretical studies carry the obvious risk

that model results may be extrapolated too far and the test of resulting hypotheses may be hampered by the inherent ambiguity of crude

Biological correlations. Nevertheless, considerable evidence is at

hand proving the relative success of theoretical approaches in elucidating specific mechanisms of drug action, in providing a basis

for rational drug design, and in leading to conclusions about the

nature of drug-receptor complexes and about the consequences of

drug-receptor interactions (see Green, Johnson and Kang 1974;

Christoffersen 1976). Yet more recently, new theoretical methods

have been developed and applied to biologically active substances.

This continuous develovpment, combined with more rigorous quantum

chemicals methods (e.g.; ab-initio) for calculating the wave function, has provided new insight into the basic molecular events

determining drug activity, and, at the same time, furnished a useful tool in drug design.

LINEAR CORRELATIONS BETWEEN BIOLOGICAL ACTIVITY AND ELETRONIC

CHARGES: TRYPTAMINE DERIVATIVES

QSAR studies including electronic parameters from total valence

(INDO) calculations on nine ring-substituted tryptamine derivatives

that contract the rat fundus strip, demonstrated several correlations of potency with charge densities and frontier electron densities at the N-1, C-4, and C-5 positions of the indole ring (f1,

f4 and f5, respectively). It was suggested that the indoles react

with the fundus receptor (see Green et al. 1978 for a review of the

biological data used in the correlations) through charge-transfer

336
forces involving these atoms, and that the greater potency of 5-HT

was due to the effect of the hydroxyl substitution at the C-5 position on the electronic structure of the ring (Green and Kang 1970).

These correlations predicted accurately the potencies of the 4-NH2,

5-F and 5-OH-7-Cl derivatives measured subsequently for their ability to contract the fundus. In the correlation with f4, the carbon,

sulfur and oxygen isosteres of tryptamine and 5-HT are predicted to

be less active than 5-HT on the fundus, in agreement with measurements. To test further the validity of the prediction we expanded

the study to eight new tryptamine derivatives which we tested on the

same biological system. The activity of the new compounds was found

to correlate with the old indices f1, f5 and with another parameter,

f3. In addition, evidence was obtained for a specialized interaction

with the receptor of substituents at the 7-position, probably involving hydrophobic forces (Johnson and Green 1974). Figure 1 shows the

correlation between the measured activity on the fundus strip and the

activity calculated from the regression equation vith f3 + f5. The

main deviant, 5,6-dihydroxytryptamine (circled) was not used on the

regression equation, for it was shown by spectrofluorometric analysis

to be destroyed in the assay medium probably by oxidation (even in the

presence of ascorbic acid). Similar deviation of the 7-OCH3 derivative from the correlation with the experimentally measured ionization

potential was reported at this symposium by Domelsmith and Houk

(1978).

The rank order of potencies of these tryptamines in contracting the

fundus was nearly identical to their potencies in blocking high affinity binding of LSD to brain membranes, implying that the two receptors

are very similar (Grew et al. 1978). Not surprisingly, the potencies

in this latter system correlated with the same electronic indices, fl,

f3 and f5.

These correlations prwided additional support for the hypothesis of

an "electron-donor" role for tryptamines in the drug-receptor complex.

The QSAR approach, Based on indices derived from molecular orbital

calculations, therefore successfully accounts for the potencies of

tryptamines acting on an LSD receptor, and thereby offers both a

guide for the synthesis of new compounds and a hypothesis for the

molecular basis of their activity. However pleasing and practical

these results are, they are subject to the fundamental question that

pharmacological activity (and chemical reactivity) may not be determined by such derived quantities as point charge dietributione,

frontier densities, etc. This consideration prompted us to introduce to our studies reactivity criteria that are based on the combined contribution from electrons and nuclei for the molecule as a

whole. These led to a representation of the interaction characteristics of the molecule such as the mutual orientation in space of

its active sites, orientation of the molecular active sites towards

reacting agents representing receptor sites, and the response of the

molecule, on an electronic level, to perturbations caused by the

environment or by the receptor itself. Clearly, this is a more

realistic attempt towards understanding molecular properties that

determine biological activity but its success requires overcoming

complex theoretical and numerical problems. At the present stage

it can be dealt with within the frame of the approximations that

337
Figure 1. Correlations between the observed and predicted values

for the activity of tryptamine derivatives on the rat fundus. The

correlation equation is pMR = 1.542 (±0.129)π7 + 20.63 (±1.23)f3+

33.14 (±2.46)f5 - 6.28 for n = 14. The correlation coefficient is

r = 0.990. The 5,6-(OH)2 and the 7-OCH3 derivatives (circled in

the figure) were not used in the regression. See Green et al.

1978 for additional details.

338
are inherent even in the most elaborate theoretical approaches. In

view, however, of what could be considered early signs of progress

obtained with the use of such theoretical approaches and because

they represent some of the most recent and powerful advances in the

analysis of molecular processes, we apply them to the elucidation

of structure activity relationships.

Since molecular systems represent a spatial distribution of electronic and nuclear charges (an averaged time independent distribution within the workable approximations of quantum mechanics), the

natural approach is to seek a description of the molecular reactive

sites and interaction ability based on the long-range intermolecular

electrostatic forces. By describing the electrostatic potential

field generated by the distribution of charge in the molecule, a

"picture" could be expected representing the forces that the molecule

can apply on another charge or on the continuous electron density

distribution of another molecule. Regions of space in which the

molecule generates negative potentials (i.e., where the contributions from all the electrons in the molecules are being felt stronger

than that from all its nuclei) should attract positively charged

species or fragments. A positive region (where the nuclear contribution is stronger) would repulse positive, and attract negatively

charged fragments. A molecular specie approaching the investigated

molecule should therefore adopt an orientation that will bring its

charged fragments into matching attractive interaction with the

positive and negative potential pattern. On this basis, the structural requirement for the interaction with the receptor can be inferred from the potential maps of active drugs, and used as criteria

for the ability of other compounds to mimic this reactive pattern

when approaching the receptor. These molecules should thereby be

able to achieve the same mutual orientation as the active drug and

interact with the same sites of the receptor. The pattern of the

potential can therefore be regarded as an "interaction pharmacophore"

and should prove to be a basic analytic tool in the study of molecular determinants of pharmacological activity. Since the criterion

established by the interaction pharmacophore is independent of the

nature of the groups generating the potential, it can be applied to

molecules which do not have atom-to-atom correspondence thereby

broadening the scope of the analysis to compounds derived from unrelated basic structures. These properties have been used by us

in the characterization of the physicochemical basis and the molecular mechanism of action of several drugs of abuse such as phencyclidine (Weinstein et al. 1973) and psychotomimetic anticholinergics

(Maayani et al. 1973; Weinstein et al. 1975; Weinstein et al. 1977);

morphine-like narcotic analgesics (Loew et al. 1974; Loew, Weinstein,

and Berkowitz 1976) and tryptamine congeners acting on an LSD/serotonin receptor (Weinstein et al. 1976; Green et al. 1978). Other

laboratories have also begun to approach specific pharmacological

problems with methods based on electrostatic potentials (Petrongolo

et al. 1974; Politzer, Daiker, and Donnelly 1976; Hayes and Kollman

1976; see also Anderson and Kollman, and Kaufman - in this volume.

339
The Relation to Rigorous Quantum Chemical Calculations

On the basis of newly developed quantum chemical methods (Weinstein

1979) it becomes possible to formulate a more rigorous link between

the electronic structure of the active compounds and the mechanisms

of action proposed on the basis of QSAR studies as described above.

Thus, the excellent correlation observed between frontier densities

and the ability of the tryptamines to contract the rat fundus strip

and to inhibit high affinity LSD binding to brain membranes indicated

that the tryptamines might interact with the receptor through a complex involving mainly electron polarization from the drugs toward the

receptor. The importance of polarizability and electrostatic forces

in complexation of indoles has been emphasized before (Green and

Malrieu 1965). The QSAR results (Johnson and Green 1974) indicate

that the main sites of interaction [besides the side-chain amino

group, which is essential] would be the indole nitrogen; a region

including the C-4 and C-5 bond; and the C-3 atom (see numbering

scheme). Strong indications for the importance of these sites for

complexation are also obtained from experimental studies in a variety

of media. Thus, C-3 end C-5 are sites of close contact in crystalline complexes of 5-HT with both picrate and creatinine sulfate

(Thewalt end Bugg 1972). NMR studies on complexes of methylated

indoles and trinitrobenzene in solution show that the 3 position of

the indole (especially in 3-methylindole) makes the greatest contribution to complexation (Foster and Fyfe 1966). Proton NMR studies

(Daly and Witkop 1967; Kang, Witherup, and Gross 1977) indicate that

the proton at the C-4 position is exchanged fastest in 5-HT and

5-methoxytryptamine.

We have shown that the positions at which a high frontier electron

density was correlated with high biological potency, correspond to

the sites at which the density of the highest occupied molecular

orbital is localized in 5-HT (Weinstein et al. 1976; Green et al.

1978). We explained these correlations in terms of a multiple perturbation expansion (Bartlett and Weinstein 1975; Chang, Weinstein,

and Chou 1976) of the interaction energies of 5-HT with positively

charged reagents: the highest contribution to the polarization term

in the interaction energy comes from the HOMO (Weinstein et al

1976; Green et al. 1978). Since the electronic density is localized

on specific centers in the HOMO of the various 5-HT congeners, this

finding explained the relation between high frontier density at the

same sites at which HOMO is localized in 5-HT, and a more potent

5-HT-like activity. Similarly, it became clear why earlier studies

found a negative correlation between potency and the frontier density

on certain atoms (Johnson and Green 1974): the localization of HOMO

and the orbital Next to HOMO (NHOMO) differs, and a requirement for

high frontier density on the atoms on which the HOMO of 5-HT is

localized is tantamount to a requirement for low frontier density

on the atoms on which the NHOMO of 5-HT is localized. We have

shown (Green et al. 1978) that the localization pattern in the 5-HT

NHOMO is precisely on those sites on which Johnson and Green (1974)

found a negative correlation between frontier density and biological

activity.

340
The relation between the different localization patterns of HOMO

in the various congeners and the biological activity also indicate

some of the reasons for the apparent failure to find a direct correlation between the energy of HOMO (or the molecular ionization

potential) and 5-HT-like biological activity (Green et al. 1978).

The predictive value of the HOMO localization pattern was further

demonstrated in a study of the relation between experimentally

obtained C-13 chemical shifts of tryptamine derivatives and the

biological activity of the compounds: When the total electronic

charges on the indole moieties in a series of 5-HT congeners were

compared, the two most similar compounds were found to be 5-HT and

5-fluorotryptamine (5-FT). This similarity in total charges (calculated with the ab-initio method) was echoed by a comparison of the

C-13 chemical shifts for the atoms in the indole fragments of the

same molecules. Table 1 shows that the highest correlation is obtained between the shifts on 5-HT and 5-FT. These similarities do

not correlate with the biological potencies of the compounds: figure 1 shows that 5-FT is closer in its activity to tryptamine than

to 5-HT. This similarity in potency is predicted by the correlation with frontier charges: the distribution of HOMO (and NHOMO)

charges on the atoms of the indole fragments of 5-FT is much more

similar to that of tryptamine than to the distribution of these

charges in 5-HT. This is indicated here by the high correlation

coefficient between the distribution of HOMO (and NHOMO) charges

in 5-FT and tryptamine, as shown in table 2.

The important insight prwided by these results from quantum chemical studies into the basic relation between QSAR parameters, molecular structure, and biological activity prompted a more thorough

analysis of the molecular mechanisms of action of 5-HT congeners

on the LSD/serotonin receptor.

MOLECULAR REACTIVITY CRITERIA FOR THE ACTION OF TRYPTAMINES ON THE

LSD/SEROTONIN RECEPTOR

Our quantum chemical studies aim to establish end compare the molecular determinants for the rank order of the affinity of tryptamine

congeners for the LSD/serotonin receptor. Some molecular structural

factors that may be responsible for the differences between measured

affinities of molecules in the tryptamine series for the LSD/serotonin receptor were identified from the reactivity characteristics

of these molecules (Weinstein et al. 1976). These characteristics

were obtained from ab-initio LCAO-SCF-MO calculations with (5s,3p/

2s,p) minimal gausian basis sets.

The studies shoved that as a result of the interaction of the protonated side chain amine with a negative site or an electron donating group, the indole portion of the tryptamine congener cations

would assume the same reactivity characteristics that this moiety

would have in the neutral species (free base form). The importance

of this primary interaction with an anionic site has been stressed

by the experimental data: pharmacological SAR studies demonstrated

the necessity of a cationic side chain for activity on the rat fundus (Vane 1959; Johnson and Green 1974), while proton NMR experiments

341
Table 1. CORRELATION COEFFICIENTa)

Table 2. CORRELATION COEFFICIENTa)

342
and fluorescence spectroscopy studies indicated that the ringstacking interaction of these molecules vith the bases in poly-A,

DNA or ATP is dependent upon the ionic interaction of the side chain

(Helene, Dimicoli, and Brun 1971; Nogrady, Hrdina, and Ling 1972).

The theoretical finding on the effect of such an interaction is not

peculiar to the tryptamine series and has been shown to be important

in the determination of the physicochemical and biological properties

of other substances such as histamine (Weinstein et al. 1976). The

relevant reactivity characteristics of the tryptamine derivatives

were therefore determined from the electronic structures of the

isolated neutral drugs, or from the structure of the complex between

the cationic form of the drug and an anionic group.

A comparison of the interaction pharmacophores (obtained from the

molecular electrostatic potentials) of the tryptamine derivatives

indicated that there will be differences in the preferred orientation that each of the congeners will assume towards a polar receptor

site following the primary interaction with the anionic site. It

was shown, for example, that after the interaction with the anionic

site, the vector describing an optimal electrostatic alignment between 5-hydroxytryptamine (5-HT) and other parts of a putative receptor would be almost perpendicular to the vector describing the

optimal alignment of 6-HT with the same receptor site. Figure 2

shows the electrostatic potentials in planes at 1.6Å above the indole

portions of 5-HT and 6-HT, respectively. Figure 2 also shows the

directions of the orientation vectors obtained from the gradients of

the electrostatic-potential maps (i.e., the lines connecting the

sites of strongest negative potentials through the areas of steepest

change in the potential). These discrepancies in optimal electrostatic orientations for the various tryptamine derivatives (see

Weinstein et al. 1976 for further examples) are fully consistent

with NMR data on the complexes of these molecules: different geometries are observed for the different congeners (Sapper and Lohman

1976). Consequently, when the cationic head of the tryptamines is

anchored at the same anionic site, the difference in optimal alignment will necessitate a change in the conformation of the side chain

if optimal interaction is to be achieved by both 5-HT and 6-HT with

the same reactant (such as another part of a receptor). The energy

expenditure required for such a conformational change affects the

probability for optimal binding and should be measurable as "lover

affinity" or "lower potency" of one drug relative to another. If

6-HT is to assume an orientation that is electrostatically equivalent

to that of 5-HT at the receptor, its apparent affinity should be

lower. This is in fact the experimental observation. A hypothesis

for the interaction of tryptamine congeners with the LSD/serotonin

receptor resulted from these findings: "the difference between the

affinity of 5-hydroxytryptamine (5-HT) andthatof any other congener is related to the discrepancy between the preferred electrostatic orientation of 5-HT and that of the congener in the field of

the receptor" (Weinstein et al. 1978).

The definition of molecular reactivity parameters that have such a

proved predictor function is not only of immediate practical value

in the design of specific drugs, but is a most important element in

343
Fig. 2. Molecular electrostgtic potential maps for 5-HT and 6-HT.

The potential in a plane 1.6Å above the indole fragment of the

neutral (free base) forms of the molecules was calculated from

ab-initio wave functions with the (5s, 3p/2s, p) basis set. Values

ar kcal/mole. the arrows indicate the orientation of optimal

electrostatic alignment ("orientation vectors"). See Weinstein

et al. 1976 for details on the choice of planes, the geometry of

the molecules and methods of calculation.

344
the elucidation of the molecular structural factors that are responsible for the biological activity of the molecules. It was therefore reassuring to find that the reactivity criteria proposed on the

basis of calculations on the isolated molecules were validated by

calculations on complexes of 5-HT with imidazolium cation (Weinstein

and Osman 1977). Calculations on supermolecular models of complexes

with imidazolium were therefore carried out to test the working hypothesis for the interaction with the LSD/serotonin receptor and to

identify a possible electronic basis for the activation mechanism.

MODELS FOR MOLECULAR MECHANISMS

Methods and Background for the Simulation

The studies of Shinitzky, Katchalski and coworkers on the complexes

between indole and imidazole derivatives (for an elegant review see

Shinitzky and Katchalski 1968) have described inter- as well as

intramolecular complexes that are of particular relevance to the

reactivity of indoles in biological systems. These careful studies

have demonstrated the formation of complexes between imidazole,

histidine, or histamine, and indole, tryptophan, aminotryptophan,

or methoxytryptophan. All the complexes are formed when the imidazole ring protonated and "the binding forces between the indole

and imidazole moieties are weak and strongly dependent on pH and

orientation" (Shinitzky and Katchalski 1968). Although "no final

proof as to the nature of the complex" was provided by the authors,

they interpreted their finding as suggestive of "a complex of the

charge-transfer type". The complexes of tryptamine derivatives with

imidazolium cation were therefore chosen as models for the test of

the reactivity criteria and the related hypothesis. For the theoretical simulation of interactions in systems that have also been

studied experimentally can provide additional insight into the properties of the molecules and their mechanisms of interaction. Because the reactivity of a substituted tryptamine cation interacting

with an anionic site has been shown to be identical to that of the

neutral tryptamine (free base) (Weinstein et al. 1976), we studied

the interaction of the neutral tryptamine derivatives with the imidazolium cation. Such a model simulates the simultaneous interaction

of the cationic tryptamine derivatives (all these molecules would be

almost fully protonated at pH = 7.4) with an anionic site and with

other molecules (or "receptor sites") that can form complexes with

the indole portion.

The first calculations on these models (Weinstein and Osman 1977)

were performed with a near ab-initio pseudopotential (model potential) method devised by Van Wazer coworkers (Ewig and Van Wazer

1975; Ewig, Osman, and Van Wazer 1977). The applicability of this

method to molecules such as tryptamine derivatives was tested by

comparisons with full ab-initio calculations using the same gaussian basis set (5as,3p2,s) contracted to minimal basis (Osman and

Weinstein 1977).

These calculations indicated that the 5-HT complex with imidazolium

cation (IMID) is basically electrostatic in nature: the amount of

345
charge transferred from 5-HT to imidazolium was very small (0.0046e)

and the molecular orbitals of the complex could be assigned back to

either molecule based on their very pronounced localization. It

appeared, therefore, that there would be a valid theoretical basis

for the we of reactivity criteria obtained from the electrostatic

potential maps of the molecules (in the form of the electrostatic

orientation vectors). This possibility was tested by calculations

on the [5-HT + IMID] and [6-HT + IMID] complexes in mutual configurations that align the electrostatic vectors of IMID (see figures 4

in Weinstein et al. 1976, and 1 in Weinstein and Osman 1977) with

that of the corresponding tryptamine derivative. In order to test

the relative importance of vector alignment for the stabilization

of complexes with IMID, we studied the following supermolecular

systems:

a) [5-HT + IMID] in a mutual configuration that aligns the

vector of IMID with that of 5-HT (to be referred to as A-5);

b) [6-HT + IMID] in a configuration that aligns the vector

of IMID with that of 6-HT (referred to as A-6);

The geometrical arrangements of the imidazolium (IMID) relative to

the indole portion of the 5-HT congeners is shown in figure 3 for

configurations A-5 and A-6. We have shown that an optimal interaction between a relatively positive hydrogen and the indole nitrogen may be important for the enhancement of the polarization interaction (Chou and Weinstein 1978), due to the special properties of

nitrogen as a "charge transducer." Consequently, the configurations

are chosen according to the additional criterion that one of the

hydrogens in IMID is positioned above N1 in both the A-5 and the

A-6 configuration. Note, however, that due to geometrical constraints the hydrogen placed above N1 differs in the two configurations: in configuration A-5 it is H2; in configuration A-6 it is

H4. It should be noted that H2 carries a more positive charge than

H4 and is therefore expected to have a stronger effect on the polarization at the indole nitrogen.

Stabilization Energies and the Nature of the Complexes

Table 3 presents the stabilization energies of the complexes of 5-HT

and 6-HT with IMID in the geometries described in figure 3. It is

clear that in the complex with 5-HT, the preferred geometry is that

in which the imidazolium cation aligns its most positively charged

regions with the electrostatic orientation vector of 5-HT (i.e.,

configuration A-5), whereas the A-6 configuration is preferred for

the [6-HT + IMID] complex. These results are in full agreement with

the predictions that evolved from the reactivity critertia regarding

the differences in the preferred configurations that 5-HT and 6-HT

would adopt towards the same polar site. It appears now that the

electrostatic orientation vectors that were generated as force vectors from the molecular electrostatic potentials are useful indicators of the reactivity characteristics of the 5-HT congeners in

interactions with molecules of the type of imidazolium cation.

The underlying reason for the validity of these reactivity criteria

was suggested by our findings (Weinstein and Osman 1977) that the

complexes [5-HT + IMID] and [5-HIND + IMID] are electrostatic in

346
Figure 3. The orientation of imidazolium (IMID) in complexes with

5-HT and 6-HT: a) [5-HT + IMID] in the A-5 configurations; b) [6-HT +

IMID] in the A-6 configuration. See text for the definition of A-5

and A-6. ©1978, John Wiley & Sons, Inc.1

nature. A more complete elucidation is given by the results of the

decomposition of the interaction energies in the complex that was

performed according to the scheme proposed by Morokuma and coworkers (Morokuma 1971; Kitaura and Morokuma 1976). Table 3 shows that

for all the complexes, in all the configurations, the electrostatic

interaction (ES) is the largest term. Moreover, it is evident from

table 3 that the total energetic preferences for a given configuration (i.e., A-5 for the 5-HT complex, and A-6 for the 6-HT complex)

is determined by the electrostatic contribution (ES): for a given

configuration, either A-5 or A-6, the only energy term that is different in the 5-HT complex from the 6-HT complex is ES, while all the

others (PL, EX, CT + mix) are virtually identical. Also, for the

same complex (e.g., [5-HT + IMID]), only the ES values differ significantly between the A-5 and A-6 configurations. The very small contribution of the (CT + mix) term indicates that the main changes in

the electron distributions of the molecules upon complex formation

are in the form of charge polarization (see below).

The predictive value of the reactivity characteristics is thus confirmed and explained by the calculation of the supermolecular model

complexes: The anchoring of the side chain in the tryptamine molecule

347
Table 3. Energy Decomposition in Complexes with Imidazoliuma)

Type of 5-HT + IMID 5-HT + IMID 6-HT +IMID 6-HT +IMID

contribution at A-5 at A-6 at A-6 at A-5

E S -4.52 -3.70 -4.51 -3.93

P L -1.00 -0.98 -0.96 -1.00

E X

CT(+ mix)

2.39 2.27 2.28 2.38

Total

-0.59 -0.38 -0.38 -0.59

stabilization -3.72 -2.79 -3.57 -3.14

a

Energy values are from calculations with the STO-3G set.

Units are Kcal/mole. © 1978, John Wiley & Sons, Inc.1

determines a fixed direction with respect to which the substituted

indole portion can orient itself as determined by the electrostatic

orientation vector, in order to achieve optimal electrostatic interaction. The preferred orientations are different in different congeners, and can be predicted by the orientation vectors.

Since the orientation vectors are defined only on the indole portion

of the neutral (or neutralized) tryptamine congeners, it was interesting to find that the same configurational preferences hold for

the complexes between indole derivatives (i.e. 5-hydroxy and 6-

hydroxyindole: 5-HIND and 6-HIND) and imidazolium. The energetic

differences between the tryptamine and the indole complexes with

imidazlium are insignificant both in the total stabilization energies and in most of the components. The only noticeable difference,

albeit small, is in the polarization term (PL) which is greater in

the tryptamine complexes than in the corresponding indole models.

This difference is directly attributable to the inductive effect of

the saturated, neutrel side chain. It appears, therefore, that in

addition to its role in defining the directionality constraints of

the interaction of the indole portions in these molecules, the side

chain reinforces the electrostatic nature of the interaction. The

preferred orientation is, however, determine by the electrostatic

reactivity charecteristics in the idole portion. Table 4 shows how

the complex stabilization energy, and its decomposition, very with

the interplanar distance for the [5-HIND + IMID] complex. The optinal interplanar distance in the A-5 geometry is 3.5Å i.e., slightly

greater than the "close contact" distance from crystallographic data,

but very close to the average interring distance in the complexes of

5-HT (Thewalt and Bugg 1972). It is evident from the energy decomposition presented in table 4 that the electrostatic nature of the complex is preserved at all relevant distances, as the ES component is

the determinant factor in the total stabilization energy at distances

greater than 3.1Å Identical conclusions hold for the [6-HIND +

IMID] complex in the A-6 configuration.

The Electron Charge Redistribution in the Complexes as an Element of

the Activation Mechanism.

The electrostatic nature of the complexes and the relative contribution of polarization, exchange repulsion and "charge transfer" terms

to the stabilization energies indicated that the chargere disttibution

348
Table 4. Energy Decomposition in complexes 5-HIND + IMID at A-5a)

accompanying complex formation would be mainly "intramolecular" in

character: the polarization within each molecule will enhance the

electrostatic interaction but there will be little "overlap" or actual transfer of charge. With the STO-3G basis set in an ab- initio

calculation, the Mulliken population analysis (which at an interplanar distance of 3.3Å can be expected to reproduce well the charges

within each component of the complex) indicates a minimal amount of

charge transferred from 5-HT to IMID (0.0029 at A-5, and 0.0018 at

A-6). The same results are obtained for the [6-HT + IMID] complex:

0.0029 and 0.0017 for the A-5 and A-6 configurations; respectively.

In order to study the detailed characteristics of the charge redistribution we used partial integrations of the total electron density

distribution function (ρ). The "planar density" (ρ Ρ), obtained by

integrating (ρ ) in a given plane, has been calculated with Module PA60

of the POLYATOM program. An expression of the charge redistribution

upon complex formation is obtained from the difference between the

ρΡ values for the complex and the sum of the ρΡ values of the separated molecules. This difference function (ΔρΡ) is plotted in

figure 4, and indicates that the charge in 5-HT has been strongly

polarized towards IMID, while the charge in imidazolium cation is

polarized away from 5-HT. This result is in full agreement with

the basic idea that the polarizability of the 5-HT molecules is a

major determinant of its ability to form molecular complexes, especially with positively charged species. The polarization of

the electronic eharge in IMID will cause an increase in the positive electrostatic field generated by the molecule in the direction of 5-HT; this further increases the interaction with the

excess electronic charge introduced in the interplanar region by

the polarization of 5-HT.

Figure 4 also shows that the polarization pattern of the [5-HT +

IMID] complex in the A-6 configuration will be very similar to that

in the A-5 arrangement (although very slightly less accentuated).

This is in full agreement with the contribution of polarization

(PL) to the stabilization of the complexes in the two configurations (see tables 3 and 4). Since the contributions of the PL terms

to the total stabilization of the [6-HT + IMID] and [6-HIND + IMID]

complexes were nearly identical to those for the corresponding

349
Fig. 4. Differences in planar densities showing the electron density

redistribution in [5-HT + IMID] complexes in the A-5 and A-6 configurations. At each distance (r = n x 0.6238 a.u.), ρΡ(r) is the total

(integrated) density in a plane parallel to the indole portion of

5-HT. ΔρΡ(r) is the difference (at each point r) between the total

density in the plane calculated from the wave function of the complex,

and the sum of densities in that plane calculated from the wave

functions of 5-HT and IMID, separately.

©1978, John Wiley & Sons, Inc.1

350
complexes of 5-HT and 5-HIND, it is not surprising that the same

polarization pattern is observed in the complexes of 6-HT. This

near identity holds not only for the integrated values of the

charge distribution function, but also for the charge redistribution pattern in localized parts of the complex. This chatacteristic is exemplified in figures 5 and 6 in which the changes in

the total density upon complex formation (Δ ρ) are calculated in

equivalent planes for the [5-HT + IMID] complex (figure 5), and

the [6-HT + IMID] complex (figure 6).

Figure 5. Density difference map in a plane perpendicular to the

rings in the [5-HT + IMID] complex, in the A-5 configuration, The

insert in the upper right corner shows the position of the plane

intersecting the complex. ©1978, John Wiley & Sons, Inc.1

351
This finding is most important in view of the biological activity

exhibited by 5-HT and 6-HT: the mechanism of receptor activation

must be the same for these compounds since they have the same

maximal activation ability ("intrinsic activity").

Figure 6. Density difference map in a plane perpendicular to the

rings in the [6-HT + IMID] complex, in the A-6 configuration. The

plane intersects the complex through the two nitrogens in the imidazolium ring, and is perpendicular to the rings. See figure 5.

©1978, John Wiley & Sons, Inc.1

352
It appears therefore that polarization complexes of the type analyzed

here provide a good model for the interaction mechanism of these molecules with the LSD/serotonin receptor: the considerable difference in

affinity is a direct result of the molecular reactivity pattern of

the molecule and is expressed in the difference of preferred interaction geometries with the same reactant. However, once a congener

(e.g., 6-HT) adopts au interaction geometry that aligns its vector

with that of the common reactant (albeit with lover probability), the

consequences of the interaction are identical. Finally, it is not

difficult to realize that the other members of the congeneric series

of singly substituted tryptamine derivatives will all exhibit the

same type of reactivity characteristics: i) formation of polarization

complexes with a variable degree of probability (dependent on their

molecular structure and predictable by the reactivity parameters);

ii) generation of charge redistribution patterns that are very

similar to those of 5-HT once an equivalent interaction geometry

is adopted with the receptor site. This hypothesis can be tested

at the present level of biological experimentation by the prediction,

based on the reactivity parameters identified above, of structures

that can have similar electrostatic orientation preference to 5-HT

and generate the same charge redistribution in a model reactant

(such as IMID) without necessarily sharing the molecular structure

of tryptamine derivatives.

FOOTNOTE

1. Figures 3-6 aNd tabie 3, © 1978 by John Wiley & Sons, Inc.,

are reprinted by permission of the publisher, from Weinstein, H.,

Osman R., Edwards, W.D., and Green, J.P. Theoretical models for

molecular mechanisms in biological systems: tryptamine congeners

acting on an LSD/serotonin receptor. Int J Quant Chem, QBS5, 1978.

In press.

ACKNOWLEDGMENTS

This work was supported by the National Institute on Drug Abuse

(DA 01875). The computations were supported in part by a grant of

computer time from the University Computer Center of the City University of New York. Analysis of results in this work was carried

out in part on the PROPHET System, a national computer resource

sponsored by the NIH through the Chemical/Biological Information -

Handling Program, Division of Research. H.W. is recipient of a

Career Scientist Award from the Irma T. Hirschl Trust.

353
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357
AUTHORS

Harel Weinstein, D.Sc.

Jack Peter Green, M.D., Ph.D.

Roman Osman, Ph.D.

W. Daniel Edwards, Ph.D.

Department of Pharmacology

Mount Sinai School of Medicine

of The City University of New York

New York, New York 10029

358
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. WiIIette, eds.

National Institute on Drug Abuse, 1978

Conformational Energies and

Geometries of Narcotics, Using a

Potential Function Method

Mark Froimowitz

INTRODUCTION

As anyone who reads the scientific literature must be aware,

conformational enerygy calculations are an increasingly popular

approach to the analysis of biologically interesting molecules. The

apparent objective of much of this work is to characterize possible

conformations for molecules which have significant rotations about

single bonds. Sometimes, it is also explicitly or implicitly assumed

that the conformation with the lowest computed energy should be the

one that is responsible for biological activity. However, this is

incorrect for a number of reasons. First and most importantly,

there is no evidence that the results of these calculations are

quantitative. This problem is worsened by the recent demonstration

that bond angle bending and stretching, which certainly occurs in a

molecule, can significantly affect the quantitative results of an

energy calculation (Gelin and Karplus 1975). In contrast to this,

almost all calculations make the assumption of rigid bond angles and

lengths. Moreover, it can be documented that the semiempirical

quantum mechanical PCILO, CNDO and INDO methods may produce

unrealistic results under certain conditions (see below). A second

difficulty with these calculations is that they are performed with

unrealistic molecular environments, since they generally consider

only a single isolated molecule. The computed energy surface in

such an environment is unlikely to be quantitatively relevant for

a molecule in its biological system where there are unknown and

unspecified intermolecular interactions. Finally, these calculations

generally compute conformational energies rather than the thermodynamically relevant free energies. For all of these reasons, it is

clear that calculated conformational energy differences of several

kilocalories/mole should not be considered to be significant. However, the results of energy calculations can provide useful

qualitative information about the likelihood of various molecular

conformations. In particular, it should be possible to exclude

those regions of conformation space that are unlikely to be

important for a molecule due to the energetically unfavorable

overlap of nonbonded atoms.

359
Conformational energy calculations can be performed with methods

that are based on either quantum mechanics or on a more classical.

approach using potential functions. Some of the most commonly used

methods are the semiempirical quantum mechanical PCILO, CNDO, and

INDO computer programs whose results can be unrealistic under

certain conditions. In comparing the results of an INDO calculation on acetylcholine (Beveridge and Radha 1971) with that obtained

by a potential function method (Froimowitz and Gans 1972), it was

noted that the former produced low energy minima for conformations

in which there should have been serious energetically unfavorable

overlaps of nonbonded atoms. Indeed the lowest energy minimum

found by the INDO calculation, at τ(C6-01-C5-C4) = 270° and

τ(01-C5-C4-N) = 50° ([270°, 50°]), appears to be a totally

fictitious result. Two PCILO conformational energy maps for

acetylcholine using different molecular geometries (Pullman,

Courrière, and Coubeils 1971; Pullman and Courrière 1972) show the

corresponding extra pair of minima at [270°, 60°] and [90°, 300°].

In the second of these two calculations, these are computed to be

the global minima. That these minima are fictitious is reinforced

by the results of ab initio calculations on acetylcholine which do

not show these conformations as minima (Genson and Christoffersen

1973; Pullman and Port 1973). A more recent study on model systems

has also indicated that the CNDO and INDO methods produce insufficient repulsion between nonbonded atoms (Gregory and Paddon-Row

1976). It was also found that extended Hückel, which is a full

overlap method although it is the most simple of the various semiempirical quantum mechanical methods, does have the corect

repulsive behavior between nonbonded atoms, though it is well known

to have other deficiencies.

A more recent illustration of these difficulties is provided by the

PCILO investigation of the methadone molecule (Loew, Berkowitz, and

Newth 1976). Using a mathematical search algorithm to locate

minima in the conformational energy surface, a total of twelve minima were reported, along with some of their associated interatomic

distances. Of the twelve, eight have interatomic distances that are

within the van der Waals' radii of those atoms (table 1). There are

likely to be additional overlaps which were not reported. While

nonbonded atoms can approach to within their van der Waals' radii

under some conditions, such as when hydrogen bonding occurs or if

other molecular interactions are so favorable that they can counterbalance the expected repulsion, the severity and ubiquity of the

atomic overlaps argues against this explanation. Thus, it appears

that these semiempirical quantum mechanical programs consistently

report low energies for conformations which should be energetically

unfavorable. This is not to say that these methods do not provide

a great deal of useful information. However, it would be helpf'ul

if users of these program excluded those conformtions which are

clearly unrealistic.

These computional difficulties may also provide an explanation

for the results of a recent investigation of the enkephalins, in

which conformational energies were computed by both the PCILO method

360
TABLE 1

Conformation Atom Reported Van der Waals'

Pair interatomic radii for atom pair*

distances (A)

(A)

4 N-O 2.46 3.05

4 H-O 1.58 2.70

5 H-O 1.76 2.70

6 C-C 2.40 3.47

7 C-N 3.27 3.32

8 C-N 2.67 3.32

I N-O 2.97 3.05

I N-C 2.58 3.25

III N-O 2.65 3.05

III N-C 2.46 3.25

I V C-N 2.84 3.32

IV N-C 2.91 3.32

The short interatomic distances of various atoms of some "low

energy conformations that were reported from a PCILO investigation of methadone (Loew, Berkowitz, and Newth 1976). These

conformations are unlikely to be of low energy since many of

the atoms are within their van der Waals' radii, which is the

interatomic distance at which atoms begin to repel each other.

*It should be noted that there are different van der Waals' radii

for phenyl and alkyl carbon and hydrogen atoms (Bondi 1964).

and a potential function method (Loew and Burt 1978). It was

reported that local minima found by the potential function method

were also minima for the PCILO method but that the latter produced

additional minima. It would appear that the likely cause of these

extra "minima" is the qualitatively incorrect behavior that is

associated with the PCILO method when nonbonded atom overlap.

In contrast to these difficulties, potential diction methods do

appear to give qualitatively correct conformational energies. For

361
example, in regions of conformation space in which there are no

atomic overlaps, a potential function method gave similar results

to an INDO calculation in that minima and a maximum appeared in

similar locations (Froimowitz and Gans 1972). similarly, the

results detained for the dopamine molecule are consistent with

intuitive expectations that there are minima associated with a

trans and two gauche conformations (see below). Using this method,

it was also possible to account for the crystal conformations of

a large number of phenothiazine derivatives relative to a restricted

dihedral angle range (Froimowitz and Matthysse, to be published).

That is, all of the observed values of the dihedral angle were

close to the three computed minima for this dihedral angle.

Another very important advantage to using a potential function

method is the relative speed of the calculation. It has been

noted that this method is 10,000 times as fast as an INDO calcula

tion on the same molecule and computer (Froimowitz and Gans 1972).

This enormous increase in speed allows one to compute conformational

energies on a finer grid for larger molecules with increased numbers

of variable dihedral angles.

Conformational Geometries

While the relative energies of various conformations are undoubtedly

important for the analgesic activities of the narcotics, an additional factor which should also be important are conformational

geometries, since the narcotics have been shown to interact with

receptors (Gyang and Kosterlitz 1966; Pert and Snyder 1973). In

order for them to do so, they must be satisfying specific molecular

and geometrical requirements that allow them to mimic the characteristics of an endogenous substance and successfully compete with

it for its receptors. One such requirement is that the narcotics

must be the correct size and shape to fit into the receptor site.

A second requirement is that they and their endogenous equivalents

must contain specific atom or atomic groups which attract complementary atoms or atomic groups in the receptor. This molecular and

geometrical specificity insures that biologically active substrates

only bind to certain receptors in the complex regulation of

biological processes. Therefore, in addition to computing the

conformational energy as a function of variable dihedral angles,

various intramolecular geometrical paramters will also be

monitored. This should allow the characterization of the various

conformational geometries.

METHODS

Potential Function Method

A number of different possible interactions should be considered

in computing intramolecular conformational energies using potential

functions (Wilson 1972). For the molecules to be considered here,

these include nonbonded, electrostatic, torsional, and bond bending

and stretching forces.

362
The nonbonded interaction between a pair of atoms i and j that are

at a distance of rij can be simulated by a Lennard-Jones 6-12

potential function

(1)

where aij and bij are constants which depend on the atomic types

(Scott aij Schersga 1966a; Brant, Miller, and Flory 1967;

Froimowitz and Gans 1972). The parameter bij which controls the

attractive van der Waals' forces can be calculated from

(2)

where e and m are the electron charge and mass, is Planck's

constant, and αi and Nj are the atomic polarizability and effective

number of valence electrons of atom i. Using this formulation for

the van der Waals' forces, it was possible to account for the heats

of formation and energies of isomerization of small hydrocarbon

chains (Pitzer 1959). Since the "effective" number of valence

electrons is unclear for most atoms, the actual number will be

used in this work. The parameter aij which controls the repulsive

part of equation (1) that is attributed to the overlap of nonbonded

atoms can be obtained by minimizing the nonbonded energy at the

van der Waals' radii of the pair of interacting atoms. Thus,

(3)

where rvdw is the sum of the van der Waals' radii of atoms i and j

(Scott and Scheraga 1966b; Froimowitz and Gans 1972). The

qualitatively correct behavior of equation (1) is to have a sharply

repulsive energy within rvdw, an energy minimum at rvdw, and a

slight attractive energy outside of rvdw. Atomic polarizabilities

and van der Waals' radii to be used in equations (2) and (3) can

be obtained as before (Ketelaar 1958; Bondi 1964). We have

previously shown that equation (1) tends to define the permissible

regions of conformation space (Froimowitz and Gans 1972). In

particular, it assigns high energies to those regions in which

there is significant overlap of nonbonded atoms.

A second force which should also be important for the conformational

energies of many molecules is the electrostatic interactions that

can occur between partially charged atoms. The usual method of

representing this is by

(4)

where qi and qj are partial charges that are centered on atoms

i and j at a distance of rij and where D is the dielectric constant

(Scott and Scheraga 1966b; Brant, Miller, and Flory 1967; Froimowitz

and Gans 1972). There are uncertainties associated with the values

and locations of the partial charges and the appropriate dielectric

363
constant (Froimowitz and Gans 1972; Momanyy et al 1975). For these

reasons, a quantitative number will not be assigned for the electrostatic interaction. However, it will be possible to recognize

molecular conformations that could be electrostatically favored.

A third set of terms that is typically added to these computations

is used to represent torsional forces. While the theoretical

basis for these forces is uncertain, they are added to give the

correct barrier height for the rotation about a single bond, since

the use of equation (1) alone generally underestimates the

experimentally observed barrier (Scott and Scheraga 1966a). However, terms for the torsional forces will not be included in this

work since they add at most only one or two kilocalories/mole to

the rotation about a single bond. As with the electrostatic

interactions, certain molecular conformations such as gauche

trans along a hydrocarbon chain, can be identified as being

preferred.

The effect of bond bending and stretching on the computation of

conformational energies using potential functions has recently

been examined (Gelin and Karplus 1975). Their results indicate

that structural flexibility can affect the quantitative results of

such calculations, especially in regions in which there is steric

hindrance. This is due to the very steep repulsive nature of the

first term of equation (1) since it is possible for a molecule to

decrease this energy by bond bending and strecthing. However,

it was also found that the location of local minima were unchanged

by these forces. For this work, bond bending and stretching will

be neglected. However, to ensure that possibly important conformations are not missed, regions of conformation space that are

within 20 kilocalories/mole of the global minimum will be considered

in our analysis.

To summarize the above discussion, the conformational energy will

be computed by summing the nonbonded interactions that are

represented by equation (1) over all atoms whosee distances can

change relative to each other. This equation defines the permissible regions of conformation space, since it assigns high

energies to conformations in which there is significant atomic

It should be noted that other interactions that are

discussed above are not being explicitly included, since there is

uncertainty in the correct quantitative assignment of their values.

Instead, these interactions will be incorporated qualitatively by

the direct examination of the conformations. It should be

realized that, for this work, we are only interested in computing

qualitatively accurate energies since our primary concern is

elimination of those conformations which cannot be significant

due to the overlap of nonbonded atoms.

Intramolecular Geometrical Parameters

The minimal features that are common to all narcotics appear to be

a basic nitrogen separated from an aromatic ring, which is almost

364
always benzene, by several bonds (Beckett and Casy 1954). Very

often, the presence of a hydroxy group in a particular position of

the benzene ring leads to enhanced activity (Jacobson, May, and

Sargent 1970). Thus, it appears that the critical intramolecular

geometrical parameters would include the distance of the basic

nitrogen atom (1) from the center of the aromatic ring, (2) to the

plane of the aromatic ring, and (3) to the hydroxy oxygen on the

ring. Another structural feature which is particularly important

for the potencies of the meperidines and the methadones is a

carbonyl group (Jacobson, May, and Sargent 1970). Therefore,

distances between the electronegative carbonyl oxygen and the

positively charged nitrogen may be important and will also be

monitored.

The tyrosine is the most likely portion of the enkephalins

to be relevant to the structures of narcotics, since it contains the

phenyl hydroxy group and basic nitrogen appear to be important

for analgesic activity. The only other phenyl group in the enkephalins is that of phenylalanine while there are no other basic

nitrogens or hydroxy groups. While it will not be possible to

analyze the enkephalins in detail because of the large number of

single bonds about which rotation can occur, analysis of the tyrosine

fragment will be feasible.

RESULTS

While we are only beginning our calculations on conformationally

flexible narcotics, our general method is illustrated by our

investigation of the phenothiazine and thioxanthene classes of

neuroleptic (antipsychotic) drugs, some of whose results are

briefly presented here. More detailed results will be published

elsewhere (Froimowitz and Matthysse, to be published). As with the

narcotics, neuroleptic drugs are believed to compete with an

endogenous substrate in the central nervous system. There is

considerable evidence that the relevant endogenous substrate for the

neuroleptics may be dopamine (Matthysse 1977).

The conformational energy surface for dopamine (figures la and lb)

contains two equivalent sets of minima due to molecular symmetry.

Each set consists of one trans and two gauche conformations, which

is the expected result for the rotation about the C7-C8 bond.

Various intramolecular geometrical parameters, which happen to be

identical to those defined above for the narcotics, were measured

for these preferred conformations of dopamine, and the conformational

volumes of the neuroleptics were then searched for possible

geometrical analogues to them.

Chlorpromazine (figure 2a), which is the prototypical phenothiazine,

contains three single bonds about which rotation can take place and

which can affect the relative orientation of the sidechain nitrogen

to the phenothiazine structure. Of the three, rotation about the

C13-Nl bond was found to be the most hindered with two relatively

broad minima at τ (C14-C13-Nl-C12) = 210° and 300° and a very narrow

365
FIGURE 1

1a. Crystal conformation of dopamine (Bergin 1b. Variation of the conformational energy for

and Carlström 1968. Hydrogen atom (unlabeled), dopamine. G and T indicate gauche and trans

which were not determined, have been added. conformations. Contour levels are 1, 3, 5, 10,

20, and 100 Kilocalories/mole. Unlabeled extrema

are maxima.
one at 75°. A number of possible geometrical analogues to the

dopamine conformations were found, some of which are noted in

figure 2b, which is the energy surface for chlorpromazine with

τ (C14-C13-Nl-C12 = 210°. The two analogues in the lower right

hand side of the figure occur when the N2 atom approaches the Cl

substituent, with the S atom in an equivalent position to one of

the dopamine oxygens. These conformations would be expected to

be electrostatically favored since the N2 atom, which would be

protonated at physiological pH, approaches the electronegative

Cl atom. The trans analogue is also very close to the conformation

that chlorpromazine assumes in the crystal state (McDowell 1969;

Horn and Snyder 1971), though different conformations occur in the

crystal structures of other phenothiazines which are also potent

neuroleptics (Horn, Post, and Kennard 1975). The two dopamine

analogues which appear in the upper left side of figure 2b occur

when the N2 atom swings toward the other benzene ring and the Cl

atom assumes an equivalent position to one of the dopamine oxygens.

We have considered the latter to be possible dopamine analogue

even though the N2-benzene plane distance for them is about 2 Å

further than the equivalent parameter in dopamine. However, it

appears that this parameter is sensitive to slight tilts of the

benzene ring. In addition to these, equivalent analogues also

appear at the minima at τ (C14-C13-Nl-C12) = 75° and 300°. However,

as shall be seen from our results for the thioxanthenes, it is

unlikely that the latter are crucial for antipsychotic activity.

The thioxanthenes, of which α-flupenthixol(figure 3a) is an

example, are neuroleptics that are structurally related to the

phenothiazines, with the only difference between them being that the

first sidechain N-C single bond of the latter (the Nl-C13 bond in

chlorpromazine) is replaced by a C-C double bond. The restricted

rotation about a double bond results in a pair of geometrical

isomers with cis isomers (relative to a substituent on the tricyclic

structure) consistently having greater potency as neuroleptics than

the equivalent trans isomer (Kaiser, Warren, and Zirkle 1974).

Since cis isomers, such as the relatively potent neuroleptic αflupenthixol, have a value of τ (C17-C16-C9-C14) near 180°, this

suggests that the crucial dopamine analogues occur at the equivalent

dihedral angle of 210° in the phenothiazines. The similarity in

these dihedral angles also shows up in the geometrical and conformational energy results (figure 3b) for a molecule based on the crystal

structure of α-flupenthixol (Post et al. 1975). However, the gauche

dopamine analogue in which the protonated Nl atom approaches the

tricyclic substituent is absent for steric reasons. Thus, there are

still three possible dopamine analogues for neuroleptics of this

class.

Diethazine (figure 4a) is typical of low potency neuroleptics in which

there are only two carbon atoms between the sidechain nitrogen and

the phenothiazine structure. The possible dopamine analogues

figure 4b) for such a molecule (with a Cl atom in the approptiate

position) are ones in which the Cl atom is in an equivalent position

to one of the dopamine oxygens. Other analogues found previous, in

367
FIGURE 2

2b. Variation of the conformational energy

for molecule based on crystal structure of

2a. Crystal conformation of chlorpromazine chlorpromasine with τ (C14-C13-N1-C12) = 210°

(MoDowell 1969). Hydrogen atoms (unlabeled), contour levels are 1, 3, 4, 10, 20 and 100

which were not determined, have been added. kilocalories/mole. Unlabeled extrema are

maxima. G and T indicate the location of

analogues to gauche and trans dopamine conformations while C indicates the approximate

crystal state conformation.
FIGURE 3

3a. Crystal conformation of α-flupenthixol

(Post et at. 1975).

3b. Variation of the conformational energy

for molecule based on crystal structure of

α-flupenthixol. Contour levels are 1, 3, 5,

10, 20, and 100 kilocalories/mole. Unlabeled

extema are maxima. G and T indicated the location of analogues to gauche and trans

dopamine conformation while C indicates the

crystal state conformation.
FIGURE 4

4a. Crystal conformatio of disthazine

(Marsau 1971).

4b. Variation of the conformational energy

for molecule based on crystal structure of

diethazine. Contour levels are 1, 3, 5, 10,

20, and 100 kilocalories/moles. G and T

indicate location of analogues to gauche

and trans confromations of dopamine, while C

indicates the crystal state conformation.
which the protonated sidechain nitrogen approaches the phenothiazine

substituent, do not appear because the N17 atom cannot swing over far

enough to assume that position. The two analogues that do appear

should be preferred since they are close to τ (N17-C16-C15-N10) =

60° and 300°, which are preferred conformations for a hydrocarbon

chain, though the gauche analogue is about 4 kilocalories/mole over

the global minimum. Since these possible analogues appear with

reasonably low energies for phenothiazines like diethazine, which

have very weak neuroleptic activity, it suggests that the remaining

trans analogue of chlorpromazine, in which the sidechain nitrogen

approaches the phenothiazine substituent and in which the sulfur

atom takes an equivalent position to one of the dopamine oxygens,

is the relevant one for antipsychotic activity. This is the same

chlorpromazine conformation that was suggested previously on the

basis of geometrical similarities between the crystal structures

of chlorpromazine and dopamine (Horn and Snyder 1971).

In summary, a number of possible geometrical analogues to dopamine

can be found in the low energy regions of chlorpromazine. Because

of the uncertainties associated with conformational energy calculations, it is not possible to clearly differentiate which of these

analogues is responsible for the antipsychotic activity of the

neuroleptics. However, by relating low energy geometries of

structurally related molecules with varying pharmacological

potencies, it was possible to make a prediction as to the

biologically active conformation.

ACKNOWLEDGMENTS

I would like to thank the attendees at the Conference for their

many useful comments. Special thanks go to Milan Trsic for his

critical reading of the original manuscript.

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AUTHOR

Mark Froimowitz, Ph.D.

Ralph Lowell Laboratories

McLean Hospital

115 Mill Street

Belmont, MA 02178

373
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Conformational Study of Lysergic

Acid Derivatives in Relation to their

Hallucinogenic and Antiserotonin

Activities

Mahadevappa Kumbar

Conf'ormational analysis of five lysergates in six different

solvents has been carried out by an empirical method. The energy

maps are developed for all molecules in all solvents. Further,

the receptor site conformations are identified for both hallucinogenic and antiserotonin activities. It appears that all the molecules do not act on the same site. Effect of solvent is examined

by comparing the solvated conformation with that of vacuum and

it can be concluded that this effect definitely exists, but depends

on both solute and solvent. Various properties such as free

energy, entropy, percent population, dipole moment, translational

and rotational diffusion coefficients, and relaxation time are

obtained for receptor site conformations and correlated with the

observed activities. It is concluded that the mode of action in

eliciting the hallucinogenic response is different from that of

antiserotonin response.

INTRODUCTION

The ergot alkaloids have always been a fascinating class of

drug molecules due to their diversified effects and unusual structure activity relationships. There have been numerous investigations regarding the pharmacological actions of LSD (d-lysergic

acid diethylamide) and other similar compounds. Among all the

phamracological actions, the hallucinogenic (H) action has dram

most of the attention (Sankar 1975). The main interest lies in

understanding the mechanism of hallucinogenic and/or antiserotonin

(S) actions in terms of molecular structure under existing physiological conditions. Earlier molecular orbital studies (Karreman

et al. 1959; Snyder and Merrill 1965; Kang and Green 1970) have

indicated a charge transfer reaction for the hallucinogenic activity of LSD (being a donor) due to high degree of correlation between the actitity and the highest occupied molecular orbital

energy which was later found to be not true (Johnson et al. 1975;

Kumbar and Sankar 1973; Sankar and Kumbar 1974). In recent years,

however, attention has been turned to utilize the concept of

"structural resemblance" in explaining the activities of various

hellucinogens other than LSD. This concept has been examined by

374
using molecular models (Snyder 1970), x-ray crystallography (Baker

et al. 1973), and molecular orbital methods (Kier 1971; Green et

al. 1974; Pullman et al. 1974). All these studies, however, have

been confined to congeners of LSD, but not to the derivatives of

d-lysergic acid.

The quantitative values of hallucinogenic and antiserotonin

effects are known for about 20 lysergates (Cerletti and Doepfner 1958)

These include mono and disubstituted amides, cycle and 1, 2, and

8 substituted derivatives of LSD. In the present study, only five

lysergates ( figure 1) have been selected due to their structural

differences in side chains which might help to assess the role of

the side chain in producing the pharmacological response. Any

change in the drug molecule and in the medium in which the molecule

exists can dramatically effect the active (receptor site) conformations. Therefore, these molecules are studied in different solvents varying in physiochemical properties to see how the conformational behavior changes from solvent to solvent. In the course

of this action, the empirical method in conjunction with reaction

field theory (Sinanoglu and Abdulnur 1965) has been used. Within

the framework of reaction field theory, an approach to solvation

effect involves the computation of the interaction energy of

solute with its surrounding, making use of a continuum model for

solvent. However, considerable doubt exists (Linder 1967) about

the applicebility of this continuum model, because of its treatment

of the solvent as a uniform medium inside and outside the cavity

of the molecule. In reality, the dielectric constant inside and

outside of the cavity may be different. Previously (Kumbar 1976),

it was pointed out that the effective dielectric constant (inside

the cavity) is about one half of the bulk dielectric constant (outside the cavity). With this in mind, a conformational analysis is

carried out and properties such as free energy, entropy, percent

population, dipole moment, translational and rotational diffusion

coefficients, and relaxation times are deduced for active conformations, with the hope of obtaining a quantitative relation between

structure and activity.

The translational diffusion coefficient describes how the

molecule travels in a given medium, while the rotational diffusion

coefficient gives an indication of how the molecule orients itself

at the receptor site. When an interaction between the drug molecule and the receptor site begins, the molecule that is present in

a random state in a solution near the receptor site mist come to

an order state (specific orientation) at the receptor site for a

proper interaction. Therefore the relaxation time may be defined

as the time required for the molecule to relax itself from an ordered state into a random state. In other words, the relaxation

time may be considered as the time spent by the drug molecule at

the receptor site.

375
METHOD OF CALCULATION

(a) Conformational energy: The total energy of a molecule for a

particular geometry under the influence of a solvent is calculated

as

(1)

where Esolute is the total energy of a solute molecule in the free

space approximation and Esolvation is the energy of solute solvent

interaction. Esolute is computed as the sum of various contributions as given in the following equation:

(2)

where φ,ψ, and τi are the dihedral angles defined in figure 1.

The nonbonded interaction energy between the pair of interacting

atoms is calculated by means of the Lennard-Jones 6-12 potential

function, which is,

(3)

The first term in this equation represents the repulsive forces

and the second term describes the attractive forces. The term

rij is the distance between the ith and the jth interacting atoms,

and eij and dij are coefficients which have been determined according to the method described previously (Scherega 1968; Kumbar 1975)

The torsional potential energy about C-C single bonds and the C-N

single bond is added via the following equation

(4)

where , , and are the energy barriers which are respectively set equal to 1.0 kcal mole-1, 1.9 kcal mole-1, and 0.58

kcal mole-1 (Volkenstein 1963; Flory 1969; Scheraga 1968). In

recent years various crystallographic studies (Cody et al. 1973)

and theoretical investigations (Renugopalkrishnan and Rein 1976)

have indicated that the peptide bond deviates from its planar

structure. Besides, the assumption of the planar structure in

d-lysergic acid diethylamide introduces considerable steric

interaction between the side chain and the ring portion. In

order to understand how the side chain behaves if this constraint

is relaxed, and how this effects the orientation of -C=O bond; the

peptide bond is treated as a single bond. In addition, the potential barrier is lowered to about one tenth of its true value.

Therefore, the results obtained here should be used cautiously, due

to hypothetical nature of the present model. The electrostatic

energy is evaluated using the formula

(5)

376
where ε' is the effective dielectric constant that is applicable

inside the cavity and ei is the charge on the ith atom, which is

the sum of π and σ charges. The π and σ charges are computed

respectively by the Huckel method (Pullman and Pullman 1963) and

the procedure suggested by Del Re (1958).

Esolvation energy is the sum of three contributions, namely,

(6)

where Ees is the electrostatic solute solvent interaction, Edis

is the solute solventt interaction energy due to dispersion forces,

and Ecav is the energy required to create a solvent cavity. The

Ees is computed by treating the solute as a sphere of radius a

with a point dipolar ion of charge q and the total dipole moment

μ positioned at the center. The solute sphere is imbeded in a

cavity of identical size, making the dielectric constant of the

medium inside and outside the cavity different. According to

Onsager's reaction field theory (Onsager 1936), the electrostatic

energy is given by

(7)

(8)

where ε is the bulk dielectric constant of the solvent outside

the cavity and α is the average polarizability of the medium,

which is α = 1/3 ( + 2 ). The solute-solvent interaction

energy due to dispersion forces is

(9)

where ρ is the number density of the solvent, g

(2)(r) is the

radial distribution function for solvent molecule about a central

solute, whlch is taken as zero for r<a and unity for r>a. The

effective pair potential between a solute A and a solvent B at a

separation r is

(10)

where VAB(r) and B'AB(r) are pairwise interaction potentials for

a molecule in a gas phase and a liquid phase correction factor

(Sinanoglu 1967) respectively. The VAB(r) is taken in the form

of a Kihara potential (Kihara 1953), assuming each molecule as a

spherical core of diameter 1. This potential function is

(11)

In the above equation, CAB is the London dispersion coefficient,

PAB is the intercore distance, and σAB is the collision diameter.

377
The intercore distance is given by

(12)

These core size parameters are evaluated from the semiempirical

relations

(15)

(16)

where wA is Pitzer's "accentric factor" (Dannon and Pizer 1962),

as (Amos and Burrows 1973)

ß 1.15, and a is an effective solute cavity radius which is taken

(17)

The rA is generated from the radius of gyration for a particular

conformation and rB is determined from the critical densities of

solvents. The liquid phase correction factor for the gas phase

potential in equation (10) (Sinanoglu 1967) is

(18)

where ΔAB is a function of ionization energies IA and IB, as shown

in the following equations:

(19)

(20)

(21)

where nB is the refractive index of the solvent. The London dispersion coefficient CAB is set equal to

(22)

Finally, the energy required to create a solvent cavity to accomodate a spherical solute of volume v is expressed as

378

(23)
(24)

(25)

where kB is the constant dependent of the volume fraction, vB/vA,

T is the thermal expansion of the solvent, γB is the surface

tension of the solvent, and T is the absolute temperature of the

system which is set equal to 310°, a physiological temperature.

(b) Conformational properties: The Helmnoltz free energy is

calculated by

(26)

where E is the conformational energy in a particular solvent, and

S is the entropy, which is computed by the following set of equations:

(27)

(28)

(29)

The dipole moment, on the other hand, is predicted by using the equation, μq = Σ qiri, where qi is the charge on the ith atom and ri

is the distance of the ith atom from the origin of the chosen reference coordinate system. The translation and rotational diffusion coefficients are obtained from the well known equations applicable to spherical molecules in an unbounded fluid of viscosity

η. These equations are

(30)

Here, k is the Boltzmann constant. Further, the rotational diffusion coefficient is related to the relaxation time (τ ) through

the equation, τ = 1/2 Dr. The percent population of the nth minimum is estimated according to the equation:

(31)

where is the energy of the ith conformation in the vicinity of

the nth minimum. The most probable properties associated with a

given energy valley are determined by

(32)

In addition, the most probable properties averaged over various

solvents are computed through the following equation:

(33)

379
(34)

RESULTS AND DISCUSSION

Energy maps were obtained for all five molecules, (figure 1)

in six diffferent solvents: heptane, carbon tetrachloride, aniline,

ethanol, methanol, and water which have a wide range of dielectric

constants. The LSD molecule has a bulky side chain with six

degrees of freedom. It is difficult (see figure 1) to rotate all

six dihedral angles simultaneously, due to computer time. Hence, a

different approach was taken. Firstly, the angles τ3 and τ4 in

all the molecules were fixed at 60° in a staggered position with

the preceeding group (minimum interactions). Secondly, the angles

τ 1 and τ2 were rotated in 10° interval to determine the most

stable values by considering only the interactions in the side

chain. The energy maps produced as a result of rotations of τ1

and τ 2 contained two minima centered around τ1 = τ2 = 90°, and τ1

= 70° and τ2 = 250°. The minimum energy conformations may not

represent the energy valley in which they exist. Therefore, most

probable conformations were determined as indicated in equation

(32). It happened that the most probable values did not deviate

considerably from their respective minimum values. So these

values were used in further computations in LSD and dimethylamide

The same strategy was also adopted for ethylamide and found that

τ2 had three different most probable values, namely 60°, 90°, and

180°. Similarly, the most probable value of τ2 in methylamide

was determined to be 180°. Using these values, the rotations

around φ and ψ were carried out in 10° increment in various solvents mentioned above. The most probable conformations for each

molecule in all six solvents are shown in figures 2(a) to 2(e).

Effect of solvation

In order to understand the influence of the solvent medium or

to gain some information about the conformational behavior in presence of external forces (solvent medium), the vacuum (gaseous

phase) conformations were also generated by setting ε' = 1.0 in

Esolute equation (2). The most probable conformations are also

indicated in figures 2(a) to 2(e). These vacuum conformations

may be considered as the most ideal situation conformations

(ideal conformations). Some of the properties of ideal and solvated conformations are compared in figure 3. It is appparent

that the solvated conformation is quite different from an ideal

conformation. The degree of influence of the solvent medium

depends on the nature of the solute as well as the nature of the

solvent. In all cases, the conformational energies of ideal conformations are lower than the corresponding solvated conformations.

The conformational entropy decreases when molecule is solvated, due

to a loss of conformational freedom. A further decrease in entropy is observed when the dielectric constant of the medium is increased. The dipole moment is higher when the molecule is solvated except for dimethylamide, where it is lower. This property

380
slightly varies as the dielectric constant of the medium changes.

The volumes of all the molecules except dimethylamide are smaller

in a given solvent and also to some extent depend on the dielectric constant of the medium. Therefore, it is clear that the solvent definitely influences the conformational behavior of the solute, depending upon the nature of the solute as well as that of the

solvent.

Solvated conformations

For a given molecule, number of minima appearing in energy

maps depends on the nature of the solvent. As the dielectric constant of the medium increases, some of the minima disappear due to

an increased interaction between the solute and the solvent that

favors certain conformational regions. As a result, the conformational entropy decreases. Even though the most probable values

of the dihedral angles φ and ψ have altered to some extent from

solvent to solvent, they have grouped together into a small region

as shown by the circles in figures 2(a) to 2(e). This behavior

is observed in spite of the fact that τ1 and τ2 have been set at

different values. This might be an indication that these subsidiary angles have less influence on the main two dihedral angles,

φ and ψ. On the whole, the LSD has eight conformational regions,

dimethylamide has four, ethylamide has four, methylamide has two,

and an amide has four. In reality, the LSD should have only 4 conformational regions, the dimethylamide should have only 2, and the

amide should have only 2 due to the symmetry nature of the side

chain over the dihedral angle ψ. The asymmetry over the angle ψ

is due to fixing of τ1 and τ2 at certain values when φ and ψ were

rotated.

Free energy, entropy, dipole moment, percent population,

translational and rotational diffusion coefficients, and relaxation times are important aspects of drug receptor interactions.

All these properties truly depend on the nature of the solvent

medium. Some of these properties of each molecule as a whole,

without differentiating various minima, are considered in figure 3.

It is obvious that these properties are functions of the nature of

the medium and the molecule. For a given property, all molecules

except dimethylamide have behavior similar to the dielectric

constant of the medium increases. These changes inproperties

point out that the solvent medium that exists around the receptor

site(s) may play an important role. The effect of the solvent

depends on the nature of the energy valley. If the valley is

deeper, for example, IV(VIII) in LSD, the effect is smaller. On

the other hand, if the valley is shallow like I or III or IV in

ethylamide, the effect is greater. The shallowness, however, is

dictated by the conformational flexibility. It means that a molecule with a considerable flexibility might experience a greater

effect of the solvent.

Receptor site conformations

Among the five molecules studied here, the LSD is highly

381
potent in terms of hallucinogenic and antiserotonin activities.

The magnitudes of these activities are also shown in figure 1.

The differences in the activities of these molecules may be attributed to differences in behaviors of side chains. The structural

differences, together with differences in various physiochemical

factors, might account for the variability of their activities.

Therefore, it is essential to identify those conformations which

are believed to be present at the receptor site(s). This is, of

course, a diffcult task. To get around this problem, the following approach is taken. Since the LSD possesses the maximum activities (H=100 and S=100), it is assumed that all four conformations (figure 2(a)) are involved in producing these activities.

However, it is difficult to assess their degree of contribution

towards the total activity. When a drug molecule binds to the

receptor site, the molecular conformation might be complementary

to the receptor site(s). On this basis, it is assumed that the

LSD binds to four different sites corresponding to four sterically

different conformations labelled as I to VIII in figure 2(a).

Region III(VII) appears only in high dielectric constant medium

as a result of dissappearce of region II(VI). It means that

there are only three sites in low dielectric medium and three

in high dielectric medium. If there is a continous fluctuation

in dielectric constant, or if the receptor site region exists in

an inhomogeneous medium, then all four sites might be activated

simultaneously as shown below:

In order to obtain the receptor site conformations for other

molecules, the conformational regions of each molecule are compared with those of the LSD in figures 4(a) to 4(d). It is clear

that some of the regions lie close to those of the LSD. Thus it

appears that there exists a conformational resemblance between

LSD and other molecules. Therefore, it is conceivable to assume

that these molecules ellcit their pharmcological response by

resembling the conformations of LSD. However, in methylamide the

resemblance is found only for regions II and IV, and in amide only

for I and IV. In addition, all molecules do not act on the same

site (table I). To have some kind of overall representation of

each region shown in figure 2(a) to 2(e), a further averaging over

all solvents is carried out end resulting diherdal angles, together

with various properties, are tabulated in table II. These overall

conformations are presented in figures 5(a) to 5(e).

A close look at the LSD molecule reveals that it contains

various centers for interactions as schematically shown in figure

6. Various intracenter distances for all the overall conformations as

are listed in table III. These distances might be complementary

382
to the interacting centers existing in receptor site(s). Since

the ring portion of the LSD is common to all molecules, the interactions in this portion are retained in all molecules. Therefore,

the variation in antiserotonin and hallucinogenic activities might

be explained in terms of the variability that exists in side chain

interactions such as the hydrogen bonding, hydrophobicity, free

energy, etc. The hydrophobicities for these molecules have been

calculated by Dunn and Bederka (1974) which are 2.16, 1.16, 1.16,

0.66 and 0.16 respectively for LSD, dimethylamide, ethylamide,

methylamide, and amide. This property decreases as the activities

decrease. In addition to hydrophobicity, other factors might also

influence the drug receptor interaction. We have looked into

the possibility of hydrogen bonding by the carbonyl oxygen of the

side chain and the receptor site. Since the LSD molecule has the

maximum activities among the molecules considered here, it is

assumed that the oxygen atom in all the receptor site conformations

(I . . . IV, table II) of LSD is situated in an optimum position.

The deviations of this atom from the optimum positions (measured

in terms of the angle, θ, figure 6) for only receptor site conformations (shown in figures 4(a) to 4(e)) are also tabulated in

table III. This angle increases as activities decrease, suggesting

that the strength of hydrogen bonding is also an important factor.

This strength decreases as activities decrease. Further, it is

possible to distinguish the conformations responsible for hallucinogenic and antiserotonin activities using the angle θ. The antiserotonin activity is always higher than the hallucinogenic activity for a given molecule, which might be due to a close congruity

between the molecule and the receptor site(s). It means that a

stronger hydrogen bonding is necessary for antiserotonin activity

compared to hallucinogenic activity. Thus, the conformations with

smaller θ values might be responsible for antiserotonin activity

while larger θ for the hallucinogenic activity. This distinction

is noted in table III by the letters S and H. This is further

supported by a good correlation obtained between the θ and the

differences in activities (table IV).

In order to investigate the role of other properties in drug

receptor interaction, a correlation is also carried out between

these properties and the activities. From the correlation coefficient, few conclusions may be drawn. Entropy and percent population do not seem to play any role. On the contrary, the free

energy, rotational diffusion (relaxation times), hydrophobicity,

and the strength of the hydrogen bonding appear to be an important

determinants. The dipole moment and translational diffusion coefficients to sane degree are also important factors only in

hallucinogenic activity. The amide has no hallucinogenic activity.

Therefore, we assume that the conformations falling in region

II(IV) are responsible (figure 2(e)) for this activity. Hence, the

properties of this region are used in correlating the hallucinogenic activity. From the correlation coefficients, one can see

that even among the correlated properties, the degree of correlation is not the same for both activities. Besides, the dipole

moment is better correlated in hallucinogenic activity. Therefore,

it is concluded that the mode of action in eliciting the hallucinogenic action is quite different from that of antiserotonin activity.

383
TABLE 1

Receptor site conformations.

The Roman numerals indicate the conformational regions shown in

figures 2(a) to 2(e) and 4(a) to 4(d)

Molecule LSD Sites

I(V) II(VI) III(VII) IV(VIII)

d-lysergic acid

dimethylamide II(IV)

d-lysergic acid

ethylamide I III II IV

d-lysergic acid

methylamide I II

d-lysergic acid

amide I(III) II(IV)

384
TABLE II

Over all conformations (averaged over all solvents) with their various properties
Table II. Continued
TABLE III

Various intracenter distances and the angle θ (see Fig. 6)

for various sites.
TABLE IV

Summary of the properties of the receptor site conformations for both

activities along with correlation coefficients
FIGURE 1

Definition of dihedral angles. φ=0 when C7-C8 bond is

in planar cis position with C-N bond, ψ=0 when C8-C

bond is in planar cis position with N-C bond. τ1 (τ 2 )

=0 when C-N bond is in planar cis position with C-C

bond, τ3 (τ 4)=0 when N-C bond is in planar cis position

with C-H bond. the calculations were performed for

τ 3 = τ4 = ω = 60°. The magnitude of the hallucinogenic

(H) and antisertonin (S) activities is also indicated.

389
FIGURE 2

(a) d-lysergic acid disthylamide (*)

The most probable conformations in all six solvents. Each minimum is identified by Roman numeral. The

vacuum conformation is shown by: (a) d-lysergic acid diethylamide, (b) d-lysergic acid dimethylamide

(c) d-lysergic acid ethylamide (d) d-lysergic acid methylamide, and (e) d-lysergic acid amide.
FIGURE 2

(b) d-lysergic acid dimethylamide
FIGURE 2

(c) d-lysergic acid ethylamide
FIGURE 2

(d) d-lysergic acid methylamide
FIGURE 2

(e) d-lysergic acid amide
FIGURE 3

Comparison of various properties in various solvents relative to the vacuum conformations:

(a) LSD, (b) dimethylamide, (c) ethylamide, (d) methylamide, and (e) amide.
FIGURE 4

(a) LSD (*) and dimethylamide (x)

Comparison of various most probable conformational regions of a given molecule with those of LSD.

.- LSD x - a given molecule: (a) LSD and dimethylamide, (b) LSD and ethylamide, (c) LSD and

methylamide, and (d) LSD and amide.
FIGURE 4

(b) LSD and ethylamide (x)
FIGURE 4

(c) LSD and methylamide (x)
FIGURE 4

(d) LSD and amide (x)
FIGURE 5

Over all conformations (see text and also table III) of molecules

studied here. The conformation(s) responsible for each activity

(both activities) is (are) indicated by S and/or H: (a) LSD,

(b) dimethylamide, (c) ethylamide, (d) methylamide, and (e) amide.

400
FIGURE 5

(b) dimethylamide

401
FIGURE 5

(c) ethylamide

402
FIGURE 5

(d) methylamide

403
FIGURE 6

Schematic representation of various centers for interaction in LSD. The intracenter distances are listed

in Table III. The angle θ measures the deviation of -C=0 bond in receptor conformations (see figures

4(a) to 4(d) It is zero for LSD conformations. The values may be found in table III.
ACKNOWLEDGMENT

I am thankful to Dr. Gene Barnett of the National Institute On Drug

Abuse for valuable discussions, This work is supported by the

National Institute on Drug Abuse grant number 1R01 DA01542-01A1.

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AUTHOR

Mahadevappa Kumbar, Ph.D.

Department of Chemistry

Adelphi University

Garden City, New York. 11530

407

Section IV

Spectroscopic Methods

The power of the many and varied techniques of spectroscopy needs to be fully applied in the QuaSAR area.

The papers presented here have been primarily concerned

with properties of the isolated drug molecule and

questions of molecular conformations and solvent interactions. It is hoped that studies will be applied to

more complex systems to model the drug at the site of

the receptor. These techniques have been extensively

applied to the problem of biomembrane structure and the

next step in complexity will be to obtain some physicochemical parameters for the drug receptor complex at the

physiological site of action.

409
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute of Drug Abuse 1978

Carbon-13 Nuclear Magnetic

Resonance Study of the a- and ßIsomers of Methadol and

Acetylmethadol Hydrochlorides

F. Ivy Carroll, Charles G. Moreland, George A.

Brine, and Karl G. Boldt

INTRODUCTION

In 1954 Beckett and Casy (Beckett and Casy 1954) proposed that the

opiate receptor topography consisted of an essentially inflexible

site possessing a flat surface, a cavity, and an anionic site to

accommdate an aromatic ring, a hydrocarbon moiety, and a protonated nitrogen, respectively. Over the years this proposal has been

modified in attempts to account for the high activity of certain

synthetic narcotics including some acyclic compounds which did not

fit the receptor area proposed by Beckett and Casy. More recently,

the proposal has been further modified to account for the morphine

like activity of the pentapeptides (Met-enkephalin, H-Tyr-Gly-GlyPhe-Met-OH and Leu-enkephalin, H-Tyr-Gly-Gly-Phe-Leu-OH) isolated

from pig brain (see Horn and Rodgers 1977, Smith and Griffin 1978).

Thus, any structure activity relationship proposal will have to

account for the analgesic activity of flexible acyclic narcotics

and enkephalins, as well as the rigid opiates.

The flexibility of the acyclic narcotics makes it very difficult to

derive definitive structure activity relationships. However, it is

apparent that the conformation(s) of the acyclic narcotics will be

required to arrive at such a relationship.

Both 1H- and 13C-NMR spectral studies have been used to determine

the conformation of morphine and similar compounds in solution

(F. I. Carroll et al. 1976). Since these compounds possess rigid

structures, it is likely that the conformation indicated by the

spectral studies are the actual conformers of the drug at the

"opiate receptor."

There have also been a number of pKa, 1H-NMR, and other physical

chemical studies conducted on the acyclic narcotics, methadone,

α-methadol, and ß-methadol, in an attempt to define the conformer(s) that are present in solution (Henkel, Bell, and Portoghese

1974; Portoghese and Williams 1970; Portoghese and Williams 1969).

From the 1H-NMR data reported it appears that methadone and α- and

ß-methadol in solution exist as mixtures of interconverting

410
conformers. Due to limitation of the low field 1

H-NMR technique,

it was not possible to determine accurately the conformational

population or the structure of the individual conformers.

The solid state structure [conformation(s)] of methadone hydrobromide (Hanson and Ahmed 1958), isomethadone hydrochloride,

α-methadol hydrochloride and α-acetylmethadol hydrochloride (Shefter

1974) have been determined by X-ray single crystal analysis. The

most interesting feature of these studies was the lack of conformational similarity among these acyclic narcotics. Of the compounds

studied only methadone hydrobromide and isomethadone hydrochloride

showed similar conformations.

The present study was undertaken to ascertain whether 13C-NMR could

provide additional information about the conformation of narcotic

agonists in different solvents and, furthermore, to determine

whether or not specific differences between the conformation of αand ß-methadol hydrochlorides and α- and ß-acetylmethadol hydrochloride could be defined. Since chemical shift differences are generally larger for carbon resonances than those of protons, it was

also anticipated that low temperature 13C-NMR would show separate

resonances for the individual conformers.

CARBON-13 NMR STUDIES

a-Methadol Hydrochloride

Table 1 gives the 13C-NMR chemical shifts of α-methadol hydrochloride and ß-methadol hydrochloride in DMSO-d6 and in CD2Cl2.

A comparison of the chemical shifts for α-methadol hydrochloride in

DMSO-d6 with those in CD2Cl2 shows large differences for both C-3

( 7 ppm) and C-5 ( 7 ppm). The change that is observed for C-3

could be due to the fact that DMSO-d6 would, by means of hydrogen

bond formation with the C-3 hydroxyl group, interact more strongly

than CD2Cl2 However, the fact that the shift at C-3 is accompanied by an analogous shift at C-5 leads one to believe that DMSO-d6,

through such hydrogen bonding interactions, has effectively reduced

the ratio of internally hydrogen bonded cyclic conformer(s) to

acyclic conformer(s). Thus, it is proposed for α-methadol hydrochloride that acyclic conformer(s) are dominant in DMSO-d6 and that

cyclic conformer(s) are dominant in CD2Cl2.

These propositions are supported by the observations of two N-methyl

carbon resonances for α-methadol hydrochloride in CD2Cl2 and one

N-methyl carbon resonance in DMSO-d6 at 30°C. Further evidence to

support these arguments can be seen in figures 1A, B and C. As

shown in figure 1C, two conformers of α-methadol hydrochloride in

CD2Cl2 have been "frozen out" at -95°C and, furthemore, the conformer present in the larger amount has two N-methyl carbon signals

while the one present in the smaller amount has only one. In the

low temperature 13C spectrum the lesser conformer has resonances

for C-3 at 73.8 ppm, for C-S at 39.5 ppm, and for the N-methyl

carbons at 39.5 ppm. The close similarity of these chemical

shifts to those for the same carbons of α-methadol hydrochloride in

411
TABLE 1

13C-NMR Chemical Shiftsa

 for α-Methadol Hydrochloride and

ß-Methadol Hydrochlorideb in Dimethylsulfoxide-d6 and in

Methylene Chloride-d2

CH3 -CH2 -CHOH-C(C6 H5 )2 -CH2 -CH(CH3 )-N(CH3 )2 •HCl

1 2 3 4 5 6 7 8 9

α-Methadol•HCl ß-Methadol•HCl

Carbon

1 11.41 11.56 11.43 11.24

2 25.75 26.15 26.16 25.80

3 81.70 74.80 73.21 73.62

4 55.67 54.96 55.17 54.45

5 46.50 39.53 41.61 39.31

6 60.51 58.93 58.88 58.54

7 14.66 15.87 12.94 14.16

8 36.71c

38.21 35.52c

39.31

9 41.83c

38.21 42.30c39.31

C D 2 C l 2 DMSO-d 6 C D 2 C l 2 DMSO-d 6

a

All values are in ppm downfield from TMS at 30°C.

b

The α- and ß-methadol hydrochloride samples were dried at 90°C

under vacuum.

c

The N-methyl chemical shifts cannot be assigned to a particular

methyl group.

412
DMSO-d6 (table 1) along with the presence of one N-methyl carbon

resonance indicates that the lesser conformer is acyclic. If

temperature effects on the chemical shifts can be ignored, then the

13CNMR chemical shifts of each signal at 30°C (figure 1A) should

be the weighted average of the chemical shifts of the corresponding

signals observed for the conformers at -95°C (figure 1C). Using

chemical shifts for C-3, C-5, and the N-methyl carbons to calculate

a mole ratio of cyclic to acyclic conformers gives an average value

of approximately 5 to 1.

ß-Methadol Hydrochloride

In the case of ß-methadol hydrochloride an examination of the

chemical shifts (table 1) shows a very small change with solvent

for C-3 (0.4 ppm) and a somewhat larger change for C-5 (2.3 ppm).

In addition, two N-methyl carbon resonances are observed for ßmethadol hydrochloride in CD2Cl2 as compared to one in DMSO-d6.

In order to be consistent with the conclusions reached for the

α-isomer, the latter result should be taken to mean that the

ß-isomer is acyclic in DMSO-d6 and mainly cyclic in CD2Cl2.

Additional evidence in support of acyclic conformers for the α- and

ß-isomers in DMSO-d6 is that there are small differences in their

chemical shifts in DMSO-d6 (table 1). One is then left to explain

why the changes in chemical shifts with solvent at C-3 and C-5

for the ß-isomer are much less than the changes observed for α-isomer. The only rationale that seems plausible is that the chemical

shifts for the acyclic conformer(s) are indeed very similar to the

chemical shifts of the cyclic conformer(s) for the ß-isomer but not

for the α-isomer. This could possibly indicate that the acyclic

conformers of the α- and ß-isomers and the cyclic conformer(s) of

the ß-isomer are similar.

The low temperature spectra of ß-methadol hydrochloride in CD2Cl2

are presented in figures 2B and 2C. As can be seen, C-2, C-3 and

C-5 (and other carbon resonances) have broadened and shifted in

comparison to the same signals in the spectrum at 30°C (figure 2A).

The N-methyl resonances, on the other hand, have shifted and

broadened only sightly. Unfortunately, due to low temperature

limitations on both the 13C probe and the solvent it was not possible to reach a low enough temperature where separate signals from

the contributing conformers could be observed. Even with this

limitation it is possible to suggest the following explanations.

There is little or no acyclic conformer(s) for the ß-isomer in

CD2Cl2 since all carbon resonances including the N-methyl carbons

should have broadened and shifted. There is a strong possibility

that the ß-isomer is a mixture of cyclic conformers that have

N-methyl groups with very similar 13C-NMR chemical shifts and

dissimilar chemical shifts for the other carbon resonances such as

C-3, C-5, C-2, etc. As expected, another more definite conclusion

is that the cyclic conformer(s) of the ß-isomer is different from

the cyclic conformer of the α-isomer. This is based primarily on

the large difference in chemical shifts between the α- and ß-isomers

in CD2Cl2.

413
FIGURE 1

13C-NMR Spectra of α-Methadol Hydrochloride in CD2Cl2

at Various Temperatures

414
FIGURE 2

13C-NMR Spectra of ß-Methadol Hydrochloride in CD2Cl2

at Various Temperatures

415
a-Acetylmethadol Hydrochloride

The 13C-NMR spectra of α-acetylmethadol hydrochloride and ß-acetylmethadol hydrochloride have been run in CD2Cl2 at 30° and -90°C.

figures 3A and 3B show the spectra of α-acetylmethadol hydrochloride

at 30° and -90°C respectively. Examination of figure 3 shows that

C-3 appears as one signal at 30° and two signals at -90°C. If the

chemical shifts of the two peaks obtained at -90°C are weighted

80/20, the calculated chemical shift for C-3 is identical to that

in the 30°C spectrum. If the two C-S peaks in the -90°C spectrum

are also weighted 80/20, the observed chemical shift for C-5 in

the 30°C spectrum is obtained. The N-methyl groups appear as two

signals separated by 2.9 ppm in the 30°C spectrum. The -90°C

spectrum shows two major N-methyl peaks separated by 4.0 ppm and in

addition a third smaller peak which is located between the two

major peaks. If the 13C chemical shift of the smaller N-methyl

signal is weighted by 20% and the larger N-CH3 resonances are

weighted by 80%, the-result gives two signals separated by ~3.0

ppm which is the separation in the 30oC spectrum. Combining these

observations with the conclusions reached for α-methadol hydrochloride, it is proposed that the α-acetylmethadol hydrochloride in

CD2Cl2 is a 4 to 1 mixture of cyclic to acyclic conformers.

The 13C-NMR spectra of α-acetylmethadol hydrochloride in deuterium

oxide (D2O) were also obtained at 30° and +5°C and are shown in

figure 4. The most interesting feature of these spectra is the

appearance of two distinct N-methyl resonances when the temperature

is reduced to +5°C. This is different from what was observed for

α-methadol hydrochloride in D2O or in DMSO-d6 where only one

N-methyl resonance was observed (see table 1). By comparison to

the results discussed previously for α-methadol hydrochloride and

the observation of a single N-methyl resonance for the acyclic

conformer in CD2Cl2 at -90°C for the α-acetylmethadol hydrochloride, one is led to the conclusion that the α-acetylmethadol

hydrochloride is not totally acyclic in D2O. Furthermore, analysis

of the separation of the N-methyl signals for α-acetylmethadol

hydrochloride in D2O compared to that in CD2Cl2 predicts that it is

about a 1 to 1 mixture of cyclic to acyclic conformers.

ß-Acetylmethadol Hydrochloride

Figures 5A and 5B show the 13C-NMR spectra of ß-acetylmethadol

hydrochloride in CD2Cl2 at 30° and -90°C respectively. Inspection

of the low temperature spectrum shows two signals for the C-1,

C-2 and the acetyl methyl group indicating the presence of two

conformers. However, since C-3 is not well resolved, and the

N-methyl and C-5 carbons give a complicated set of signals, it is

not possible to analyze the date in relation to cyclic and acyclic

structures. The picture is further complicated by the 13C-NMR

spectrum of ß-acetylmethadol hydrochloride in D2O (figure 6) which

shows two N-methyl resonances and very sharp signals for all

carbons. Additional experiments at higher fields and lower temperatures will be required in order to explain these results.

416
FIGURE 3

13C-NMR Spectra of α-Acetylmethadol Hydrochloride

in CD2Cl2 at 30° and -90°C

417
FIGURE 4

13C-NMR Spectra of α-Acetylmethadol Hydrochloride

in D2O at 30° and + 5°C

418
FIGURE 5

13C-NMR Spectra of ß-Acetylmethadol Hydrochloride

in CD2Cl2 at 30° and -90°C

419
FIGURE 6

13C-NMR Spectra of ß-Acetylmethadol Hydrochloride

in D2O at 30°

420
SUMMARY

The 13C-NMR spectra reported in these studies give direct evidence for

the presence of two contributing conformers for α-methdol hydrochloride, α-acetylmethadol hydrochloride and ß-acetylmethadol hydrochloride. Indirect evidence is also available for the presence of

more than one conformer for ß-methadol hydrochloride. However, in

order to be more descriptive about the structures of the conformers,

it is necessary to obtain 13C-NMR spectra that have a higher degree

of resolution than is available from our present NMR system. Such

systems are available, and plans are currently in progress to

obtain 13C-NMR spectra on these compounds at high enough magnetic

fields and low enough temperatures to give us the necessary data.

REFERENCES

Beckett, A. H., and Casy, A. F. Synthetic analgesics: Stereochemical considerations. J Pharm Pharmacol, 6:986, 1954.

Carroll, F. I., Moreland, C. G., Brine, G. A., and Kepler, J. A.

Carbon-13 nuclear magnetic resonance spectra of morphine alkaloids.

J Org Chem, 41:996, 1976.

Hanson, W. A., and Ahmed, F. R. The crystal structure and absolute

configuration of the monoclinic forms of d-methadone hydrobromide.

Acta Crystallogr, 11:724, 1958.

Henkel, J. G., Bell, K. H., and Portoghese, P. S. Stereochemical

studies on medicinal agents. 16. Conformational studies of methadone and isomethadone utilizing circular dichroism and proton

magnetic resonance. J Med Chem, 17:124, 1974.

Horn, A. S., and Rodgers, J. R. The enkephalins and opiates:

Structure activity relationship. J Pharm Pharmacol, 29:257, 1977.

Portoghese, P. S. and Williams, D. A. Stereochemical studies on

medicinal agents. VII. Absolute stereochemistry of methadol

isomers and the role of the 6-methyl group in analgesic activity.

J Med Chem, 12:839, 1969.

Portoghese, P. S. and Williams, D. A. Stereochemical studies on

medicinal agents. VIII. Absolute stereochemistries of isomethadol

isomers. J Med Chem 13:626, 1970.

Shefter, E. Crystal structures of analgesics. Hydrochlorides of

(-)-isomethadone, α-methadol and α-methadyl acetate. J Med Chem,

17:1037, 1974.

Smith, G. D., and Griffin, J. F. Conformation of [Leu5

]enkephalin

from X-ray diffraction: Features important for recognition at

opiate receptors. Science, 199:1214, 1978.

421
AUTHORS

F. Ivy Carroll, Ph.D., George A. Brine, Ph.D., and Karl G. Boldt, B.S.

Chemistry and Life Sciences Group

Research Triangle Institute

Research Triangle Park, North Carolina 27709

Charles G. Moreland, Ph.D.

Deparment of Chemistry

North Carolina State University

Raleigh, North Carolina 27650

422
‘QuaSAR’ Research Monograph 22

G. Barnet, M. Trsic, and R. Willette, eds.

National lnstitute on Drug Abuse, 1978

Photoelectron Spectroscopic

Studies of Hallucinogens: The Use

of Ionization Potentials in QSAR

L. N. Domelsmith and K. N. Houk

Studies of the electronic structures of hallucinogenic drugs (Snyder

and Merril 1965; Snyder 1970; Green and Kang 1970; Weinstein et al.

1976) have provided various lines of evidence for the role of electronic

characteristics, such as molecular orbital energies and electrostatic

potentials, in the causation of hallucinogenic activity. Because of

the power of photoelectron spectroscopy as a tool for elucidation of

electronic structure (Rabalais 1977), we have studied a variety of

hallucinogenic substances and inactive analogs by this technique.

Here, we wish to: (1) demonstrate the utility of photoelectron spectroscopy, in conjunction with various types of quantum mechanical

calculations, for the determination of orbital energies and shapes,

as well as conformations of side chains and substituents, even for

relatively larger molecules ranging in size from phenethylamine to

LSD; (2) describe the results of our studies of phenethylamines,

amphetamines, tryptamines and ß-carbolines (hamines); (3) show that

ionization potentials are important empirical parameters useful in

the quantitative correlation between physical properties and hallucinogenic activities, as well as other types of potencies in biological

systems; and, (4) suggest that these correlations are not fortuitous,

but, instead, confirm the role of complexation by the aromatic moiety

at biological amine receptor sites.

ELECTRONIC INFORMATION FROM PHOTOELECTRON SPECTROSCOPY

Photoelectron spectroscopy is a technique for measurement of ionization

potentials, or orbital energies, of molecules. Our instrument is

equipped with a He(I) discharge source, which produces photons of

21.21eV energy. Upon collision with vapors of a sample, the photons

cause excitation to a free electron and various states of the radical

cation of the molecule. The energy required for ionization, the

ionization potential (IP), is measured as the difference between the

incident photon energy and the energy of the ejected electron. With a

He(I) source, IPs only up to 21eV can be measured, but this allows

determination of the energies of the low-lying valence IPs of organic

molecules,

The connection between the IPs and the orbital energies of a molecule

was made by Koopmans (1934). That is, the energy of an electron in a

423
molecule relative to the energy of a free electron according to SCF

molecular orbital calculation is the kinetic energy of the electron

plus the attraction of the electron for the molecular nuclei (the socalled "one electron orbital energy") minus the repulsion of that

electron for all other electrons in the molecule. Upon removal of an

electron from a molecule, all of these terms go to zero. By convention, IPs are positive, whereas orbital energies are negative.

Koopmans' theorem is based on two assumptions: (1) after ionization,

the orbitals are unchanged from those of the neutral molecule, and

(2) correlation energy (the inescapable error in SCF calculations)

is the same in ground and radical cation states. Neither of these

assumptions is correct, but they tend to cancel, and in any case

become less troublesome as the size of the molecule increases.

Throughout this paper, Koopmans' theorem language will be used: the

negatives of orbital energies will be considered synonomous with

the IPs of a molecule.

Photoelectron spectroscopy can reveal more than the IPs, or orbital

energies, of molecules. For example, vibrational structure in the

spectra can reveal considerable information about the type of orbital

involved in that ionization process, and the magnitudes of changes in

IPs caused by substitution of a molecule can provide evidence about

the shapes, or positions of electron densities, in orbitals. An example relevant to the subject of this paper is given in Figure 1.

Figure 1. Ionization potentials of benzene,

tolune, and phenethylamine

Benzene has two degenerate highest occupied molecular orbitals which

give rise to identical IPs of 9.25eV. Upon methylation of benzene,

the degeneracy of these orbitals is lifted dramatically. One is

not changed at all, since there is a node at the site of methylation,

while the HOMO is raised by about 0.35eV (8 kcal/mol) by the weakly

electron-donating methyl substituent. Two values, 8.72 and 8.9eV,

are given for the first IP of toluene, and these result from population

of different vibrational states of the radical cation ground state.

The vertical IP, 8.9eV, represents ionization to form the radical

cation in the same geometry as the ground stat of toluene. Since the

vertical IPs will represent the best indicator of reactivity, they

will be used here. Phenethylamine has IPs very similar to toluene,

and for the same reason: changes in IPs or orbital energies are

424
largest when substituents are attached at the site of highest orbital

electron density. According to perturbation theory, the change in

orbital energy is proportional to the square of the orbital coefficient at the site of substitution.

Having given this all too brief introduction to photoelectron specionization potent to chemical phenomena, namely the reactivity

troscopy, let us pause for a moment to consider the relevance of

and complexing abilities of molecules. It can be shown that IPs are

useful correlators and predictors of chemical reactivity, and this

leads us to believe that quantitative structure-reactivity relationships (QSRR) for these simple system will be paralleled by quantitative structure-activity relationships (QSAR) in biological systems.

Figure 2 shows a plot of the logs of the second-order rate constants

for acid-catalyzed hydrations of alkenes (Oyama and Tidwell 1976) and

styrenes (Schubert and Keefee 1972) versus the IPs of the alkenes

(McAlduff and Houk, unpublished). Over a range of 1018 in reactivity,

Figure 2. Plot of log k of acid-catalyzed hydrations

of alkenes versus the IPs of the alkenes

425
there is a reasonably linear plot--those molecules with the lowest

IPs react fastest and vice versa. Why should such a plot be linear?

There are two ways to think about such a plot. From the point of

view of perturbation theory, we may say that the transition state for

proton transfer from hydronium to an alkene is stabilized by interaction of the alkene HOMO and the hydronium LUMO. According to

perturbation theory, the stabilization resulting from this interaction

is inversely proportional to the HOMO-LUMO gap, or, using Koopmans'

theorem,the difference between the alkene IP and the electrophile

EA. Alternatively, the correlation between rates and IPs may be

considered to arise in the following way:

Assuming the transition state is near the carbonium ion intermediate

in structure, the energy of the transition state may be estimted in

step, First, an electron is transfered to the electrophile. The

energy of this step is equal to IP(alkene) - EA(H+). Second, the CH

bond is formed between the two radicals. If this second step has a

constant energy (-BDE), then the difference between the energies of

two transition states will be directly proportional to the difference

between the alkene IPs, for the same electrophile. Linear correlations

of the type observed in Figure 2 suggest that the constancy of this

BDE is a reasonable assumption.

An even better correlation is found for the rates of bromination

reactions of alkenes (Olah and Hockswender 1974). shown in Figure 3.

These reactions involve the formation of the bromonium, and the

transition state resembles a relatively loose molecular complex.

A third example, somewhat closer to the subject at hand, comes from

our work on cycloaddition reactions. The transition states for DielsAlder reactions are believed to be akin to π complexes, stabilized by

interaction of the diene HOMO with the alkene LUMO, and we and others

have found very good correlations between the IPs of dienes and their

rates of reactions with alkenes (Houk 1975).

Finally, and probably of greatest relevance to the use of IPs to correlate activities of hallucinogens, a correlation exists between IPs of

aromatics and stabilities of molecular complexes formed with good

acceptors. We have shown earlier that (Domelsmith, Munchausen, and

426
Figure 3. Plot of log k of bromination of

alkenes versus the IP of the alkene

Houk 1977a) there is an excellent linear correlation between the electron-donor ability of a substituted aromatic, as measured by the

average of the first and second IPs, and the stability of the complex

with TCNE. In this case, a 6 kcal/mol (or 0.25eV, in more conventional

IP units) decrease in IP is accompanied by a 1 kcal/mol increase in the

stability of the complex.

Because of the success of these quantitative structure-reactivity

relationships (QSRR), we had some hope at the outset of this work that

IPs would be useful quantities for QSAR. As will be shown later, this

is, indeed, the case.

PHOTOELECTRON SPECTRA OF HALLUCINOGENS

Tryptamines and LSD

Indole has five occupied π orbitals, two of which are considerably

higher in energy than the others, and are expected to dominate the

427
chemical characteristics of indole and its derivatives. In the photoelectron spectrum of tryptamines, three π IPs are resolved, along with

that due to the amine lone pair on the side-chain nitrogen (Domelsmith,

Munchausen, and Houk 1977a ). The two highest π orbitals of indole,

which has a similar π system, are shown in Figure 4. These pictures,

Figure 4. HOMO and SHOMO of indole (CNDO/2)

which indicate the p orbital coefficients by the size of the circles,

are obtained by CNDO/2 calculations, but qualitatively similar orbital

shapes were obtained by Weinstein et al. (1976) using higher quality

ab initio calculations. As mentioned earlier, the coefficient magnitudes at various positions in a given orbital are indicators of the

.

influence that substituents will have on the IP. The HOMO has large

coefficients at carbons 4, 6, and 7, but a very small coefficient at

carbon 5. By contrast, the second highest occupied MO (SHOMO) has

the largest coefficient at carbon 5. The first IP should not be

.

influenced much by substitution at carbon 5, while the second should

be influenced substantially. The opposite effect should be observed for

substitution at carbon 4. To date, we have only measured the effects

of substituents at the 5 position. Table I lists the first and second

π IPs of tryptamines we have studied. The first IP remains essentially

constant, with changes in apparent values due, in part, to overlapping

of bands. The second IP is influenced significantly by methyl and

even more so by methoxy substitution. The MOs of the substituted

complex will not, of course, be unchanged from those of the parent

compound. For example, Weinstein et al. (1976) have shown the significant reorientation which occurs upon substitution of tryptamine by

hydroxyl groups at various positions. It is also interesting that the

ability of tryptamines to inhibit binding of D-LSD to rat brain membranes (IC-50, see Table I) increases as the IP of the tryptamine

decreases. The first IP changes little, hut the second decreases

428
Table I. IPs (eV) of Tryptamines1

substantially upon going from parent to methyl to methoxy. So, also,

does the ability to bind to the LSD site, as shown by the IC-50.

LSD has a much lower IP than simpler tryptamines (Domelsmith,

Munchausen and Houk 1977b ). The first two IPs are due to aromatic π

orbitals. The first IP here changes most from the value for tryptamine, since conjugation of the vinyl group, which lowers IPs, is much

greater in the HOMO than in the SHOMO, the oppositetothe effect at

carbon 5. LSD, the best electron-donor in the series of tryptamines

we. have studied, is the most potent binder to rat brain membranes

(Table I).

Harmala Alkaloids

Harmine and four of its derivatives have been studied. The spectra

of these compounds, shown in Figure 5, exhibit four well resolved

bands with IPS in the range of 7-10.5eV. In addition to these bands,

which are due to π orbitals, the pyridine-like nitrogen lone pair

appears as a shoulder at 9.3eV. These IPs are given in Table II.

Table II. Ionization Potentials (±0.06eV) of Harmala Alkaloids

The substituent effects can be interpreted in a fashion similar to

that used earlier for tryptmines. That is, the two highest occupied

MOs of norharmane are such that the site of hydroxy or methoxy substitution has a near-node in the HOMO and a large coefficient in the

SHOMO, as shown in Figure 6.

429
Figure 5. Photoelectron spectra of (top to bottom)

norharmane, harmane, harmine, and harmaline
Figure 6. HOMO and SHOMO of norharmane (MINDO/3)

In harmol, substitution of a hydroxy group has caused the first IP to

increase slightly and the second to decrease substantially as compared

to the IPs of harmane. This behavior is experimental evidence for a

node in the HOMO at the site of substitution, and a relatively large

orbital electron density at this site in the SHOMO. In phenol, the

HOMO is raised substantially from the position in benzene, and the

SHOMO is slightly lowered. The methoxy group of harmine lowers both

IP1, and IP2, but the second to the greatest extent. It is interesting to note that this behavior is identical to thatobservedfor

substitution at the carbon 5 of tryptamine.

Although it has been observed that harmines and related ß-carbolines

are hallucinogenic, quantitative data are mainly available on the

ability of these compounds to inhibit MAO.

Phenethylamines

Each of these molecules has two high-lying aromatic orbitals, resembling those of benzene, and the side-chain amino lone-pair orbital.

These give rise to IPs between 8 and 10eV, and are listed in Table III

(Domelsmith, Munchausen, and Houk 1977).

Table III. Ionization Potentials of Phyenthylamines

431
In the parent and p-substituted compounds, the HOMO and SHOMO are

related to those of toluene, shown earlier in Figure 1. Both 3,4-

dimethoxyphenethylamine and mescaline have lower IPs due to the

presence of additional donor substituents. Interestingly, mescaline

us that the central methoxyl group has become non-planar, and thus a

has a higher IP than 3,4-dimethoxyphenethylamine, which indicates to

poorer electron donor. This variation in methoxyl conformations will

be discussed in more detail later.

Amphetamines

The most extensive series of hallucinogens we have stuedied are

amphetamines. The aromatic rings of phenethylamine and amphetamine

are electronically indentical, as reflected by th photoelectron spectra show in Figure 7. Indeed, the same can be said for toluene, a

Figure 7. Photoelectron spectra of

phenethylamine and amphetamine

finding which not only has implications about the activities of these

compounds, but shows that ionization potentials of one series may be

estimated very accurately from ionization potentials of of another

series. The lowest two ionization potentials of the amphetamines we

have studied are given in Table IV, along with quantities which are

to be disscussed later. These data confirmed many expectations, but

also produced some surprises. In order to investigate these compounds

in more detail, ab initio calculations using the STO-3G basis set

were carried out on polymethoxybenzenes. Figure 8 shows the results

of these calculations for dimethoxybenzenes, along with the IPs measured for these molecules.

432
Table IV. IPs (±0.06eV) Partition Coefficients, and

Hallucinogenic Activities of Amphetamines

Figure 8. Calculated IPs and orbital shapes for dimethoxybenzenes. Experimental values are given in parentheses.

433
The para-dimethoxy compound has a very large split in degeneracy due

to the fact that one orbital is raised both substituents, while

the second is essentially unaffected. The first IP is 1.35eV lower

than that of benzene, while the second is essentially identical to

that of benzene. Meta-dimethoxybenzene has a much higher IP and much

closer first and second IPs due to the smaller and more nearly equal

influence of the two methoxys on both orbitals. The meta-disubstitution pattern produces a much less potent electron-donating aromatic

ring than the para.

According to the simple analysis we have used so far, ortho- and metadimethoxybenzene should have identical IPs. Higher order effects

change this simple picture. While we have discussed this in more

detail elsewhere (Domelsmith and Houk 1978), this effect can be

summarized simply. In the meta case, the HOMO is lowered somewhat by

mixing with the LUMO, and the SHOMO is raised by mixing with the

lowest benzene π orbital. The HOMO and SHOMO of the ortho compound

are affected in opposite fashion. In the photoelectron spectrum of

ortho-dimethoxybenzene, a small shoulder is observed at 7.8eV, but

the first strong band appears at 8.44eV, considerably above the calculated IP of 7.63eV. Much better agreement between calculation and

experiment is obtained when the calculation is carried out on a geometry in which one of the methoxyls is rotated by 90°. This is shown

at the far right of Figure 8. In such a conformation, the methoxy is

a much poorer donor, since the π orbital perpendicular to the COC

plane of the ether linkage is rotated away from ideal conjugation with

the aromatic π orbitals. We do not as yet know why ortho-dimethoxybenzene prefers a conformation in which at least one of the methoxyls

is rotated out of the benzene ring plane, but the photoelectron spectrum seems to demand to this for the major conformer. It is worth noting

that the calculated IPs, although obtained from ab initio calculations,

scaled to fit experimental IPs by a least squares treatment and done

for several conformations, still give relatively inaccurate predictions

of IPs even when calculations on many different geometries are

performed. We feel that this is an important reason for obtaining

experimental IPs rather than only calculating these.

The various dimethoxyamphetamines show a similar pattern (Table IV).

The 2,5-dimethoxyamphetamine, which has the para-dimethoxy pattern,

has much lower IPs than those amphetamines with the ortho- and metadimethoxy patterns. This has some significance in understanding

hallucinogenic activities of amphetaines. The 2,5-dimethoxyamphetamines have been long recognized to be more potent hallucinogens than

isomeric analogs (Shulgin, Sargent and Naranjo 1969). The lower IPs

and higher electron-donating abilities of the 2,5-dimethoxy compounds

are possibly the origin of higher activities of these compounds.

Turning to the more highly substituted amphetamines, this special

potency of the p-dimethoxy pattern persists. Inspection of Table IV

shows that the best electron-donors are those with the 2,5-dimethoxy

substitution pattern.

THE USE OF IONIZATION POTENTIALS IN QUANTITATIVE STRUCTURE ACTIVITY

RELATIONSHIPS

Photoelectron spectral studies have revealed a great deal about the

434
electronic structures of hallucinogenic drugs. The ionization potentials obtained in this way have also been found to be very useful

indicators of activity. One of our early correlations is shown in

Figure 9.

Figure 9. Correlation between minimum effective brain

levels (MEBL) to interfere with conditioned avoidance

respnse in rats (Vogel and Evans 1977) and

average of the first and second IPs

This plot includes both phenethylamines and tryptamines and is surprising for that reason, since most workers in this area believe that

these compounds influence different receptors. More extensive structure-activity relationships have been tested for the hallucinogenic

amphetamines, on which a variety of physical measurements have been

made, and many good correlations between physical properties and

activities have been found. Of course, the availability of human

activity data measured by Shulgin and coworkers (1969) has provided, for

this series of molecules, an unprecedented fund of information with

which to test the importance of various physical properties upon

hallucinogenic activity. Correlations between these hallucinogenic

activities reported by Shulgin and coworkers (in molar mescaline

units, MMU) and the IPs of amphetamines were tested.

For the first IP alone, a moderate correlation is found:

which indicates a substantial increase in activity with a decrease in

IP, in accord with the expected increase in the complexing ability of

the aromatic ring as the IP decreases. If IP2, is also used, no sig435
nificant improvement is obtained, in light of the two independent

parameters now included in the correlation equation:

log MMU = -2.35 IP1 - 0.13 IP2 + 20.42

(n = 11, r = 0.86).

The most significant deviation from either of these equations is

observed for 4-bromo-2,5-dimethoxyamphetamine, and this molecule was

not included in the correlation. Even for those compounds fitting

this equation better, there are some notable deviations which give a

clue to how the correlation can be improved. For example, although

the first two IPs for 3,4-dimethoxyamphetamine and 3,4-methylenedioxyamphetamine are very close, 3,4-methylenedioxyamphetamines is at least

2.8 times more active. Similarly, 2,4,5-trimethoxyamphetamine and

4-methyl-2,5-dimethoxyamphetamine have essentially identical IPs, but

4-methyl-2,5-dimethoxyamphetamine is approximately 4.4 times more

active than 2,4,5-trimethoxyamphetamine. In each of these cases, the

more active drug is the one which has a significiantly higher 1-

octanol: water partition coefficient (see Table I); log P for 3,4-

methylenedioxyamphetamine is 1.38 while that for 3,4-dimethoxyamphetamine is only 1.00 and log P for 4-methyl-2,5-dimethoxyamphetamine is

2.08 compared with 1.74 for 2,4,5-trimethoxyamphetamine.

Correlations between partition coefficients and hallucinogenic activties have been shown by Barfknecht, Nichols, and Dunn (1975), and

more recently Nichols, Shulgin, and (1977) have described correlations of partition coefficients of both phenethylamines and amphetamines with 5HT stimulation in sheep umbilical artery preparations.

The Barfknecht et al. correlation, uses a set of amphetamines Similar

log MU = 3.15 log P - 0.50 log P2 - 3.17

(n = 26, r = 0.79)

to, but more exstensive than the set used in correlation. The

Nichols et al. correlation, could be modified to eliminate the unusual

log RBR = -0.89 log P + 0.95(log P)2 -0.16(log P)3 - 0.23

(n = 15, r = 0.98),

third term by using an indicator parameter to account for steric bulk

of large alkyl substituents. Since both log P and IP are of value in

Predicting activities, correlations combining both of these physical

properties were tested. The relationships between hallucinogenic

activity and IP1, IP2, and log P for the same eleven compounds are

given by the equations:

log MMU = -1.48 IP1 + 0.78 log P + 11.15

(n = 11, r = 0.94)

log MMU = -1.42 IP1, - 0.19 IP2 + 0.80 log P + 12.30

(n = 11, r = 0.94)

Once again, inclusion of IP2 gave no significant improvement. A plot

the observed versus calculated activity according to the equation

436
using only IP1 and log P is given in Figure 10. The 4-Br-2,5-

dimethoxyamphetamine is included in this figure, but was excluded from

the regression analysis.

Figure 10. Plot of predicted versus observed

hallucinogenic activities based on IP1 and log P

Ionization potentials have also been found to provide valuable indicators of potencies of various amphetamines and tryptamines. Green

and coworkers have determined the inhibition of high affinity d-LSD

binding by various amphetamines, tryptamines, mescaline and LSD in

rat brain homogenates (Green et al. 1976). The relationship between

the inhibition constant, IC50, and IP1 and IP2 for the drugs mescaline,

3,4,5-, 2,4,6- and 2,4,5-trimethoxytryptamines, 2,4- and 2,5-dimethoxyamphetamine, 5-methyl- and 5-methoxytryptamine, tryptamine, and d-LSD,

is given by

-log IC50 = -3.81 IP1 - 1.64 IP2 + 47.78

(n = 10, r = 0.85)

Similarly, Bennett and Aghajanian (1974) measured drug concentrations

required to displace d-LSD in rate brain homogenates for a wide variety

of psychotropic drugs, including several that we have investigated by

photoelectron spectroscopy: the phenothiazine tranquilizers, promethazine and chloropromazine (Domelsmith, Munchausen, and Houk 1977c) .

LSD, dimethyltryptamine, 4-methyl-2,5-dimethoxyamphetamine, mescaline,

437
and dopamine. The relationships between the effective dose necessary

to displace 50 percent o f d-LSD, ED50, and IPs are given by:

-log ED50 = -3.49 IP1 + 32.50

(n = 7, r = 0.92)

-log ED50 = -2.79 IP1 - 1.93 IP2 + 43.26

(n = 7, r = 0.97)

These relationships indicate that ionization potentials are quite

promising predictors of the ability of hallucinogens to bind to the

LSD binding site. We hope to test the generality of this conclusion

with a more extensive series of compounds.

CONCLUSION

These correlations suggest that the complexing abilities of the

aromatic rings of amphetamines are very imporant in the production

of hallucinogenic activity. Furthermore, even for the less closely

related compounds, ionization potentials can predict the ability of

molecules to bind to biological receptors.

Although only scratching the surface of compounds available for

investigation, our studies show that photoelectron spectroscopy is not

only capable of providing valuable experimental information about the

electronic structures of hallucinogenic molecules, but the ionization

potentials of the aromatic portions of these molecules can be used

profitably in developing quantitative structure-activity relationships.

From a more general point of view, the correlations we have observed

help to restore some of the lost faith in the importance of electronic

characteristics of molecules in producing biological activities.

ACKNOWLEDGEMENTS

We are grateful to the National Institutes of Health for financial

support of this research (GM-21311); to George Anderson III, A. T.

Shulgin, and Harel Weinstein for helpful discussions and unpublished

data; to Jacqueline Bryant and Albert Manian for some of the compounds

used in this work.

REFERENCES

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of psychotomimetic activity of phenethylamines and amphetamines with

1-octanol-water partition coefficients, J Med Chem., 18:208-210,

1975.

Bennett, J.L., and Aghajanian, G. K. D-LSD binding to brain homogenates: possible relationship to serotonin receptors, Life Sci,

15:1935-1944, 1974.

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Domelsmith, L.N., Munchausen, L. L., and Houk K. N. Photoelectron

spectra of psychotropic drugs. 1. Phenethylamines, tryptamines, and

LSD. J. Am Chem Soc, 99:4311-4321, 1977a.

Domelsmith, L. N., Munchausen, L. L., and Houk, K. N. Lysergic acid

diethylamide. Photoelectron ionization potentials as indices of

behavioral activity. J Med Chem, 20:1346-1348, 1977b .

Domelsmith, L. N., Munchausen, L. L., and Houk, K. N. Photoelectron

spectra of psychotropic drugs. 2. Phenothiazine and related tranquilizers; J Am Chem Soc, 99:6506-6514, 1977c.

Domelsmith, L. N., and Houk, K. N. Photoelectron spectroscopy and drug

design: ionization potentials and partition coefficients as predictors

of substituted amphetamine psychoactivities. Presented at the International Symposium on Quantum Biology and Quantum Pharmacology, March 9,

1978, Flagler Beach, Florida. Submitted for publication 1978.

Green, J.P., and Kang, S., The correlation of electronic structures of

indole derivatives with their biological activities. In: Kier, L.B., ed.

Molecular Orbital Studies in Chemical Pharmacology. Berlin: SpringerVerlag, 1970, pp. 105-120.

Green, J. P., Johnson, C. L., Weinstein, H., Kang, S., and Chou, D.

Molecular determinants for interaction with LSD receptor: biological

studies and quantum chemical analysis. NIDA Symposium, October 21-22,

1976. In press.

Houk, K. N. The frontier molecular orbital theory of cycloaddition

reactions. Accounts Chem Research, 8:361-369, 1975.

Jacob III, P., Anderson III, G., Meshul, C. K., Shulgin, A. T., and

Castagnoli Jr., N. Momomethylthio analogues of 1-(2,4,5-trimethoxy-

-phenyl)-2-aminopropane. J Med Chem, 20:1235-1239, 1977.

Koopmans, T. Uber die zuordnung von wellenfunktionen und eigenwerten

zu den einzelnen elektron eines atom. Physica, 1:104-113, 1934.

Nichols, D. E., Shulgin, A. T., and Dyer, D. C. Directional lipophilic

character in a series of psychotomimetic phenethylamine derivatives.

Life Sci 21:569-575, 1977.

Olah, G. A., and Hockswender Jr., T. R. Electrophilic reactions at

multiple bonds. I. The study of selectivity and the role of π and σ

complexes in the addition of bromine to alkenes and alkynes in 1,1,2-

trichlorotrifluoroethane solution. J Am Chem Soc, 96:3574-3580,

1974.

Oyama, K., and Tidwell, T. T. Cyclopropyl substituent effects on acidcatalyzed hydration of alkenes. Correlations by σ

+

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Rabalais, J. W. Principles of ultraviolet photoelectron spectroscopy.

New York: Wiley-Interscience, 1977.

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Schubert, W.M., and Keefee, J. R. The acid-catalyzed hydration of

styrenes. J Am Chem Soc, 94:559-566, 1972.

Sepûlveda, S., Valenzuela, R., and. Cassels, B. K. Potential psychotomimetics. New bromoalkoxyamphetamines. J Med Chem, 15:413-415,

1972.

Shulgin, A. T., Sargent, T., and Naranjo, C. Structure activity

relationship of one-ring psychotomimetics, Nature (London), 221:537-

541, 1969.

Snyder, S. H. and Merril, C. R. A relationship between the hallucinogenic activity of drugs and their electron configuration. Proc:

Nat Acad Sci U.S.A., 54:258-266, 1965.

Snyder, S. H. Psychedelic drug activity: electronic, steric and biochemical correlates. In: Kier, L.B., ed. Molecular Orbital Studies in

Chemical Pharmacology. Berlin: Springer-Verlag, 1970, pp. 238-261.

Vogel, W. H., and Evans, B. D. Structure-activity-relationships of

certain hallucinogenic substances based on brain levels, Lifes Sci

20:1629-1635, 1977.

Weinstein, H., Chou, D., Kang, S., Johnson, C. L., and Green, J. P.

Reactivity characteristics of large molecules and their biological

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Quantum Chem: (Quantum Biol Symp No. 3, 135-150, 1976.

AUTHORS

Linda N. Domelsmith, Ph.D., Department of Chemistry, Louisiana State

University, Baton Rouge, Louisiana 70803.

Kendall N. Houk, Ph.D., Department of Chemistry, Louisiana

State University, Baton Rouge, Louisiana 70803.

440
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

An Assessment of Parameters in

QuaSAR Studies of Narcotic

Analgesics and Antagonists

Robert Katz, Steven Osborne, Florin Ionescu,

Peter Andrulis, Jr., Robert Bates, William

Beavers, Paul C. C. Chou, Gilda Loew, and

Donald Berkowitz

Antinociceptive activity of narcotic agonists "in vivo," was recently quantitatively related to lipophilicity (log P). In both studies

(Kutter et al. 1970; Jacobson, Klee and Dunn 1977), the types of

analgesics included in analyses belonged to different classes with

diverse chemical structures. They were, however, carefully selected

to behave mainly as pure agonists "in vivo." The first part of our

presentation concentrates on narcotic drugs capable of showing both

agonist and antagonist effects, and the possible dependence of these

opposite effects, inherent in the same structure, on log P. On the

other hand, numerous previous studies (e.g., Kaufman and Kerman 1974;

Kaufman, Popkie, and Koski 1976; Loew and Berkowitz 1975; Loew,

Berkowitz, and Newth 1976) elaborated on the relationship between

activity and minimum energy conformations of molecules or activity

and charge densities calculated by various molecular orbital methods.

We shall discuss the correspondence between charge densities (calculated by the INDO, PCILO, and IEHT molecular orbital methods) and

the 13C NMR chemical shifts (δ ) of the protonated molecule measured

in D2O. We also extend the 13C NMR data of various mixed agonistantagonists to structure and activity studies. Chemical structures

for all the compounds included in the various studies (1a-6b) are

presented in figure 1.

LIPOPHILICITY AND BIOLOGIC ACTIVITY

Drug distribution coefficients (DDC) for compounds in table 1 were

measured by dissolving the narcotic drug in n-octanol saturated with

buffer ( pH 7.40 at 37°C). An approximately 10 fold excess of

buffer was added and the mixture vigorously agitated (2 minutes),

centrifuged (2000 rpm, 75 min.), and placed in a water bath (37°C,

90 min.). Five ml. aliquots were examined at 284 nm. against standard (20 mg. of sample in pretreated octanol). pKa values for (-) BC

2888 and for norketobemidone. HCl were determined in double distilled

water under N2 at 37°C. Values were calculated from the titration

curve by using a computer program entitled PHPAIR. All other pKa

values from table 1 were determined by others (see legend to table 1).

Partition coefficients were calculated from DDC and pKa values by

use of the following formula:

441
As for the biological activity data, receptor bindtng affinity in

rat brain homogenates (KRB) was measured by replacing specifically

bound [3H] dihydromorphine following the method of Klee and Streaty

(1974). KRB (nmoles/i) is the concentration of dru required to inhibit 50 percent of the stereospecific binding of [3

H] dihydromorphine in the rat brain homogenates. Antagonist potency in the

guinea pig ileum (Ke) and agonist binding (ID50)(equivalent to

agonist potency in the same "in vitro" system) were used as previously determined by Kosterlttz and his group (Kosterlitz and

Watt 1968; Kosterlitz, Lord, and Watt 1973

Plots of log P vs. log Ke and log P vs. log ID50 are shown in figures 2 and 3. A parabolic dependence of both agonist and antagonist activities on log P is obvious from these plots. Naloxone

(2e). naltrexone (2a), nalorphine (la), oxymorphone (2b), and norketobemidone (5a) are members of a group with log P < 2.0, while

the rest of the compounds (see table 1) are members of a group with

log P > 2.5. The plot of log P vs. log agonist or antagonist

potency shows good linear dependence in this second group. Optimum

log P values between 2 and 2.5 can be approximated for both activittes.

Linear Hansch type correlations between log 1/Ke vs. log P and log

1/ID50 vs. log P provided extremely low correlation coefficients

(0.46 and 0.066 respectively). The quadratic correlation with log P

is presented in table 2. Equations 1 and 5 from this table are

statistically significant, but still explain only 67 or 60 percent

respectively of the variance present. A similarly "mediocre" correlation results when log 1/Ke is correlated with log 1/KRB (eq. 3).

However, combining log P and log 1/KRB results in a significant

improvement (eg. 2) R = 0.91, R2 = 0.82.

It can-be said that a correlation coefficient of 0.88 for log ID50

vs. log 1/KRB tends to support the fact that there are fewer inherent possibilities for errors in measurement of ID50 than in case

of Ke. Thus, It is not surprising that eq. 4, combining both log P

and log 1/KRB, provides the best correlation in table 2 (although

statistically significant only at the 92 percent level). Eq. 2 and

4 yielded an ideal log P = 2.25 ± 0.44 for log 1/Ke and of 2.42 ± 

for log 1/ID50 in accordance with plots fmm figures 2 and 3.

The correlation reported by Kutter et al. (1970) is quadratic in

log P. The biological activity correlated with log P is a ratio

of two different "in vivo" agonist effects (in rabbits), and is considered to be a measure of different capability of analgesics to

cross the blood brain barrier. The correlation is given below:

log (Civentr/Civ) = 0.673 + 0.036 log P - 0.090 (log P)2

n = 11, s = 0.297, R

2

 = 0.941, R = 0.970 (eq. 8)

In this equation, as pointed out by Jacobson, Klee, and Dunn (1977),

the log P term is statistically not warranted. However, a modified

correlation, statistically valid, still contains the (log P)2 term.

442
Unfortunately, log P values in this study were measured in heptanewater system and therefore the calculated ideal log P = 0 is not

directly comparable with ours. As mentioned above, Jacobson et al.

obtained a correlation between antinociceptive activity (as measured by the standard hotplate assay in mice) and log P. Addition

of a binding term quantitattng the binding affinity of the narcotic

agonist to rat brain homogenate(identical with our KRB), to log P,

provided a good, statistically valid linear equation:

log 1/C = 4.254 + 1.107 log 1/KRB + 0.317 log P

n = 10, S = 0.62, R2 = 0.82, R = 0.91 (eq. 9)

As the authors state, this correlation suggests that besides lipophilicity, antinocicepttve activity depends on other parameters

adequately expressed by "in vivo" receptor binding affinity. Returning to table 2, we can see that equations 2 and 4, the best for

"in vitro" agonist as well as antagonist activities, are quadratic

in log P and also contain the term KRB which quantitates 'in vitro"

receptor binding affinity.

There is no contradiction between our findings and those discussed

in eq. 8 and 9. While log P "in vivo" is synonymous with transport,

"in vitro" it could be interpreted to represent a hydrophobic

"pocket" on the receptor. Equations 2 and 4 could thus be considered extensions of eq. 8 and 9 to the receptor level. As reflected

by eq. 2 and 4, the parallelism between agonist and antagonist

behaviour at the receptor level is remarkable. No major differences

am discernible, although by comparing equations 5, 1, 2, and 4, it

appears that KRB is more relevant to agonist activity than to antagonist activity.

In summary, we can state that similarities between agonist and antagonist behaviour of a drug. as expressed by eq. 2 and 4, tend to

support the presently accepted view of a receptor possessing agonist

and antagonist conformations in equilibrium.

ELECTRONIC FACTORS AND BIOLOGICAL ACTIVITY

We tntended to compare atomic charges (Q) calculated by means of

various molecular orbital methods for compounds in figure 1 to their

13C NMR chemical shifts (δ ) (D20). This comparison could validate

the use of δ values in structure and activity studies, as well as in

Hansch type correlations, instead of Q values. Naloxone•HCl (2e),

naltrexone•HCl (2a), nalbuphone•HCl (2c), and nalmexone•HCl (2g),

all oxymorphone type antagonists or mixed agonist-antagonists, were

Selected for study.

A recent crystal structure of naloxone (Karle 1974) pmvided the

oxymorphone fused ring structure. Hydrogens were placed by using

equalized standard bond length (Pople and Beveridge 1970) and bond

angles (CCH, HCH, etc.). For methyl groups, tetrahedral geometry

was used. The phenyl hydroxyl group was set in plane, trans to the

furan oxygen at C4, a minimum energy conformation calculated by

PCILO (Diner et al. 1969). The C14 hydroxyl group is ß and trans to

C9. Heavy atoms were positioned as in recent naloxone crystal structures with the cyclopropyl methyl group of naltrexone . HCl following

the cyclazocine•HCl geometry. The N-side chain geometries were

443
based on the following two torsion angles: τ1 defined as C9-N-C17-

C18 and τ2 as N-C17-C18-19, (τ 1 ABCD = clockwise rotation angle

Cyclazocine•HCl (4a) was the only benzomorphan derivative included

to superimpose A on D while looking from atom B to atom C).

in the study.

PCILO charges were calculated for the six lowest local minimum

energy conformations. The Same conformations were chosen for both

naltrexone and cyclazocine. INDO calculations were performed on

the two lowest local minimum energy conformations of both molecules,

while IEHT calculations were performed only on the lowest energy

conformation of each molecule. Calculations for nalbuphone•HCl

and nalmexone•HCl followed a similar pathway. The internal error

far net charges iS not clearly defined for INDO and PCILO results,

although it appears smaller than that in IEHT, where charge inconsistency is the controlling force in the iteration procedure.

For IEHT, the ΔQ due to internal error was set to be no greater

than O.005 electrons/atom.

13C NMR measurements for all of the above compounds, as well as

others included in table 6 were taken on the protonated base

in D2O, as opposed to previous reported work (Terui et al. 1975;

Carroll et al. 1976; Brine et al. 1976) where free bases and organic

solvents were used, Broadband decoupled and offresonance NMR

spectra were run on each sample at 22.63 MHz, on a Bruckner WH-90

spectrometer at 30-33°. The chemical shifts obtained were referenced to internal dioxane (at δ = 67.9). The assigning of carbons

to peaks was greatly aided by comparison with similar substances

and by well established substituent effects (Levy and Nelson 1972;

Wehrit 1973).

Comparison of the shifts measured in this study on protonated amines

in D20 with shifts an related free amines in DCCl3 and d6-DMSO shows

small differences, and no cases which suggest revision of assignments. In the case of naltrexone•HCl and naloxone•HCl (Carroll

et al. 1976) the largest differences come in the sp2 carbons of the

N-allyl group; C19 is shifted downfield by protonation of the nitrogen, while C18 is shifted upfield. The shifts for the free allyl

amine are similar to those in allyl ethyl ether, whereas those

in the corresponding ammonium salt are much closer to those of

ethyl acrylate (Levy and Nelson 1972).

13C NMR chemical shifts in D20 for naltrexone•HCl are compared in

table 3 with atomic charges calculated by the INDO, PCILO, and IEHT

methods in one minimum energy conformation (Martin, Martin, and

Idiot 1975). When 13C-δ values of nonaromatic carbons with sp3

hybridization (C5, C7-C16) are plotted against atomic charges

(Krandall and Sojka 1972), it becomes apparent that 13C-δ vs. Q

correlates better in the order INDO, PCILO, and IEHT. This improvement is evident from figures 4. 5, and 6. Only the atomic charges of

the two quaternary carbons C13 and C14 are underestimated by IEHT.

PCILO overestimates atomic charge on C5, C13, and C7 while underesti-.

mates C14 and C16. INDO does well only with C8, C9, C1O, and C15.

444
Table 4 summarizes 13C-δ and QIEHT values for naloxone•HCl, nalbuphone•HCl, and nalmecone•HCl. If plotted, these data show consistently good correlations comparable to the one for naltrexone•HCl

from figure 5.

Results obtained for cyclazocine•HCl are shown in table 5. In

this case net charge densities calculated by IEHT are shown to correlate best with the 13C-δ values of this benzomorphan derivative.

The above study suggests that 13C-δ values are valid measures of

atomic charges possibly for a broad variety of narcotic structures.

It is of interpst to point out that such 13C-δ vs. atomic charge

plots have been used recently to validate consistency of CNDO/2

charges for carbonyl carbons in a series of neuroleptic drugs (Riga

et al. 1977).

Let us then look at 13C-NMR data of various mixed agonist-antagonists as shown in table 6. C9, C16 and C17 are the three carbons

neighbouring the nitrogen atom required for the activity of these

compounds. C18-C21 are various side chain carbon atoms starting

ß from the nitrogen. We could find no meaningful relationship

between any of these chemical shifts, taken individually, and

antagontst potency Ke, whether compounds are compared according

to similar side chains or similar ring structures. The same lack

of correlation was found when the sum C9, 16, 17-δ was compared

with Ke.

However, across several classes with similar side chains, a certain

pattern emerges. Thus, benzomorphans 4c, 4d, 4e, and 4f, with an

isobutenyl moiety contained in the side chain, show an increase in

ΣC9, 16, 17-δ, which paralleled an increase in the respective Ke

values (see table 7). This could mean that an increased shielding

of the carbon atoms α to nitrogen (as evidenced by the lower sum

of chemical shifts) results in an increased antagonist potency

of the drug. This can be rephrased to say that the less the inductive release of electrons by C9, C16 and C17 (presumably toward

the nitrogen), the better antagonist the drug is,

Two other subgroups of compounds exist, each with a different side

chain, but bath possessing the 146 hydroxyl group. It appears that

both in subgroup one (2a and 3b, in table 8) and in subgroup two

(3c, 2c, and 2d, in table 8), with a 148 hydroxyl group present, it

is a decrease shielding that corresponds to increased antagonist

potency. Thus, the dependence of potency on 13-δ is reversed for

compounds with 148 hydroxyl group. The special role of this hydroxyl

group in drastically reducing agonist effect of mixed agonistantagonists, while increasing their antagonist effect, has been

recently established (Gordon et al. 1974). These effects could result from the electron withdrawing properties of this group as they

appear in table 8. Assumtng that the 148 hydroxyl group is the only

reason for the above reversed dependence of potency on ΣC9,16,17- δ ,

a direct involvement of the 14ß hydroxyl in receptor binding could be

445
then tentatively hypothesized. Such an involvement has been recently

considered a possibtlity by another group, based on considerations of

conformational nature (Loew and Berkowitz 1977). We would like

to emphasize that these conclusions should be considered extremely

tentative until a more vigorous study with larger sets of compounds will be undertaken.

446
Figure 1. Structures for morphine (1a-2g) and morphinan (3a-3e)

derivatves

447
Figure 1. (continued) Structures for benzomorphans (4a-4g), norketobemidone (5a) and phenylpyrrolidines (6a-6b)

448
Figure 2. Dependence of log antagonist

potency the guinea pig ileum (log Ke)

on octanol-water partition coefficients

(log P)

449
Figure 3. Dependence of log agonist potency

in the guinea pig ileum (ID50) on octanolwater partition coefficients (log P)

450
Figure 4. Correlation between atomic

charges (by INDO) and chemical shifts

of nonaromatic carbon atoms in

Naltrexone•HCl

451
Figure 5. Correlation between atomic

charges (by PCILO) and chemical shifts

(in D2O) of nonaromatic carbon atoms

in Naltrexone•HCl

452
Figure 6. Correlation between atomic

charges (by IEHT) and chemical shifts

(in D2O) of nonaromatic carbon atoms

in Naltrexone•HCl

453
Table 1. Drug distribution coefficients (DDC), pkα , octanol-water partition coefficients (log P), antagonist and agonist potency in the guinea pig ileum (Ke and ID50) and binding to the rat brain

homogenate (KRB) for 14 narcotic agonists, mixed agonist-antagonists and antagonists
Table 2. Hansch type correlations between biological activities (Ke, ID50, KRB) and octanol-water

partition coefficients of narcotic durgs from table 1.
Table 3

13C NMR chemical shifts in D2O and atomic charges for Naltrexone

•HCl (Q) as calculated by the INDO, PCILO, and IEHT molecular

orbital methods

456
Table 4. 13C NMR chemical shifts and atomic charges for other mixed mixed agonist-antagonist

members of the oxymorphone family
Table 5. 13C NMR chemical shifts (in D2O) and net charge densities

for the benzomorphan derivative Cyclazocine•HCl in the

respective minimum energy conformation

458
Table 6. 13C NMR data for mixed narcotic agonist-antagonists (in D2O)
Table 7

Dependance of antagonist potency (Ke), on the sum of C-9,16,17

chemical shifts in compounds without 14ß-OH substituent

Number ΣC-9,16,17

(δ in ppm)

Ke

4c 156.2 6.05

4d 158.8 6.83

4 e 161.4 10.40

4 f 165.3 151.00

Table 8

Dependence of antagonist potency (Ke), on the sum of C-9,16,17

chemical shifts in compounds with 14ß-OH substitent

Number ΣC-9, 16, 17

(δ in ppm)

Ke

2a* 174.3 0.38

3b* 171.3 0.86

3c** 174.9 2.14

2c** 174.5 3.00

2d** 172.5 9.00

*Compounds which have cyclopropyl methyl substituents on the

nitrogen atom

**Compounds which have cyclobutyl methyl substituents on the

nitrogen atom

460
ACKNOWLEDMENT

Financial sup ort from the PHS (grants DA-1037 to R.K. and CA10944 to R.B. are gratefully acknowledged. The authors wish

to thank Drs. A. E. Jacobson and S. A. Sojka for useful discussions.

REFERENCES

Brine, G.A., Prakash, D., Hart, C.K., Kotchmar, D.J., Moreland,

C.G., and Carroll, F. I. Ring C Conformation of 6ß-Naltrexol and

6α-Naltrexol. Evidence from Proton and Carbon-13 Nuclear Magnetic Resonance. J Org Chem, 41:3445-3448, 1976.

Carroll, F.I., Moreland, C.G., Brine, G.A., and Kepler, J.A.

Carbon-13 Nuclear Magnetic Resonance Spectra of Morphine Alkaloids. J Org Chem, 41:996-1001, 1976.

Diner, S., Malrieu, J.P., Jordan, F., and Gilliert, M. Localized

Bond Orbitals and the Correlation Problem. Theor Chim Acta 15:

100-110, 1969.

Gordon, M., Pircio, A.W., Caruso, F.S., and Pachter. I.J.,

Committee on Problems of Drug Dependence, Mexico City, 1974.

Ionescu, F., Klee, W.A., and Katz, R. Antagonist Potency and

Recptor Binding. Life Sciences, 16:1793-1794, 1975.

Jacobson, A.E., Klee, W.A.. and Dunn, W.J., Jr. A Quantitative

Relationship Between Antinociceptive Activity, Opiate Receptor

Affinity and Lipophilicity. Eur J Med Chem, 12:49-52, 1977.

Karle, I.L. On the conformation of Naloxone, a Narcotic Antagonist. Acta Crystallogr, Sect. B, 30:1682-1686, 1974.

Kaufman, J.J., Semo, N.M., and Koski, W.S. Microelectrometric

Titration Measurement of the pKa's and Partition and Drug Distribution Coefficients of Narcotics and Narcotic Antagonists

and Their pH and Temperature Dependence. J Med Chem, 18:647-655,

1975.

Kaufman, J.J. and Kerman, E. Quantum Chemical, Other Theoretical

and Physicochemical Studies on Narcotic Antagonists to Understand Their Mechanism of Action. Int J Quantum Chem, Quantum Biol

Symp, 1:289-313, 1974.

Kaufman, J.J., Popkie, H., and Koski, W. Ab-Initio LCAO-MO-SCF

Calculations of Morphine and Nalorphine and Measurement of Their

Photoelectron Spectra. J Am Chem Soc, 98:1342-1345, 1976.

461
Klee, W.A., and Streaty, R. Narcotic Receptor Sites in Morphinedependent Rats. Nature, 248:61-63, 1974.

Kosterlitz, H.W., and Watt., A.J. Kinetic Parameters of Narcotic

Agonists and Antagonists, with Particular Reference to N-Allylnoroxymorphone (Naloxone). Br J Pharmacol Chemother 33:266-276,

1968.

Kosterlitz, H.W., Lord, J.A.H., and Watt, A.J., In: Agonist and

Antagonist Action of Narcotic Analgesic Drugs, Baltimore: University Press, 1973, pp 45-61.

Krandall, J.K.. and Sojka, S.A. Carbon-13 Spectra of Allenes.

J Amer Chem Soc, 94:5084-5086, 1972.

Kutter, E., Herz, A., Teschenmacher, H.J., and Hess, R. StructureActivity Correlation of Morphine-like Analgesics Based on Efficiencies Following Intravenous and Intraventrfcular Application.

J Med Chem, 13:801-807, 1970.

Levy, G.C., and Nelson, G.L. Carbon-13 NMR for Organic Chemists,

New York: Wiley-Interscience, 1972.

Loew, G.H., and Berkowitz, D.S. Quantum Chemical Studies of Morphine-like Opiate Narcotics. Effect of N-Subttituent Variations.

J Med Chem 18:656-662, 1975.

Loew, G.H., Berkowitz, D.S., and Newth, R. Quantum Chemical Studies of Methadone. J Med Chem, 19:863-869, 1976.

Loew, G.H., Berkowitz, D.S., private communication 1977. See

this monograph.

Martin, G.J., Martin, M.L., and Idiot, S. Theoretical and Empirical Calculations of the Carbon Chemical Shift in Terms of the

Electronic Distribution in Molecules. Org Magn Res, 7:2-27,

1975.

Parke, T.W., and Davis, W.W. Use of Apparent Dissociation Constants in Qualitative Organic Analysis. Anal Chem, 26:642-645,

1954.

Theory. New York: McGraw Hill, 1970.

Pople, J., and Beveridge, D., Approximate Molecular Orbital

Riga, J., Verbist, J.J., Degelaen, J., Tollenaere, J.P., and

Koch, M.H.J. Electron Densities In Neuroleptics by X-ray Photoelectron Spectroscopy, 13C Nuclear Magnetic Resonance and

Quantum Calculations on Model Compounds. Mol Pharmacol, 13:

892-900, 1977.

462
Teruf, Y., Tori, K., Maeda, S., and Sawa, Y.K. C-13 NMR Spectra

of Morphine Alkaloids, Codeine, Thebafne, and Sinomenine, and

Related Compounds. Tetrahedron Lett, 2853-2856, 1975.

Wehrli, F.W. Carbon-13 Nuclear Magnetic Resonance Spectral

Assignments in Large Molecules with the Aid of Spin-Lattice

Relaxation Data. J C S Chem Commun, 379-380, 1973.

AUTHORS

Robert Katz*, Ph.D., Steven Osborne, Florin Ionescu, Ph.D. and

Peter Andrulis, Jr., Ph.D, Mid-Atlantic Research Institute,

Bethesda, MD. 20014

Robert Bates, Ph.D., William Beavers, Ph.D., and Paul C. C. Chou,

Ph.D., Department of Chemistry, University of Arizona, Tuscon, AZ.

85721

Gilda Loew, Ph.D. and Donald Berkowitz, Ph.D., Department of Genetfcs, Stanford University School of Medicine, Stanford, CA.

94305

*Present address: NIH, Bethesda, MD. 20014.

463
‘QuaSAR’ Research Monograph 22

G. Barnett, M. Trsic, and R. Willette, eds.

National Institute on Drug Abuse, 1978

Conformational Studies on

Phenethylamine Hallucinogens: The

Role of Alpha Alkyl Substitution

Alexandros Makriyannis and James Knittel

INTRODUCTION

A large number of methoxyphenethylamine analogs have been synthesized (Shulgin 1970; Barfknecht and Nichols 1971) and evaluated in

humans (Shwlgin, Sargent, and Naranjo 1969; Shulgin 1973) and animals (Aldous, Barrass, and Brewster 1974; Smythies, Bradley, and

Johnston 1967) for psychotomimetic activity. The overall picture

which emerges from these evaluations shows that small structural

variations can produce drastic changes in the psychotropic potency

of these compounds, making them particularly suitable for studies in

structure activity relationships.

The dramatic effect that alpha alkyl substitution has on the pharmacological activity of these phenethylamines is of particular

interest. It is thus well known (Shulgin 1970) that the presence of

an alpha methyl group increases considerably the psychotomimetic

potency of the corresponding phenethylamine. On the other hand,

alpha ethyl substituted analogs are devoid of psychotomimetic activity and act as functional antagonists for their alpha methyl

counterparts (Shulgin 1968; Standridge et al. 1976). Activity

is recovered if the alpha ethyl group is tied back on the phenyl

ring to give tetralin analogs which are weaker agonists (Violland,

Violland-Duperet and Pacheco 1971; Nichols, Barfknecht et al.

1974).

In the present study we have used model methoxyphenethylamine analogs (1-5; figure 1) to study the effect of alpha alkyl substitution on the conformational properties of the phenethylamine component in these molecules. The information thus obtained was used to

explain their observed divergent pharmacological properties.

The conformation of a drug is relevant to any consideration of its

interaction with the receptor. Like many small molecules, phenethylamines and their alpha alkyl analogs are potentially flexible, and

thus, may exist in solution in a number of conformations in equilibrium with one another. Of these, the most stable are the perfectly staggered conformations shown in figure 2, Since it is generally

recognized that a flexible drug has only one pharmacophoric conformation in its complex with the receptor, the formation of this com464
FIGURE 1

plex must involve a conformational selection which will influence

the kinetics and energetics of complex formation. If the drug is

present in more than one conformation, the extent to which the drug

exists in the pharmacophoric conformation should influence its reactivity.

To study the conformational distribution of the model methoxyphenethylamine salts in solution, we used nuclear magnetic resonance

(NMR) spectroscopy, which is clearly the experimental method of

choice. We have also sought to obtain information on the role of

the solvent in the conformational behavior of phenethylamines. by

conducting our studies in two different solvents,

whenever possible. Because of its lower polarity, CDCl3 can be exD20 and CDCl3,

pected to represent more accurately the hydrophobic environment of

the nerve membrane.

It has been argued (Makriyannis 1974; Mautner 1974) that, in addition to conformation, molecular flexibility may play an important

role during the drug-receptor interaction. To better evaluate the

role that the conformational distribution of phenethylamine salts

in solution plays on their biological activity, it is important to

have information on the rate of conformational interconversion. If

the compound exists predominantly in its pharmacophoric conformation, restricted flexibility will enhance its interaction with the

receptor. On the other hand, if the predominant drug conformation

is not the pharmacophoric one, the restricted rotation will hinder

the conformational change necessary for the drug to achieve its

proper conformation and will thus affect adversely the drug-receptor interaction. Such an observation (Gannelin 1973) served to explain the different pharmacological activities of histamine and

4-methylhistamine.

In this study, we have obtained a reasonable semiquantitative index

for the molecular flexibility of phenethylamine salts in D20 solu465
tion by measuring the 13C spin-lattice relaxation times (T1) of

individual carbon atoms. The information on molecular flexibility

was then integrated into the results obtained from conformational

analysis. The outcome was a more accurate dynamic picture for the

conformational behavior of each molecule in solution. Such a picture gives us more insight into the possible mode of drug-receptor

interaction (figure 3).

METHODS

A. Conformational Analysis

No single measurement can provide enough information for the unambiguous conformational analysis of potentially flexible molecules

in solution. We have thus sought more than one criterion in our

conformational assignments. As a result, we were able to clear.

some remaining ambiguities existing in the literature related to the

conformational analysis of alpha methylphenethylamines in solution

(Neville et al. 1971).

1. 1H Coupling Constants

The measureument of vicinal 1H-1H coupling constants is the most direct and still the most important method used in the conformational

analysis of small molecules in solution. The principle of this

method is described in the Karplus equation (Karplus 1959) which

states that the spin-spin coupling constant between proton attached to two adjacent carbon atoms is proportional to cos2

φ , the

dihedral angle about the C-C band. In our system, the dihedral

angle of interest is the one about the central Ar-C-C-N bond of the

arylphenethylamine.

The two benzylic protons in the alpha alkylphenethylamine analogs

(2, 3, 4) and those on the C1 carbon of compound 5 constitute the AB portion of an ABC spin system. The four protons on

the ArCH2CH2CH2N fragment of the phenethylamine analog are of the

AA'BB' type. Initial estimates of the spectral parameters were obtained by standard methods (Pople, Schneider, and Bernstein 1969;

Makriyannis et al. 1972) assuming that the corresponding spectra

were approximately ABX and AA'XX'. The parameters were then refined by spectral simulation and iteration using Nicolet ITRCAL

program.

The coupling constants thus obtained were considered to be averaged

values arising from a mixture of three possible perfectly staggered

rotamers. The relative population of each rotamer was then extracted from simple analogies which include Jg and Jt terms for the

coupling constants of perfectly staggered gauche and trans vicinal

protons.

While calculating the conformer distribution for the compounds

under study, we made a special effort to consider the uncertainties

inherent in the above method. The first of these uncertainties is

the spectral assignment for each of the two benzylic protons in

alpha alkylphenethylamines. Previous assignments (Neville et al.

1971; Bailey et al. 1971) in similar spin systems depended on pre466
dicting trends in the relative distribution of conformers among a

series of analogs, based on stereochemical considerations. We have,

in this study, provided additional spectral evidence which confirmed the previous assignments.

A second uncertainty is introduced with the choice of Jg and Jt

values. to be used in our calculations. These values vary and to a

large extent are dependent on the substituents of the C-C bond.

They can thus either be obtained from model compounds in which the

HCCH dihedral angle is fixed, or can be calculated from a number of

semiempirical equations. In this study we have chosen those Jg

and Jt values which gave results with the best internal consistency. We have used a combination of values (Table I, page ), some obtained from model compounds and others calculated from an empirical

relationship between vicinal coupling constants and substituent electronegativities. This relationship (Abraham and Gatti 1969) has

given us satisfactory results in the past (Makriyannis et al. 1972).

The final uncertainty considered here is related to the assumption

that measured vicinal coupling constants with intermediate values,

in between those of Jg and Jt, are the result of the populationweighted average of the Jg and Jt values in three perfectly staggered rotamers. Although perfectly staggered rotamers are generally accepted as the most stable, the possibility must be considered that an off-staggered stable rotamer could exist in solution.

The presence of such a conformer, with dihedral angles different

from the usually assigned values of 60° and 180°, could lead to

serious errors in our calculations. It could also mean that a

single distorted conformer may account for observed coupling constants with intermediate values. Such a possibility is more likely

in the case of sterically crowded compounds with large free energy

differences among individual conformers.

In order to settle this uncertainty we undertook low temperature

studies on compound 5 and other alpha ethylphenethylamine analogs.

Since the coupling constants of a rotamer are usually minimally

affected by temperature, a change in the observed vicinal coupling

constants is an indication of a change in rotamer distribution. No

change in the coupling constants with temperature indicates that,

most probably, the compound exists solely in one conformation. Our

low temperature experiments on alpha ethylphenethylamines in CD3OD

and in CDCl3 showed a considerable variation in the vicinal coupling

constants with temperature, and indicated that these compounds exist in solution as an equilibrium mixture of two or more conformers. We shall report on these experiments in greater detail elsewhere. The results can be extrapolated to the other open chain

phenethylamine analogs considered in this study and used as evidence against the presence of any of these compounds in solution as

a single distorted conformer. Further evidence is, however, necessary to rule out the possibility that any of the compounds could

exist as an equilibrium mixture of two or more off-staggered conformers.

2. Chemical Shifts

A careful examination of the -CH2CH- component in the 1H NMR spec467
FIGURE 2

FIGURE 3

Selected spectra from a 13C spin-lattice relaxation

tine (T1) experiment on 2-amino-6-methoxytetralin hydrochloride. Determinations were made with complete 1

H decoupling using a (-180°-τ-90°-T-)n inversion recovery

pulse sequencewhere t is experimentally varied

(τ=0.05, .1, .2, .5, .7, 1.0, 5 and 10 sec) and T is

equal to at least five times the longest T1 to be

measured.

468
trum of the alpha alkyl arylethylamines under consideration revealed that valuable information could be learned from the chemical

shift differences between the two H2, H3 benzylic protons in this

ABC system. These AB protons, which frequently give rise to two

individual "quartets", have different chemical environments as a

result of their different positions relative to their two vicinal

substituents: the NH3 and the alkyl groups. These substituents

exert respectively deshielding and shielding influences with the

more pronounced effects being experienced by the proton situated

gauche to the substituent. The population-weighted average of these

effects in the three possible rotamers determines the relative chemical shifts for each of the benzylic protons.

Additional useful information was gained by observing the spectra

of the phenethylamine analogs in D20 and in CDCl3 solutions. Solvent variation was usually accompanied by considerable changes in

the chemical shifts and the coupling constants of the side chain

protons. Chemical shift changes can be attributed to a reduction

in the deshielding influence of the NH3 group upon the neighboring

gauche proton when CDCl3 is replaced with D2O. Such a modification

of the deshielding influence of a charged nitrogen by solvent has

been already reported (Reynolds and Priller 1968). The highly

polar D20 presumably (Reynolds and Priller 1968) decreases the

effect of the positive charge through direct solvation of the positive ion. The result is that, in the less polar CDCl3, we observe a

downfield shift relative to the chemical shift in D2O. We have

recently demonstrated (Makriyannis and Hite 1978), using a tropane

derivative, that the magnitude of the chemical shift differences in

the two solvents can be related to the relative distances between

the protonated nitrogen and the neighboring protons. We were able

to successfully apply this principle here, in the phenethylamine

systems under investigation.

The information obtained from chemical shift measurements was used:

a. to distinguish between the two diastereotopic H2,H3 benzylic protons in the spectra of the alpha alkylphenethyl analogs; b. to provide supporting evidence for the results on rotamer populations

which were calculated from the vicinal coupling constants.

B. Molecular Dynamics

The method we have used to study the microdynamic behavior of the

phenethylamine analogs in solution is based on the determination of

effective correlation times (τ eff) for individu 1 carbon atoms

which can be calculated from the corresponding 13C spin-lattice relaxation times (T1). The correlation times measure the period of

molecular reorientation of a C-H vector through a given angular

displacement. They can also serve as a measure for the motions of

individual 13C atoms and thus provide. a description of the molecule's dynamic behavior in solution. Such measurements allow us to

make semiquantitative comparisons on the flexibility of closely related molecules in solution and also give us information about

specific intramolecular interactions which affect their flexibility.

In intermediate sized molecules, spin-lattice relaxation, for carbons directly attached to protons, is usually dominated by the

469
13C-H dipole-dipole mechanism. In such instances, the T1 of a

13C nucleus can be related to its effective correlation tine using

the following equation (Allerhand et al. 1971) which assumes isotropic motion:

where n = number of protons directly attached on the 13C nucleus

under consideration. The only possible exceptim in our systems

are fast rotating methyl groups where spin-rotation can be a contributing mechanism in the relaxation process. In such instances,

the calculated τeff values should be considered only as upper

limits.

RESULTS AND DISCUSSION

The conformational and dynamic properties in solution for each of

the compounds examined, are discussed, with special emphasis on the

effects of alpha alkyl substitution. Chemical shifts, coupling constants and relaxation parameters are listed in tables I, II and III.

4-Methoxyphenethylamine hydrochloride (1)

This compound exists in solution as a mixture of two conformers.

The conformational preference is slightly in favor of the trans conformer, which makes up slightly over me third of the total population. One third is the expected ratio for the trans conformer,if

there is no conformational preference.

In p-nethoxyphenethylamine hydrochloride, the side chain carbons have

similar correlation times indicating that both carbons are moving

in unison. The movement of the side chain appears to be faster than

the tumbling of the phenyl ring, as indicated by the longer correlation tine values for the phenyl carbons. This is an indicatim that

rotation along the C-Ph bond is faster than along the C-C bond of

the side chain. The methyl carbon in the para methoxy group has a

T1 value considerably longer than that of the rest of the protonated carbons and indicates very fast motion. Its calculated correlation time is probably only an upper limit, because of a possible

contribution of spin-rotation in the relaxation of this carbon.

4-Methoxyamphetamine (2) and 3,4,5-trimethoxyamphetamine (3) hydrochlorides.

Amphetamines can occur as mixtures of two major (fig. 2, I. II) and

one minor (fig. 2, III) conformers. In D20 compound 2 and compound 3 (which was included in this study because of its solubility

in chloroform) occur as a mixture of equally populated conformers I

and II with mly a modest cmtribution from conformer III. This

last conformer has all three vicinal substituents gauche to one

another and is, therefore, understandably not favored. When the

solvent was changed to CDCl3 in the trimethoxy analog (3) the conformational distribution was shifted strongly in favor of rotamer I

which has the ammonium group trans and the methyl group gauche to

the aromatic ring. This is an indication that hydrophobic media

favor a trans phenethylamine configuration.

470
TABLE I

Proton Chemical Shiftsa for the

Phenethylamine Hydrochloride Analogs

Com- Sol- Chemical Shift (ppm) DifferenceC

pound vent Hl H2 H3 R4 [CH2 ] [CH3] V3-V2 (HZ)

1

2 D 2 O

b

D 3.199 2.910 2 0

b

3.442 2.725 1.154 0

3 D 2 0

b

3.425 2.733 1.183 0

CDCl3 3.573 2.816 3.166 1.406 94.7

4 D2 0 3.442 2.789 2.998 (1.659,1.710) 1.006 56.4

CDCl3 3.375 2.880 3.183 (1.708,1.727) 1.092 81.7

5 D2 0 3.449 2.639 2.962 (1.667,2.020) 87.3

CDCl3 3.613 3.044 3.266 (2.041,2.379) 60

a

Chemical shifts were measured from TMS which was used as an internal standard in the CDCl3 solutions and as an external standard in a

concentric tube in the D20 solutions. Unless stated otherwise spectra were obtained at 270 MHz using 0.1OM solutions at an ambient

temperature of 20°; bSpectra taken at 60 MHz using 0.3OM solutions

at 20°; cFrequency differences for spectra measured at 270 MHz.

The accidental equivalence of the H2 and H3 benzylic protons in both

amphetamine analogs when examined in D20 reflects the weighted deshielding influence of the ammonium group and the shielding influence of the methyl group on each of the two protons, in all three conformers. The net sum of these effects, in the above two examples,

is apparently equal for both protons. The change to a hydrophobic

solvent, in compound 3, is accompanied with a downfield shift for

both H2 and H3 protons. This behavior reflects the increased deshielding influence of the positively charged nitrogen when going

from aqueous to hydrophobic media. Furthermore, examination of the

spectrum in CDCl3 revealed that the H3 proton undergoes a stronger

downfield shift an the H2 proton. he presence of the H2-frequencies at higher field relative to the H3 signal is compatible

with the observed increase in the population of rotamer I. In this

rotamer, the H2 proton experiences the preferential shielding influence of the gauche methyl group. The above observed solvent effect

is therefore evidence that the spectral assignments for the H2 and

the H3 protons are correct.

The addition of an alpha methyl group on the side chain of the

phenethylamine salt analog appears to slow down the overall movement of the molecule, as indicated by the general increase in the

correlation times. In this amphetamine analog, the alpha methine

carbon is the slowest moving carbon in the side chain and has a

correlation time very similar to those of the ring carbons. The beta

methylene carbon moves only slightly faster, indicating some flexibility around the C-Ph bond. Finally, the alpha methyl carbon ro471
tates cmsiderably faster than any other carbon on the side chain.

Nevertheless, this methyl carbon has a correlation time at least

three times as long as that of the freely rotating O-methyl group

carbon. This indicates serious restriction in its rotation.

3,4-Dimethoxy-a-ethylphenethylamine Hydrochloride 4

This compound occurs in two principal conformations (I,II). Of

these, the trans phenethylammonium conformation which has the ethyl

group gauche to the aromatic ring is favored. As with the alpha

methyl analog, the change to hydrophobic solvents enhances this conformation. The change of solvent also causes the signals for the H2

and H3 protons to shift to lower fields; the strongest effect is experienced by the H3 proton. All of the above effects can be explained using the same arguments used for 4-methoxyamphetamine.

In this molecule (4), all but the three methyl carbons have very

short relaxation times which correspond to long correlation times

and indicate sluggish overall reorientation. Again the alpha methine carbon is the slowest, while the benzylic carbon has a slightly

shorter correlation time. Such τeff values are compatible with

slightly freer mobility around the C-Ph bond compared to the central

C-C phenethylamine bond. The two carbons of the alpha ethyl substituent differ greatly in their dynamic behavior. While the

methylene carbon is cmsiderably restricted, the methyl carbon has

a very fast rotation, as shown by its very short correlation time.

The free rotation of the above mentioned methyl group gives us some

TABLE II

Spin-Spin Coupling Constants and Conformational Distributions

for the Phenethylamine Hydrochloride Analogs

472
TABLE III

Carbon-13 Spin-Lattice Relaxation Timesa (sec) and Effective

Correlation Times (x10-12 sec) for Phenethylamine Hydrochloride

Analogs

Compound

C2 C3 C4C5 C6 Cß Ca CH2 CH3 OCH3

1 1.76 1.76 - 1.76 1.76 1.02 1.02 - - 2.31

(26) (26) (26) (26) (22) (22) (<6.5)

2 1.07 1.04 - 1.04 1.07 .58 1.04 .74 2.06

(42) (44) - (44) (42) (39) (44) (20) (<7.7)

4 .34 - - .39 .33 .27 .44 .40 1.33 0.93 .93

(133) (116) (137) (84) (103) (57) (<11) (<16)(<16)

5. - .68(C5) .80(C7).74(C8).42(C1).68(C2).41(C3).41(C4) 1.76

(67) (57) (61) (54) (67) (55) (55) (<8.6)

a

 Measurements were made using .75M solutions of the compounds in D20.

The results given are the average of three determinations.

insight into the conformation around the HC-CH2CH3 bond. Of the three

possible conformers, only one will allow the methyl group to experience free rotation. This conformer is one in which the methyl group

is gauche to the ammonium group and trans to the benzylic methylene.

According to the evidence, this conformation is the most favored.

The chosen conformation is compatible with the downfield shift experienced by the methyl group when the solvent is changed from D20 to

CDCl3. The slightly increased value in the correlation tines of the

two ortho 0-methyl groups reflects some mutual interference in the

rotation of the two groups.

2-Amino-6-methoxytetralin Hydrochloride (5)

In addition to the coupling constants shown in table II, we have obtained approximate estimates for the vicinal coupling constants of the

protons on carbon C3 when coupling with the methine proton on carbon C2 of this compound. These coupling constants were found to be

equal to: Jvic=1OHz; 4Hz. The four vicinal coupling constants at

hand are consistent with the two Hax,-Hax and two Hax-Heq couplings.

Such coupling indicates that the saturated ring exists in a chair in

which the methine proton on C2 occupies an axial position and the

ammonium group is equatorial.

The accurately measured vicinal coupling constants between the two

benzylic protons on carbon Cl and the methine proton on C2 indicate

that this chair conformation is slightly deformed. If we assume that

compound 5 exists in one dominant conformation in solution, we can

then evaluate the ring distortion. This can be done by comparing the

measured vicinal coupling constants with the coupling constants of

473
FIGURE 4

474
The evaluation of the results obtained in this study is given below:

1.

2.

3.

4.

This study has helped to put the conformational assignments for

alpha alkylphenethylamine salts on more solid ground, with the

use of variable temperature and solvent experiments. It was

found that, in all of the compounds studied here, the trans

phenethylammonium conformer makes up a sizeable portion of the

total conformer population in solution.

The trans phenethylammonium conformer appears to be enhanced

a) by the presence of a small alkyl group in the alpha position;

b) by hydrophobic solvents.

The addition of alpha alkyl substituents introduces a considerable degree of rotational restriction. This restriction is more

pronounced around the central C-C bond, while the C-Ph bond

appears to maintain more rotational freedom in all the open chain

compounds studied here. The deceased flexibility in the molecule allows a more important role to be played by the conformer

distribution in the determination of the drug's biological

activity.

It is tempting to suggest, on the basis of the evidence provided

here, that the trans phenethylammonium conformation is essential

for psychotomimetic activity. Such a suggestion is compatible

with the finding that in the 2-(3.4,5-trimethoxyphenyl) cyclopropylamines, psychotomimetic activity resides with the trans

isomer (Walters and Cooper 1968; Cooper and Walters 1972).

cyclohexane (Jax,ax=13.31, Jax eq=3.59; Abraham and Gatti 1969) which

is considered to be a perfectly staggered chair. The following

are the calculated dihedral angles for the C1H2C2H fragment of

2-amino-6-methoxytetralin hydrochloride in D20 and in CDCl3 respectively: φ(HaxC1C2Hax)155°,158°; φ(HeqC1C2Hax)=55°, 56°.

The differing relaxation time values of the phenyl ring carbons in

this tetralin analog are an indication of a certain degree of anisotropic motion. In this molecule, rotation appears to be favored

around an axis which runs very near the C6-0 and the C2-N bonds.

The consistently shorter correlation times of the aliphatic carbons

are an indication of some flexibility in the ring.

CONCLUSION

Stick as well as space-filled models for compounds 1, 2, 4,and 5, in

their preferred conformations, are shown in figure 4. The stick

models provide a more accessible representation of the molecular

conformation, while the space-filled models give a realistic portrayal of the structural features that may affect the drug-receptor

interaction. The conformations around the C-O and the C-Ph bonds

are only predicted conformations. Work is currently under progress

for their experimental verification.

NO conclusions can be drawn from our experiments about the

pharmacophoric conformation around the C-Ph bond.

475
5.

This question will be investigated using ortho substituted

phenethylamine analogs. A trans pharmacophoric conformation

does not rule out the possibility that these drugs may produce their effects by interacting with more than me receptor.

The presence of an alpha methyl group gauche to the phenyl ring

does not seem to obstruct the interaction of the phenethylammonium component with the receptor. On the contrary it may

enhance its affinity through some hydrophobic interaction of the

methyl group with the receptor site. The possibility of such

a specific interaction is strengthened by the existing evidence

about the stereospecific requirements of methoxyamphetamines for

activity (Shulgin 1973; Standridge et al. 1976). When the

alpha methyl is substituted with an ethyl group, the steric requirements of the receptor for productive interaction are not

satisfied any longer. As can be seen from the models, the preferred conformation of the ethyl group is such,that the methyl

group is protruding beyond the level of the ammonium group and

probably obstructs its interaction with the receptor site. However,the molecule can still bind to the receptor although not

productively because of its other structural features and therefore acts as an antagonist (probably competitive). The conformation of the alpha CH2CH2 chain is quite different in the tetralin analog. Such a conformation allows the interaction between

the protonated nitrogen with the receptor site to occur and accounts for the psychotomimetic properties of this molecule.

We have currently extended our studies to include other phenethylamine analogs. We are seeking to understand how other structural modifications can affect the conformational properties of

phenethylamines and how these conformational changes correlate

with their psychotomimetic activity.

REFERENCES

Abraham, R. J., and Gatti, G. Rotational isomerism. Effect of substituents on vicinal coupling constants in XCH2•CH2Y fragments. J

Chem Soc (B),.(8), 961-968, 1969.

Aldous, F. A. B., Barrass, B.C., Brewster. K., Buxton, D. A.,

Green, D.M., Pinder, R. M., Rich, P., Skeels, M., and Tutt, K. J.

Structure activity relationships in psychotomimetic phenylalkylamines. J Med Chem, 17(10):1100-1111, 1974.

Allerhand, A., Doddrell, D., and Komoroski, R. Natural abundance

carbon-13 relaxed Fourier transform nuclear magnetic resonance spectra of complex molecules. J Chem Phys, 55(1):189-198, 1971.

476
Barfknecht, C. F., and Nichols, D. E. Potential psychotomimetics.

Bromomethoxyamphetamines. J Med Chem, 14(4):370-372, 1971.

Bailey, K., By, A. W., Graham, K. C., and Verner, D. Proton magnetic resonance spectra of some amphetamines and related compounds

and observations in rotamer populations. Can J Chem, 49(19):3143-

3151, 1971.

Casey, A. F. P.M.R. Spectroscopy in Medicine and Biological

Chemistry. New York: Academic Press, 1971. p 207.

Cooper, P. D., and Walters, G. C. Stereochemical requirements of

mescaline receptor. Nature, 238(5359):96-98, 1972.

Gannelin, C. R. Conformation of histamine derivatives. A relationship between conformation and pharmacological activity. J Med Chem,

20(6):620-623, 1973.

Karplus, M., Contact electron-spin coupling of nuclear magnetic

moments. J Chem Phys, 30(1):11-15, 1959.

Makriyannis, A., Sullivan, R. F., and Mautner, H. G. Conformational

relationships between analogs of acetylcholine and those of local

anesthetics in solution. Proc Nat Acad Sci USA, 69(11):3416-

3419, 1972.

Makriyannis, A. Studies on the flexibility of acetylcholine and

analogs in solution. Pharmacologist, 16(2):285, 1974.

Makriyannis, A., and Hite, G. The conformation of nortropacocaine

in aqueous and hydrophobic media. In preparation.

Maunter, H. G. Proceedings of the Seventh Jerusalem Symposium on

Quantum Chemistry and Biochemistry. In: Bergaman, E. D., ed.

Boston: Reidel Publishing Co., 1974. p 119-129.

Neville, G. A., DesLauriers. R., Blackburn, B. J., and Smith, I.

Co P. Conformatianol studies of amphetamine and medicinally

important derivatives by nuclear magnetic resonance spectroscopy.

J Med Chem, 14(8):717-721, 1971.

Nichols, D. E., Barfknecht, C. F., Long, J. P., Standridge, R. T.,

Howell, H. G., Partyka, R. A., and Dyer, D. C. Potential psychotomimetics. Rigid analogs of 2,4-dimethoxy-4-methylphenylisopropylamine (DOM,STP). J Med Chem, 17(2):161-166, 1974.

Pople, J. A., Schneider, W. G., and Bernstein, H. J. High resolution

nuclear magnetic resonance spectroscopy. New York: Academic Press,

1969. pp 91-95.

Reynolds, W. F., and Priller, U. R. Effect of solvent and anion upon

the proton magnetic resonance spectrum of the 1-methylpyridinium

ion. Can J Chem, 46(17):2787-2791 (1968).

477
Shulgin, A. T. The ethyl homologs of 2,4,5-trimethoxyisopropylamine.

J Med Chem, 11(1):186-187, 1968.

Shulgin, A. T., Sargent, T., and Naranjo, C. Structure-activity

relationships of one-ring psychotomimetics. Nature, 221(5180):

537-541, 1969.

Shulgin, A. T. Chemistry and structure-activity relationships of the

psychotomimetics. In: Efron, D. H., ed. Psychotomimetic Drugs,

New York: Raven Press, 1970. pp 21-42.

Shulgin, A. T. Stereospecific requirements for hallucinogenesis.

J Pharm, Pharmacol, 25(33):271-272, 1973.

Smythies, J. R., Bradley, R. J., Johnston, V. S., Bennington, F.,

Morin, R. D., and Clark, L. C. Structure-activity relationship

studies on mescaline. The influence of the methoxy groups.

Psychopharmacologia, 10(5):379-387, 1967.

Standridge, R. T., Howell, H. G., Gylys, J. A., Partyka, R. A., and

Shulgin, A. T. Phenylolkylamines with potential psychotherapeutic

utility. 2-Amino-1-(2,5-dimethoxy-4-methylphenyl)butane. J Med Chem,

19(12):1400-1404, 1976.

Violland, R., Violland-Duperet, N., Pacheco, H., Psychotropes

potentiels. Synthese et structures des composes derivants de

1'amino-3 tetralol-1 et apparent& a divers psychotomimetiques.

Bull Soc Chim Fr, (1) 307-311, 1971.

Walters, G. C., and Cooper, P. D. Alicyclic analogue of mescaline.

Nature, 218(5138):298-300, 1968.

AUTHORS

Alexandros Makriyannis, Ph.D. and James Knittel, University of

Connecticut, Department of Medicinal Chemistry and Institute of

Materials Science, Storrs, Conn. 06268.

478
Appendix A.

QuaSAR Program

479
AGENDA

FRIDAY, APRIL 21, 1978

COUNSEL ROOM, SHERATON SILVER SPRING

480
AGENDA

SATURDAY, APRIL 22, 1978

COUNSEL ROOM, SHERATON SILVER SPRING

481
Appendix B.

List of Participants

CHAIRMEN

Richard E. Brown, Ph.D.

University Computing Services

State University of New York

at Buffalo

Amherst, New York 14226

Albert J. Leo, Ph.D.

Department of Chemistry

Pomona College

Claremont, California 91711

Phillip S. Portoghese, Ph.D.

University of Minnesota

College of Pharmacy

Minneapolis, Minnesota 55455

Milan Trsic, Ph.D.

Deparment of Chemistry

University of Calgary

Calgary, Alberta

Canada T2N 1N4

PARTICIPANTS

George Anderson, Ph.D.

Department of Pharmaceutical

Chemistry

University of California

San Francisco, California 94143

Sydney Archer, Ph.D.

Chemistry Department

Rensselaer Polytechnic Institute

Troy, New York 12181

Charles F. Barfknecht, Ph.D.

College of Pharmacy

University of Iowa

Iowa City, Iowa 52242

F. Ivy Carroll, Ph.D.

Chemistry and Life Science

Division

Research Triangle Institute

Research Triangle Park, North

Carolina 27705

Vernon B. Cheney, Ph.D.

The Upjohn Company

Kalamazoo, Michigan 49001

Ralph E. Christoffersen, Ph.D.

Department of Chemistry

The University of Kansas

Lawrence, Kansas 66045

Robert B. Davidson Ph.D.

Department of chemistry

Amherst College

Amherst, Massachusetts 01002

Linda Domelsmith, Ph.D.

Department of Chemistry

Louisiana State University

Baton Rouge, Louisiana 70803

William J. Dunn, III, Ph.D.

Deparment of Organic Chemistry

Umea University

S 902 87, Umea, Sweden

Mark Froimowitz, Ph.D.

McLean Hospital

Belmont, Massachusetts 02178

Richard A. Glennon, Ph.D.

Department of Pharmaceutical

Chemistry

Medical College of Virginia

.

Richmond, Virginia 23298

Jack Peter Green, M.D., Ph.D.

Department of Pharmacology

Mount Sinai School of Medicine

New York, New York 10029

Kendall N. Houk, Ph.D.

Department of Chemistry

Louisiana State University

Baton Rouge, Louisiana 70803

Arthur E. Jacobson, Ph.D.

Medicinal Chemistry Aection,

NIAMDD

National Institutes of Health

Bethesda, Maryland 20014

482
Howard L. Johnson, Ph.D.

Life Sciences Division

Stanford Research Institute

Menlo Park, California 94023

Robert Katz, Ph.D.

National Institutes of Health

Bethesda, Maryland 20014

Joyce J. Kaufman, Ph.D.

Department of Chemistry

The Johns Hopkins University

Baltimore, Maryland 21218

Lemont B. Kier, Ph.D.

Department of Pharmaceutical

Chemistry

Medical College of Virginia

Richmond, Virginia 23298

GUESTS

Peter Kollman, Ph.D.

School of Pharmacy

University of California at

San Francisco

San Francisco, California 94143

Mahadevappa M. Kumbar, Ph.D.

Department of Chemistry

Adelphi University

Garden City, New York 11530

Eric J. Lien, Ph.D.

School of Pharmacy

University of Southern

California

Los Angeles, California 90033

Gilda H. Loew, Ph.D.

Department of Genetics

Stanford University School

of Medicine

Stanford, California 94305

Alexandros Makriyannis, Ph.D.

School of Pharmacy

University of Connecticut

Storrs, Connecticut 06268

William R. Martin, M.D.

Department of Pharmacology

University of Kentucky

College of Medicine

Lexington, Kentucky 40506

David E. Nichols, Ph.D.

School of Pharmacy and Pharmacal

Sciences

Purdue University

West Lafayette, Indiana 47907

Alexander T. Shulgin, Ph.D.

1483 Shulgin Road

Lafayette, California 94549

Harel Weinstein, D.Sc.

Department of Pharmacology

Mount Sinai School of Medicine

New York, New York 10029

Bruce K. Bernard, Ph.D.

G.D. Searle and Company

Chicago, Illinois 61680

Dr. Ulrich Braun

University of Bonn

Bonn, Germany

Dr. Gisela Braun

University of Bonn

Bonn, Germany

Joseph I. DeGraw, Ph.D.

Life Sciences Division

Stanford Research Institute

Menlo Park, California 94025

Ronald L. Jacoby, Ph.D.

School of Pharmacy

Ferris State College

Big Rapids, Michigan 49307

John Jacyno

Deparment of Chemistry

University of Calgary

Calgary, Canada T2N IN4

Robert Marino, Ph.D.

Department of Physics

Hunter College

New York, New York 10021

Gerald Marquardt, Ph.D.

2111 Jefferson Davis Highway

Apt. 1212N

Arlington, Virginia 22202

483
Lawrence Martin, Ph.D.

Hoechst-Roussel Pharmaceuticals

Somerville, New Jersey 08876

MODERATORS

Toni Oja, Ph.D.

Department of Physics and

Astronomy

Hunter college

New York, New York 10021

Helen Ong, Ph.D.

Hoechst-Roussel Pharmaceuticals

Somerville, New Jersey 08876

Roman Osman, Ph.D.

Department of Pharmacology

Mount Sinai School of Medicine

New York, New York 10029

Robert E. Willette, Ph.D.

Chief, Reserach Technology

Branch

Division of Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

Gene Barnett, Ph.D.

Research Technology Branch

Division of Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

484
Single copies of the monographs may be obtained free of charge

from the National Clearinghouse for Drug Abuse Information (NCDAI).

Please contact NCDAI also for information about availability of

coming issues and other publications of the National Institute on

Drug Abuse relevant to drug abuse research.

Additional copies may be purchased from the U.S Government Printing

Office (GPO) and/or the National Technical Information Service (NTIS)

as indicated. NTIS prices are for paper copy. Microfiche copies,

at $3, are also available from NTIS. Prices from either source are

subject to change.

Addresses are:

NCDAI

National Clearinghouse for Drug Abuse Information

Room 10A-56

5600 Fishers Lane

Rockville, Maryland 20857

GPO NTIS

Superintendent of Documents National Technical Information

U.S. Government Printing Office Service

Washington, D.C. 20402 U.S. Department of Commerce

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1 FINDINGS OF DRUG ABUSE RESEARCH. An annotated bibliography of

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Volume 1, 384 pp., Volume 2, 377 pp.

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in psychosocial research to achieve operational comparability. 167 pp.

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39 pp.

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7 CANNABINOID ASSAYS IN HUMANS. Robert Willette, Ph.D., editor.

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3 NARCOTIC ANTAGONISTS: NALTREXONE. Demetrios Julius, M.D., and

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GPO Stock #017-024-00571-1 $2.60 NITS PB #266 691/AS $10.75

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GPO Stock #017-024-00576-2 $1.70

perfomance impairment, particularly on driving. 137 pp.

NTIS PB #269 602/AS $8

12 PSYCHODYNAMICS OF DRUG DEPENDANCE. Jack D. Blaine, M.D., and

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GPO Stock #017-024-00642-4 $2.75 NTIS PB #276 084/AS $9

13 COCAINE: 1977. Robert c. Petersen, Ph.D., and Richard C. Stillman,

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GPO stock #017-024-00592-4 $3 NTIS PB #269 175/AS $9.25

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14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

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of marihana use. 251 pp.

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15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wn. Sharp,

Ph.D., and Mary Lee Brehm, Ph.D., editors. A broad review of inhalant

abuse, including sociocultural, behavioral, clinical, pharmacological,

and toxicological aspects. Extensive bibliography. 347 pp.

GPO Stock #017-024-00650-5 $4.25 NTIS PB #275 798/AS $12.50

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne Rittenhouse, Ph.D., editor. Task Force report on measurement of heroinnarcotics use gaps in knowledge and how to address them, improved research technologies, and research implications. 249 pp.

GPO Stock #017-024-00690-4 $3.50 NTIS PB #276 357/AS $9.50

17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik M.D., Ph.D.,

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including epidemiology, etiology, socioeconomic and physical consequences of use, and approaches to behavioral change. From a NIDAsupported UCLA conference. 383 pp.

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18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., editor. Conference papers discuss

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development of tolerance to a variety of drugs in animal and human

subjects. 151 pp.

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19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Peterson,

Ph.D, editor. A monograph based on papers presented at the World Psychiatric Association 1977 meeting in Honolulu. Emphasis is on emerging

patterns of drug use, international aspects of research, and therapeutic

issues of particular interest worldwide. In Press

20 SELF-ADMNISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D. editor. Papers from a technical review on

methods used to study self-administration of abused substances. Discussions include overview, methodological analysis, and future planning

of research on a variety of substances: drugs, ethanol, food, and

tobacco. In Press

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen, Ph.D.

and Richard C. Stillman, M.D., editors. Monograph derived from a

technical review to assess the present state of knowledge about phencyclidine and to focus on additional areas of research. Papers are

aimed at a professional and scientific audience concerned about how

to cope with the problem of PCP abuse. In Press

487

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Printed 1978