# Patent

## Title
Oligomer modified diaromatic substituted compounds

## Abstract

Disclosed are compounds comprising diaromatic substituted compound residues, namely the anti-viral (anti-HIV) drug delavirdine, covalently attached via a linkage to water-soluble, non-peptidic oligomers, specifically to poly(ethylene glycol) (PEG) oligomers. A compound of the invention, when administered by any of a number of administration routes, exhibits advantages over non-oligomer modified diaromatic substituted compounds.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 U.S.C. §371 application of International Application No. PCT/US2011/025695, filed Feb. 22, 2011, designating the United States, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/306,802, filed Feb. 22, 2010, the disclosure of which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
This invention comprises (among other things) chemically modified diaromatic substituted compounds that possess certain advantages over diaromatic substituted compounds lacking the chemical modification. The chemically modified diaromatic substituted compounds described herein relate to and/or have application(s) in (among others) the fields of drug discovery, pharmacotherapy, physiology, organic chemistry and polymer chemistry.
BACKGROUND OF THE INVENTION
According to UNAIDS, about 33.3 million people were living with human immunodeficiency virus (HIV) at the end of 2009, up from 26.2 million people in 1999. HIV is the etiological agent of acquired immunodeficiency syndrome, AIDS. Since the beginning of the epidemic, more than 60 million people have been infected with HIV and nearly 30 million people have died of HIV-related causes. In an effort to decrease the morbidity and mortality associated with HIV infection, a variety of pharmacological interventions have been proposed.
On Mar. 20, 1987, the FDA approved the use of the compound, AZT (zidovudine), to treat AIDS patients who present with an initial episode of Pneumocystis carinii pneumonia, AIDS patients with conditions other than Pneumocystis carinii pneumonia or patients infected with the virus with an absolute CD4 lymphocyte count of less than 200/mm3 in the peripheral blood. AZT is a known inhibitor of viral reverse transcriptase, an enzyme necessary for human immunodeficiency virus replication. U.S. Pat. No. 4,724,232 claims a method of treating humans suffering from AIDS utilizing AZT.
As disclosed in U.S. Pat. No. 6,043,248, certain antibiotics and polyanionic dyes inhibit retroviral reverse transcriptase (an enzyme necessary for HIV replication). In addition, publications have reported the ability of various sulfated compounds to inhibit virus replication, including HIV. The use of benzodiazepine derivatives have been described as being potent and selective inhibitors of HIV replications. See Pauwels et al. (1990) Nature 343:470-474 and Merluzzi et al. (199) Science 250:1411-1413.
Building on some of these early attempts (such as 2′,3′ dideoxynucleoside analogues such as AZT) to address HIV infection, much effort has been focused providing superior agents. In the case of 2′,3′-dideoxynucleosides, ddC and ddI have shown potent activity against HIV in vitro and have been evaluated in clinical trials. See Drug News & Perspectives 5(3):153-169 (1992). Based on this further research, the FDA has approved ddI for the treatment of HIV-1 infections in adults and pediatrics patients who are intolerant to, or whose health has significantly deteriorated while on, AZT treatment.
Additional research has lead to the discovery of non-nucleoside reverse transcriptase inhibitors (or NNRTIs). Like the nucleoside analogs, NNRTIs block HIV's infection of via inhibiting reverse transcriptase. Combination treatment using both a nucleoside analog drug (AZT, ddI, ddC, d4T and 3TC) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are commonly used.
Despite the success experiences with some of these pharmacologic interventions, challenges remain. For example, it is known that HIV develops resistance to many drugs (some in as few as two to seven weeks when the drug is used alone). Thus, there remains a need to provide additional drugs to treat individuals infected with HIV.
Delavirdine, a diaromatic substituted compound is an approved NNRTI that has been in a number of clinical trials. Its efficacy is lower than other NNRTIs, and it also has an inconvenient schedule. The risk of cross-resistance across the NNRTI class, as well as its complex set of drug interactions, make delavirdine and other NNRTIs less suitable as therapy for HIV patients. The most common adverse event is moderate to severe rash, which occurs in up to 20% of patients. Other common adverse events include fatigue, headache and nausea. Liver toxicity has also been reported.
Therefore, pharmacotherapy with such therapeutic diaromatic substituted compounds would be improved if these and/or other adverse or side effects associated with their use could be decreased or if their pharmacology may be improved. Thus, there is a large unmet need for developing novel diaromatic substituted compounds to address not only these concerns, but the concern associated with resistance as well.
The present invention seeks to address these and other needs in the art.
SUMMARY OF THE INVENTION
In one or more embodiments of the invention, a compound is provided, the compound comprising diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer.
Exemplary compounds of the invention include those having the following structure:




wherein:

R1 is —CH2— or —CO—;
Z is




R7 is —N(R7-5)H where R7-5 is C1-C6 alkyl, —CH2-cyclopropyl, or —CH2—CH2F;
R8 is —H or —F;
R9 is —H or —F;
R10 is —H or —F;
Aryl/Heteroaryl is a substituent of formula




wherein X14 is selected from the group consisting of —H, —CH2-phenyl, —O—CH2-phenyl, —O—CH2—COOH, C1-C6 alkyl (e.g., C1-C4 alkyl), —F, —Cl, —Br, —O—SO2—(C1-C4 alkyl), —NH—SO2—(C1-C6 alkyl), —NO2, —NH2, —N3, —N═CH—N(C1-C6 alkyl)(C1-C6 alkyl), —N═C(C1-C4 alkyl)-N(C1-C6 alkyl)(C1-C6 alkyl), —NH—CO—X14-9 where X14-9 is —H, —C1-C4 alkyl or phenyl; n7 is 0 or 1; X6, when present, is —H, —OH, —(CH2)—OH, —O—CH2-phenyl, —CHO, C1-C3 alkoxy, —O—SO2—C1-C4 alkyl, —NH—SO2—C1-C4 alkyl, —O—(CH2)2-5—N(X6-3)(X6-4) where X6-3 and X6-4 are both —H or where X6-3 and X6-4 are taken together with the attached nitrogen atom to form a heterocyclic ring selected from the group consisting of 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and 1-aziridinyl;

X is a spacer moiety; and
POLY is a water-soluble, non-peptidic oligomer,
and pharmaceutically acceptable salts thereof.
The “diaromatic substituted compound residue” is a compound having a structure of a diaromatic substituted compound that is altered by the presence of one or more bonds, which bonds serve to attach (either directly or indirectly) one or more water-soluble, non-peptidic oligomers.
In this regard, any diaromatic substituted compound having anti-HIV activity can be used as a diaromatic substituted compound moiety. Exemplary diaromatic substituted compound moieties have a structure encompassed by Formula I:




wherein:

R1 is —CH2— or —CO—;
Z is




R7 is —N(R7-5)H where R7-5 is C1-C6 alkyl, —CH2-cyclopropyl, or —CH2—CH2F;
R8 is —H or —F;
R9 is —H or —F;
R10 is —H or —F; and
Aryl/Heteroaryl is a substituent of formula




wherein X14 is selected from the group consisting of —H, —CH2-phenyl, —O—CH2-phenyl, —O—CH2—COOH, C1-C6 alkyl (e.g., C1-C4 alkyl), —F, —Cl, —Br, —O—SO2—(C1-C4 alkyl), —NH—SO2—(C1-C6 alkyl), —NO2, —NH2, —N3, —N═CH—N(C1-C6 alkyl)(C1-C6 alkyl), —N═C(C1-C4 alkyl)-N(C1-C6 alkyl)(C1-C6 alkyl), —NH—CO—X14-9 where X14-9 is —H, —C1-C4 alkyl or phenyl; n7 is 0 or 1; X6, when present, is —H, —OH, —(CH2)—OH, —O—CH2-phenyl, —CHO, C1-C3 alkoxy, —O—SO2—C1-C4 alkyl, —NH—SO2—C1-C4 alkyl, —C≡N, —O—(CH2)2-5—N(X6-3)(X6-4) where X6-3 and X6-4 are both —H or where X6-3 and X6-4 are taken together with the attached nitrogen atom to form a heterocyclic ring selected from the group consisting of 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and 1-aziridinyl.

Exemplary diaromatic substituted compound moieties also include N-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-1-yl}carbonyl)-1H-indol-5-yl]methanesulfonamide.
In one or more embodiments of the invention, a composition is provided, the composition comprising a compound comprising a diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer, and optionally, a pharmaceutically acceptable excipient.
In one or more embodiments of the invention, a dosage form is provided, the dosage form comprising a compound comprising a diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer, wherein the compound is present in a dosage form.
In one or more embodiments of the invention, a method is provided, the method comprising covalently attaching a water-soluble, non-peptidic oligomer to a diaromatic substituted compound moiety.
In one or more embodiments of the invention, a method is provided, the method comprising administering a compound to a mammal in need thereof, comprising a diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer.
Additional embodiments of the present conjugates, compositions, methods, and the like will be apparent from the following description, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Additional aspects and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.


DETAILED DESCRIPTION OF THE INVENTION
As used in this specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions described below.
“Water soluble, non-peptidic oligomer” indicates an oligomer that is at least 35% (by weight) soluble, preferably greater than 70% (by weight), and more preferably greater than 95% (by weight) soluble, in water at room temperature. Typically, an unfiltered aqueous preparation of a “water-soluble” oligomer transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. It is most preferred, however, that the water-soluble oligomer is at least 95% (by weight) soluble in water or completely soluble in water. With respect to being “non-peptidic,” an oligomer is non-peptidic when it has less than 35% (by weight) of diaromatic substituted compound residues.
The terms “monomer,” “monomeric subunit” and “monomeric unit” are used interchangeably herein and refer to one of the basic structural units of a polymer or oligomer. In the case of a homo-oligomer, a single repeating structural unit fonds the oligomer. In the case of a co-oligomer, two or more structural units are repeated—either in a pattern or randomly—to form the oligomer. Preferred oligomers used in connection with present the invention are homo-oligomers. The water-soluble, non-peptidic oligomer comprises one or more monomers serially attached to form a chain of monomers. The oligomer can be formed from a single monomer type (i.e., is homo-oligomeric) or two or three monomer types (i.e., is co-oligomeric).
An “oligomer” is a molecule possessing from about 1 to about 30 monomers. Specific oligomers for use in the invention include those having a variety of geometries such as linear, branched, or forked, to be described in greater detail below.
“PEG” or “polyethylene glycol,” as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a “PEG oligomer” or an oligoethylene glycol is one in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, though, the oligomer may contain distinct end capping moieties or functional groups, e.g., for conjugation. PEG oligomers for use in the present invention will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for the PEG oligomers, the variable (n) ranges from about 1 to 30, and the terminal groups and architecture of the overall PEG can vary. When PEG further comprises a functional group, A, for linking to, e.g., a small molecule drug, the functional group when covalently attached to a PEG oligomer does not result in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).
The terms “end-capped” or “terminally capped” are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety. Typically, although not necessarily, the end-capping moiety comprises a hydroxy or C1-20 alkoxy group. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. In addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned. Moreover, the end-capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active agent) of interest to which the polymer is coupled, can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric moieties (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films, spectrometers, and the like. In addition, the end-capping group may contain a targeting moiety.
The term “targeting moiety” is used herein to refer to a molecular structure that helps the conjugates of the invention to localize to a targeting area, e.g., help enter a cell, or bind a receptor. Preferably, the targeting moiety comprises of vitamin, antibody, antigen, receptor, DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell specific lectins, steroid or steroid derivative, RGD peptide, ligand for a cell surface receptor, serum component, or combinatorial molecule directed against various intra- or extracellular receptors. The targeting moiety may also comprise a lipid or a phospholipid. Exemplary phospholipids include, without limitation, phosphatidylcholines, phospatidylserine, phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine. These lipids may be in the form of micelles or liposomes and the like. The targeting moiety may further comprise a detectable label or alternately a detectable label may serve as a targeting moiety. When the conjugate has a targeting group comprising a detectable label, the amount and/or distribution/location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, gold particles, quantum dots, and the like.
“Branched,” in reference to the geometry or overall structure of an oligomer, refers to an oligomer having two or more polymers “arms” extending from a branch point.
“Forked,” in reference to the geometry or overall structure of an oligomer, refers to an oligomer having two or more functional groups (typically through one or more atoms) extending from a branch point.
A “branch point” refers to a bifurcation point comprising one or more atoms at which an oligomer branches or forks from a linear structure into one or more additional arms.
The term “reactive” or “activated” refers to a functional group that reacts readily or at a practical rate under conventional conditions of organic synthesis. This is in contrast to those groups that either do not react or require strong catalysts or impractical reaction conditions in order to react (i.e., a “nonreactive” or “inert” group).
“Not readily reactive,” with reference to a functional group present on a molecule in a reaction mixture, indicates that the group remains largely intact under conditions that are effective to produce a desired reaction in the reaction mixture.
A “protecting group” is a moiety that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protecting group may vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule. Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters (such as a p-methoxybenzyl ester), amides and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; and the like. Such protecting groups are well-known to those skilled in the art and are described, for example, in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
A functional group in “protected form” refers to a functional group bearing a protecting group. As used herein, the term “functional group” or any synonym thereof encompasses protected forms thereof.
A “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a relatively labile bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates.
An “enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.
A “stable” linkage or bond refers to a chemical bond that is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
“Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater, more preferably 97% or greater, still more preferably 98% or greater, even more preferably 99% or greater, yet still more preferably 99.9% or greater, with 99.99% or greater being most preferred of some given quantity.
“Monodisperse” refers to an oligomer composition wherein substantially all of the oligomers in the composition have a well-defined, single molecular weight and defined number of monomers, as determined by chromatography or mass spectrometry. Monodisperse oligomer compositions are in one sense pure, that is, substantially having a single and definable number (as a whole number) of monomers rather than a large distribution. A monodisperse oligomer composition possesses a MW/Mn value of 1.0005 or less, and more preferably, a MW/Mn value of 1.0000. By extension, a composition comprised of monodisperse conjugates means that substantially all oligomers of all conjugates in the composition have a single and definable number (as a whole number) of monomers rather than a large distribution and would possess a MW/Mn value of 1.0005, and more preferably, a MW/Mn value of 1.0000 if the oligomer were not attached to the therapeutic moiety. A composition comprised of monodisperse conjugates may, however, include one or more nonconjugate substances such as solvents, reagents, excipients, and so forth.
“Bimodal,” in reference to an oligomer composition; refers to an oligomer composition wherein substantially all oligomers in the composition have one of two definable and different numbers (as whole numbers) of monomers rather than a large distribution, and whose distribution of molecular weights, when plotted as a number fraction versus molecular weight, appears as two separate identifiable peaks. Preferably, for a bimodal oligomer composition as described herein, each peak is generally symmetric about its mean, although the size of the two peaks may differ. Ideally, the polydispersity index of each peak in the bimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 or less, and even more preferably 1.0005 or less, and most preferably a MW/Mn value of 1.0000. By extension, a composition comprised of bimodal conjugates means that substantially all oligomers of all conjugates in the composition have one of two definable and different numbers (as whole numbers) of monomers rather than a large distribution and would possess a MW/Mn value of 1.01 or less, more preferably 1.001 or less and even more preferably 1.0005 or less, and most preferably a MW/Mn value of 1.0000 if the oligomer were not attached to the therapeutic moiety. A composition comprised of bimodal conjugates may, however, include one or more nonconjugate substances such as solvents, reagents, excipients, and so forth.
A “diaromatic substituted compound” is broadly used herein to refer to an organic, inorganic, or organometallic compound having a molecular weight of less than about 1000 Daltons and having some degree of activity as a diaromatic substituted compound therapeutic. Diaromatic substituted compound activity of a compound may be measured by assays known in the art and also as described herein.
A “biological membrane” is any membrane made of cells or tissues that serves as a barrier to at least some foreign entities or otherwise undesirable materials. As used herein a “biological membrane” includes those membranes that are associated with physiological protective barriers including, for example: the blood-brain barrier (BBB); the blood-cerebrospinal fluid barrier; the blood-placental barrier; the blood-milk barrier; the blood-testes barrier; and mucosal barriers including the vaginal mucosa, urethral mucosa, anal mucosa, buccal mucosa, sublingual mucosa, and rectal mucosa. Unless the context clearly dictates otherwise, the term “biological membrane” does not include those membranes associated with the middle gastro-intestinal tract (e.g., stomach and small intestines).
A “biological membrane crossing rate,” provides a measure of a compound's ability to cross a biological membrane, such as the blood-brain barrier (“BBB”). A variety of methods may be used to assess transport of a molecule across any given biological membrane. Methods to assess the biological membrane crossing rate associated with any given biological barrier (e.g., the blood-cerebrospinal fluid barrier, the blood-placental barrier, the blood-milk barrier, the intestinal barrier, and so forth), are known, described herein and/or in the relevant literature, and/or may be determined by one of ordinary skill in the art.
A “reduced rate of metabolism” refers to a measurable reduction in the rate of metabolism of a water-soluble oligomer-small molecule drug conjugate as compared to the rate of metabolism of the small molecule drug not attached to the water-soluble oligomer (i.e., the small molecule drug itself) or a reference standard material. In the special case of “reduced first pass rate of metabolism,” the same “reduced rate of metabolism” is required except that the small molecule drug (or reference standard material) and the corresponding conjugate are administered orally. Orally administered drugs are absorbed from the gastro-intestinal tract into the portal circulation and may pass through the liver prior to reaching the systemic circulation. Because the liver is the primary site of drug metabolism or biotransformation, a substantial amount of drug may be metabolized before it ever reaches the systemic circulation. The degree of first pass metabolism, and thus, any reduction thereof, may be measured by a number of different approaches. For instance, animal blood samples may be collected at timed intervals and the plasma or serum analyzed by liquid chromatography/mass spectrometry for metabolite levels. Other techniques for measuring a “reduced rate of metabolism” associated with the first pass metabolism and other metabolic processes are known, described herein and/or in the relevant literature, and/or may be determined by one of ordinary skill in the art. Preferably, a conjugate of the invention may provide a reduced rate of metabolism reduction satisfying at least one of the following values: at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; and at least about 90%. A compound (such as a small molecule drug or conjugate thereof) that is “orally bioavailable” is one that preferably possesses a bioavailability when administered orally of greater than 25%, and preferably greater than 70%, where a compound's bioavailability is the fraction of administered drug that reaches the systemic circulation in unmetabolized form.
“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl” includes cycloalkyl when three or more carbon atoms are referenced. An “alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least one carbon-carbon double bond.
The terms “substituted alkyl” or “substituted Cq-r alkyl” where q and r are integers identifying the range of carbon atoms contained in the alkyl group, denotes the above alkyl groups that are substituted by one, two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C1-7 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and so forth), C1-7 alkoxy, C1-7 acyloxy, C3-7 heterocyclic, amino, phenoxy, nitro, carboxy, acyl, cyano. The substituted alkyl groups may be substituted once, twice or three times with the same or with different substituents.
“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a lower alkyl group of 2 to 6 carbon atoms having at least one carbon-carbon double bond.
“Non-interfering substituents” are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.
“Alkoxy” refers to an —O—R group, wherein R is alkyl or substituted alkyl, preferably C1-C20 alkyl (e.g., methoxy, ethoxy, propyloxy, etc.), preferably C1-C7.
“Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to component that may be included in the compositions of the invention causes no significant adverse toxicological effects to a patient.
The term “aryl” means an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthalenyl, and the like.
“Substituted phenyl” and “substituted aryl” denote a phenyl group and aryl group, respectively, substituted with one, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosen from halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g., C1-6 alkyl), alkoxy (e.g., C1-6 alkoxy), benzyloxy, carboxy, aryl, and so forth.
Chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms are also used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g., CH3—CH2—), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., —CH2—CH2—), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding multivalent moiety, arylene). All atoms are understood to have their normal number of valences for bond formation (i.e., 1 for H, 4 for carbon, 3 for N, 2 for 0, and 2, 4, or 6 for S, depending on the oxidation state of the S).
“Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of a water-soluble oligomer-small molecule drug conjugate present in a composition that is needed to provide a desired level of active agent and/or conjugate in the bloodstream or in the target tissue. The precise amount may depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and may readily be determined by one skilled in the art, based upon the information provided herein and available in the relevant literature.
A “difunctional” oligomer is an oligomer having two functional groups contained therein, typically at its termini. When the functional groups are the same, the oligomer is said to be homodifunctional. When the functional groups are different, the oligomer is said to be heterodifunctional.
A basic reactant or an acidic reactant described herein include neutral, charged, and any corresponding salt forms thereof.
The term “patient,” refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a conjugate as described herein, and includes both humans and animals.
“Optional” or “optionally” means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.
As indicated above, the present invention is directed to (among other things) a compound comprising a diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer.
The “diaromatic substituted compound residue” is a compound having a structure of a diaromatic substituted compound that is altered by the presence of one or more bonds, which bonds serve to attach (either directly or indirectly) one or more water-soluble, non-peptidic oligomers. Exemplary diaromatic substituted compounds have a structure encompassed by at least one of the structures defined as Formula I;




wherein:

R1 is —CH2— or —CO—;
Z is




R7 is —N(R7-5)H where R7-5 is C1-C6 alkyl, —CH2-cyclopropyl, or —CH2—CH2F;
R8 is —H or —F;
R9 is —H or —F;
R10 is —H or —F; and
Aryl/Heteroaryl is a substituent of formula




wherein X14 is selected from the group consisting of —H, —CH2-phenyl, —O—CH2-phenyl, —O—CH2—COOH, C1-C6 alkyl (e.g., C1-C4 alkyl), —F, —Br, —O—SO2—(C1-C4 alkyl), —NH—SO2—(C1-C6 alkyl), —NO2, —NH2, —N3, —N═CH—N(C1-C6 alkyl)(C1-C6 alkyl), —N═C(C1-C4 alkyl)-N(C1-C6 alkyl)(C1-C6 alkyl), —NH—CO—X14-9 where X14-9 is —H, —C1-C4 alkyl or phenyl; n7 is 0 or 1; X6, when present, is —H, —OH, —(CH2)—OH, —O—CH2-phenyl, —CHO, C1-C3 alkoxy, —O—SO2—C1-C4 alkyl, —NH—SO2—C1-C4 alkyl, —C≡N, —O—(CH2)2-5—N(X6-3)(X6-4) where X6-3 and X6-4 are both —H or where X6-3 and X6-4 are taken together with the attached nitrogen atom to form a heterocyclic ring selected from the group consisting of 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and 1-aziridinyl.

In one or more embodiments of the invention, a compound is provided, the compound comprising a diaromatic substituted compound residue covalently attached via a stable or degradable linkage to a water-soluble, non-peptidic oligomer, wherein the diaromatic substituted compound residue is a residue of delavirdine, the chemical or IUPAC name of which is N-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-1-yl}carbonyl)-1H-indol-5-yl]methanesulfonamide.
In some instances, diaromatic substituted compounds can be obtained from commercial sources. In addition, diaromatic substituted compounds can be obtained through chemical synthesis. Examples of diaromatic substituted compounds as well as synthetic approaches for preparing diaromatic substituted compounds are described in the literature and in, for example, U.S. Pat. No. 5,563,142. Each of these (and other) diaromatic substituted compounds can be covalently attached (either directly or through one or more atoms) to a water-soluble, non-peptidic oligomer.
Exemplary compounds of the invention include those having the following structure:




wherein:

R1 is —CH2— or —CO—;
Z is




R7 is —N(R7-5)H where R7-5 is C1-C6 alkyl, —CH2-cyclopropyl, or —CH2—CH2F;
R8 is —H or —F;
R9 is —H or —F;
R10 is —H or —F;
Aryl/Heteroaryl is a substituent of formula




wherein X14 is selected from the group consisting of —H, —CH2-phenyl, —O—CH2-phenyl, —O—CH2—COOH, C1-C6 alkyl (e.g., C1-C4 alkyl), —F, —Cl, —Br, —O—SO2—(C1-C4 alkyl), —NH—SO2—(C1-C6 alkyl), —NO2, —NH2, —N3, —N═CH—N(C1-C6 alkyl)(C1-C6 alkyl), —N═C(C1-C4 alkyl)-N(C1-C6 alkyl)(C1-C6 alkyl), —NH—CO—X14-9 where X14-9 is —H, —C1-C4 alkyl or phenyl; n7 is 0 or 1; X6, when present, is —H, —OH, —(CH2)—OH, —O—CH2-phenyl, —CHO, C1-C3 alkoxy, —O—SO2—C1-C4 alkyl, —NH—SO2—C1-C4 alkyl, —C≡N, —O—(CH2)2-5—N(X6-3)(X6-4) where X6-3 and X6-4 are both —H or where X6-3 and X6-4 are taken together with the attached nitrogen atom to form a heterocyclic ring selected from the group consisting of 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, N-morpholinyl and 1-aziridinyl;

X is a spacer moiety; and
POLY is a water-soluble, non-peptidic oligomer,
and pharmaceutically acceptable salts thereof.
Use of discrete oligomers (e.g., from a monodisperse or bimodal composition of oligomers, in contrast to relatively impure compositions) to form oligomer-containing compounds may advantageously alter certain properties associated with the corresponding small molecule drug. For instance, a compound of the invention, when administered by any of a number of suitable administration routes, such as parenteral, oral, transdermal, buccal, pulmonary, or nasal, exhibits reduced penetration across the blood-brain barrier. It is preferred that the compounds of the invention exhibit slowed, minimal or effectively no crossing of the blood-brain barrier, while still crossing the gastro-intestinal (GI) walls and into the systemic circulation if oral delivery is intended. Moreover, the compounds of the invention maintain a degree of bioactivity as well as bioavailability in comparison to the bioactivity and bioavailability of the compound free of all oligomers.
With respect to the blood-brain barrier (“BBB”), this barrier restricts the transport of drugs from the blood to the brain. This barrier consists of a continuous layer of unique endothelial cells joined by tight junctions. The cerebral capillaries, which comprise more than 95% of the total surface area of the BBB, represent the principal route for the entry of most solutes and drugs into the central nervous system.
For compounds whose degree of blood-brain barrier crossing ability is not readily known, such ability may be determined using a suitable animal model such as an in situ rat brain perfusion (“RBP”) model as described herein. Briefly, the RBP technique involves cannulation of the carotid artery followed by perfusion with a compound solution under controlled conditions, followed by a wash out phase to remove compound remaining in the vascular space. (Such analyses may be conducted, for example, by contract research organizations such as Absorption Systems, Exton, Pa.). In one example of the RBP model, a cannula is placed in the left carotid artery and the side branches are tied off. A physiologic buffer containing the analyte (typically but not necessarily at a 5 micromolar concentration level) is perfused at a flow rate of about 10 mL/minute in a single pass perfusion experiment. After 30 seconds, the perfusion is stopped and the brain vascular contents are washed out with compound-free buffer for an additional 30 seconds. The brain tissue is then removed and analyzed for compound concentrations via liquid chromatography with tandem mass spectrometry detection (LC/MS/MS). Alternatively, blood-brain barrier permeability can be estimated based upon a calculation of the compound's molecular polar surface area (“PSA”), which is defined as the sum of surface contributions of polar atoms (usually oxygens, nitrogens and attached hydrogens) in a molecule. The PSA has been shown to correlate with compound transport properties such as blood-brain barrier transport. Methods for determining a compound's PSA can be found, e.g., in, Ertl, P., et al., J. Med. Chem. 2000, 43, 3714-3717; and Kelder, J., et al., Pharm. Res. 1999, 16, 1514-1519.
With respect to the blood-brain barrier, the water-soluble, non-peptidic oligomer-small molecule drug conjugate exhibits a blood-brain barrier crossing rate that is reduced as compared to the crossing rate of the small molecule drug not attached to the water-soluble, non-peptidic oligomer. Exemplary reductions in blood-brain barrier crossing rates for the compounds described herein include reductions of: at least about 5%; at least about 10%; at least about 25%; at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; or at least about 90%, when compared to the blood-brain barrier crossing rate of the small molecule drug not attached to the water-soluble oligomer. A preferred reduction in the blood-brain barrier crossing rate for a conjugate of the invention is at least about 20%.
Assays for determining whether a given compound (regardless of whether the compound includes a water-soluble, non-peptidic oligomer or not) can act as a diaromatic substituted compound are known and/or may be prepared by one of ordinary skill in the art and are further described infra.
Each of these (and other) diaromatic substituted compound moieties can be covalently attached (either directly or through one or more atoms) to a water-soluble, non-peptidic oligomer.
Exemplary molecular weights of small molecule drugs include molecular weights of: less than about 950; less than about 900; less than about 850; less than about 800; less than about 750; less than about 700; less than about 650; less than about 600; less than about 550; less than about 500; less than about 450; less than about 400; less than about 350; and less than about 300 Daltons.
The small molecule drug used in the invention, if chiral, may be obtained from a racemic mixture, or an optically active form, for example, a single optically active enantiomer, or any combination or ratio of enantiomers (i.e., scalemic mixture). In addition, the small molecule drug may possess one or more geometric isomers. With respect to geometric isomers, a composition can comprise a single geometric isomer or a mixture of two or more geometric isomers. A small molecule drug for use in the present invention can be in its customary active form, or may possess some degree of modification. For example, a small molecule drug may have a targeting agent, tag, or transporter attached thereto, prior to or after covalent attachment of an oligomer. Alternatively, the small molecule drug may possess a lipophilic moiety attached thereto, such as a phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,” dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a small fatty acid. In some instances, however, it is preferred that the small molecule drug moiety does not include attachment to a lipophilic moiety.
The diaromatic substituted compound moiety for coupling to a water-soluble, non-peptidic oligomer possesses a free hydroxyl, carboxyl, thio, amino group, or the like (i.e., “handle”) suitable for covalent attachment to the oligomer. In addition, the diaromatic substituted compound moiety may be modified by introduction of a reactive group, preferably by conversion of one of its existing functional groups to a functional group suitable for formation of a stable covalent linkage between the oligomer and the drug.
Accordingly, each oligomer is composed of up to three different monomer types selected from the group consisting of: alkylene oxide, such as ethylene oxide or propylene oxide; olefinic alcohol, such as vinyl alcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, where alkyl is preferably methyl; α-hydroxy acid, such as lactic acid or glycolic acid; phosphazene, oxazoline, amino acids, carbohydrates such as monosaccharides, alditol such as mannitol; and N-acryloylmorpholine. Preferred monomer types include alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, and α-hydroxy acid. Preferably, each oligomer is, independently, a co-oligomer of two monomer types selected from this group, or, more preferably, is a homo-oligomer of one monomer type selected from this group.
The two monomer types in a co-oligomer may be of the same monomer type, for example, two alkylene oxides, such as ethylene oxide and propylene oxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide. Usually, although not necessarily, the terminus (or termini) of the oligomer that is not covalently attached to a small molecule is capped to render it unreactive. Alternatively, the terminus may include a reactive group. When the terminus is a reactive group, the reactive group is either selected such that it is unreactive under the conditions of formation of the final oligomer or during covalent attachment of the oligomer to a small molecule drug, or it is protected as necessary. One common end-functional group is hydroxyl or —OH, particularly for oligoethylene oxides.
The water-soluble, non-peptidic oligomer (e.g., “POLY” in various structures provided herein) can have any of a number of different geometries. For example, the water-soluble, non-peptidic oligomer can be linear, branched, or forked. Most typically, the water-soluble, non-peptidic oligomer is linear or is branched, for example, having one branch point. Although much of the discussion herein is focused upon poly(ethylene oxide) as an illustrative oligomer, the discussion and structures presented herein can be readily extended to encompass any water-soluble, non-peptidic oligomers described above.
The molecular weight of the water-soluble, non-peptidic oligomer, excluding the linker portion, is generally relatively low. Exemplary values of the molecular weight of the water-soluble polymer include: below about 1500; below about 1450; below about 1400; below about 1350; below about 1300; below about 1250; below about 1200; below about 1150; below about 1100; below about 1050; below about 1000; below about 950; below about 900; below about 850; below about 800; below about 750; below about 700; below about 650; below about 600; below about 550; below about 500; below about 450; below about 400; below about 350; below about 300; below about 250; below about 200; and below about 100 Daltons.
Exemplary ranges of molecular weights of the water-soluble, non-peptidic oligomer (excluding the linker) include: from about 100 to about 1400 Daltons; from about 100 to about 1200 Daltons; from about 100 to about 800 Daltons; from about 100 to about 500 Daltons; from about 100 to about 400 Daltons; from about 200 to about 500 Daltons; from about 200 to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75 to about 750 Daltons.
Preferably, the number of monomers in the water-soluble, non-peptidic oligomer falls within one or more of the following ranges: between about 1 and about 30 (inclusive); between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10. In certain instances, the number of monomers in series in the oligomer (and the corresponding conjugate) is one of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, the oligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, the oligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series. Thus, for example, when the water-soluble, non-peptidic polymer includes CH3—(OCH2CH2)n—, “n” is an integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and can fall within one or more of the following ranges: between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10.
When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7, 8, 9, or monomers, these values correspond to a methoxy end-capped oligo(ethylene oxide) having a molecular weights of about 75, 119, 163, 207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When the oligomer has 11, 12, 13, 14, or 15 monomers, these values correspond to methoxy end-capped oligo(ethylene oxide) having molecular weights corresponding to about 515, 559, 603, 647, and 691 Daltons, respectively.
When the water-soluble, non-peptidic oligomer is attached to the diaromatic substituted compound (in contrast to the step-wise addition of one or more monomers to effectively “grow” the oligomer onto the diaromatic substituted compound), it is preferred that the composition containing an activated form of the water-soluble, non-peptidic oligomer be monodisperse. In those instances, however, where a bimodal composition is employed, the composition will possess a bimodal distribution centering around any two of the above numbers of monomers. For instance, a bimodal oligomer may have any one of the following exemplary combinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and so forth.
In some instances, the composition containing an activated form of the water-soluble, non-peptidic oligomer will be trimodal or even tetramodal, possessing a range of monomers units as previously described. Oligomer compositions possessing a well-defined mixture of oligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) can be prepared by mixing purified monodisperse oligomers to obtain a desired profile of oligomers (a mixture of two oligomers differing only in the number of monomers is bimodal; a mixture of three oligomers differing only in the number of monomers is trimodal; a mixture of four oligomers differing only in the number of monomers is tetramodal), or alternatively, can be obtained from column chromatography of a polydisperse oligomer by recovering the “center cut”, to obtain a mixture of oligomers in a desired and defined molecular weight range.
It is preferred that the water-soluble, non-peptidic oligomer is obtained from a composition that is preferably unimolecular or monodisperse. That is, the oligomers in the composition possess the same discrete molecular weight value rather than a distribution of molecular weights. Some monodisperse oligomers can be purchased from commercial sources such as those available from Sigma-Aldrich, or alternatively, can be prepared directly from commercially available starting materials such as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can be prepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873 (1999), WO 02/098949, and U.S. Patent Application Publication 2005/0136031.
When present, the spacer moiety (through which the water-soluble, non-peptidic polymer is attached to the diaromatic substituted compound moiety) may be a single bond, a single atom, such as an oxygen atom or a sulfur atom, two atoms, or a number of atoms. A spacer moiety is typically but is not necessarily linear in nature. The spacer moiety, “X,” is hydrolytically stable, and is preferably also enzymatically stable. Preferably, the spacer moiety “X” is one having a chain length of less than about 12 atoms, and preferably less than about 10 atoms, and even more preferably less than about 8 atoms and even more preferably less than about 5 atoms, whereby length is meant the number of atoms in a single chain, not counting substituents. For instance, a urea linkage such as this, Roligomer—NH—(C═O)—NH—R′drug, is considered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkage does not comprise further spacer groups.
In some instances, the spacer moiety “X” comprises an ether, amide, urethane, amine, thioether, urea, or a carbon-carbon bond. Functional groups such as those discussed below, and illustrated in the examples, are typically used for forming the linkages. The spacer moiety may less preferably also comprise (or be adjacent to or flanked by) other atoms, as described further below.
More specifically, in selected embodiments, a spacer moiety of the invention, X, may be any of the following: “-” (i.e., a covalent bond, that may be stable or degradable, between the diaromatic substituted compound residue and the water-soluble, non-peptidic oligomer), —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —CH2—C(O)O—, —CH2—OC(O)—, —C(O)O—CH2—, —OC(O)—CH2—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH2—, —CH2—CH2—, —CH2—CH2—CH2—, —CH2—CH2—CH2—CH2—, —O—CH2—, —CH2—O—, —O—CH2—CH2—, —CH2—O—CH2—, —CH2—CH2—O—, —O—CH2—CH2—CH2—, —CH2—O—CH2—CH2—, —CH2—CH2—O—CH2—; —CH2—CH2—CH2—O—, —O—CH2—CH2—CH2—CH2—, —CH2—O—CH2—CH2—CH2—, —CH2—CH2—O—CH2—CH2—, —CH2—CH2—CH2—O—CH2—, —CH2—CH2—CH2—CH2—O—, —C(O)—NH—CH2—, —C(O)—NH—CH2—CH2—, —CH2—C(O)—NH—CH2—, —CH2—CH2—C(O)—NH—, —C(O)—NH—CH2—CH2—CH2—, —CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—C(O)—NH—CH2—, —CH2—CH2—CH2—C(O)—NH—, —C(O)—NH—CH2—CH2—CH2—CH2—, —CH2—C(O)—NH—CH2—CH2—CH2—, —CH2—CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—CH2—C(O)—NH—CH2—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—CH2—CH2—C(O)—NH—, —NH—C(O)—CH2—, —CH2—NH—C(O)—CH2—, —CH2—CH2—NH—C(O)—CH2—, —NH—C(O)—CH2—CH2—, —CH2—NH—C(O)—CH2—CH2, —CH2—CH2—NH—C(O)—CH2—CH2, —C(O)—NH—CH2—, —C(O)—NH—CH2—CH2—, —O—C(O)—NH—CH2—, —O—C(O)—NH—CH2—CH2—, —NH—CH2—, —NH—CH2—CH2—, —CH2—NH—CH2—, —CH2—CH2—NH—CH2—, —C(O)—CH2—, —C(O)—CH2—CH2—, —CH2—C(O)—CH2—, —CH2—CH2—C(O)—CH2—, —CH2—CH2—C(O)—CH2—CH2—, —CH2—CH2—C(O)—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—CH2—, bivalent cycloalkyl group, —N(R6)—, R6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl. Additional spacer moieties include, acylamino, acyl, aryloxy, alkylene bridge containing between 1 and 5 inclusive carbon atoms, alkylamino, dialkylamino having about 2 to 4 inclusive carbon atoms, piperidino, pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino, 1-piperizinyl, 4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl, 4-(methoxy-lower alkyl)-1-piperizinyl, and guanidine. In some instances, a portion or a functional group of the drug compound may be modified or removed altogether to facilitate attachment of the oligomer. In some instances, it is preferred that X is not an amide, i.e., —CONR— or —RNCO—).
For purposes of the present invention, however, a group of atoms is not considered a linkage when it is immediately adjacent to an oligomer segment, and the group of atoms is the same as a monomer of the oligomer such that the group would represent a mere extension of the oligomer chain.
The linkage “X” between the water-soluble, non-peptidic oligomer and the small molecule is formed by reaction of a functional group on a terminus of the oligomer (or nascent oligomer when it is desired to “grow” the oligomer onto the diaromatic substituted compound) with a corresponding functional group within the diaromatic substituted compound. Illustrative reactions are described briefly below. For example, an amino group on an oligomer may be reacted with a carboxylic acid or an activated carboxylic acid derivative on the small molecule, or vice versa, to produce an amide linkage. Alternatively, reaction of an amine on an oligomer with an activated carbonate (e.g. succinimidyl or benzotriazolyl carbonate) on the drug, or vice versa, forms a carbamate linkage. Reaction of an amine on an oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, forms a urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group on an oligomer with an alkyl halide, or halide group within a drug, or vice versa, forms an ether linkage. In yet another coupling approach, a small molecule having an aldehyde function is coupled to an oligomer amino group by reductive amination, resulting in formation of a secondary amine linkage between the oligomer and the small molecule.
A particularly preferred water-soluble, non-peptidic oligomer is an oligomer bearing an aldehyde functional group. In this regard, the oligomer will have the following structure: CH3O—(CH2—CH2—O)n—(CH2)p—C(O)H, wherein (n) is one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred (n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.
The termini of the water-soluble, non-peptidic oligomer not bearing a functional group may be capped to render it unreactive. When the oligomer includes a further functional group at a terminus other than that intended for formation of a conjugate, that group is either selected such that it is unreactive under the conditions of formation of the linkage “X,” or it is protected during the formation of the linkage “X.”
As stated above, the water-soluble, non-peptidic oligomer includes at least one functional group prior to conjugation. The functional group comprises an electrophilic or nucleophilic group for covalent attachment to a small molecule, depending upon the reactive group contained within or introduced into the small molecule. Examples of nucleophilic groups that may be present in either the oligomer or the small molecule include hydroxyl, amine, hydrazine (—NHNH2), hydrazide (—C(O)NHNH2), and thiol. Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol, particularly amine. Most small molecule drugs for covalent attachment to an oligomer will possess a free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.
Examples of electrophilic functional groups that may be present in either the oligomer or the small molecule include carboxylic acid, carboxylic ester, particularly imide esters, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. More specific examples of these groups include succinimidyl ester or carbonate, imidazoyl ester or carbonate, benzotriazole ester or carbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate (2,2,2-trifluoroethanesulfonate).
Also included are sulfur analogs of several of these groups, such as thione, thione hydrate, thioketal, 2-thiazolidine thione, etc., as well as hydrates or protected derivatives of any of the above moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal, thioketal, thioacetal).
An “activated derivative” of a carboxylic acid refers to a carboxylic acid derivative that reacts readily with nucleophiles, generally much more readily than the underivatized carboxylic acid. Activated carboxylic acids include, for example, acid halides (such as acid chlorides), anhydrides, carbonates, and esters. Such esters include imide esters, of the general form —(CO)O—N[(CO)—]2; for example, N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Also preferred are imidazolyl esters and benzotriazole esters. Particularly preferred are activated propionic acid or butanoic acid esters, as described in co-owned U.S. Pat. No. 5,672,662. These include groups of the form —(CH2)2-3C(═O)O-Q, where Q is preferably selected from N-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole, 7-azabenzotriazole, and imidazole.
Other preferred electrophilic groups include succinimidyl carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide.
These electrophilic groups are subject to reaction with nucleophiles, e.g., hydroxy, thio, or amino groups, to produce various bond types. Preferred for the present invention are reactions which favor formation of a hydrolytically stable linkage. For example, carboxylic acids and activated derivatives thereof, which include orthoesters, succinimidyl esters, imidazolyl esters, and benzotriazole esters, react with the above types of nucleophiles to form esters, thioesters, and amides, respectively, of which amides are the most hydrolytically stable. Carbonates, including succinimidyl, imidazolyl, and benzotriazole carbonates, react with amino groups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl or amino groups to form, respectively, carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes, ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e., aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, and ketal) are preferably reacted with amines, followed by reduction of the resulting imine, if desired, to provide an amine linkage (reductive amination).
Several of the electrophilic functional groups include electrophilic double bonds to which nucleophilic groups, such as thiols, can be added, to form, for example, thioether bonds. These groups include maleimides, vinyl sulfones, vinyl pyridine, acrylates, methacrylates, and acrylamides. Other groups comprise leaving groups that can be displaced by a nucleophile; these include chloroethyl sulfone, pyridyl disulfides (which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate, thiosulfonate, and tresylate. Epoxides react by ring opening by a nucleophile, to form, for example, an ether or amine bond. Reactions involving complementary reactive groups such as those noted above on the oligomer and the small molecule are utilized to prepare the conjugates of the invention.
In some instances the diaromatic substituted compound may not have a functional group suited for conjugation. In this instance, it is possible to modify (or “functionalize”) the “original” diaromatic substituted compound so that it does have a functional group suited for conjugation. For example, if the diaromatic substituted compound has an amide group, but an amine group is desired, it is possible to modify the amide group to an amine group by way of a Hofmann rearrangement, Curtius rearrangement (once the amide is converted to an azide) or Lossen rearrangement (once amide is concerted to hydroxamide followed by treatment with tolyene-2-sulfonyl chloride/base).
It is possible to prepare a conjugate of small molecule diaromatic substituted compound bearing a carboxyl group wherein the carboxyl group-bearing small molecule diaromatic substituted compound is coupled to an amino-terminated oligomeric ethylene glycol, to provide a conjugate having an amide group covalently linking the small molecule diaromatic substituted compound to the oligomer. This can be performed, for example, by combining the carboxyl group-bearing small molecule diaromatic substituted compound with the amino-terminated oligomeric ethylene glycol in the presence of a coupling reagent, (such as dicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.
Further, it is possible to prepare a conjugate of a small molecule diaromatic substituted compound bearing a hydroxyl group wherein the hydroxyl group-bearing small molecule diaromatic substituted compound is coupled to an oligomeric ethylene glycol halide to result in an ether (—O—) linked small molecule conjugate. This can be performed, for example, by using sodium hydride to deprotonate the hydroxyl group followed by reaction with a halide-terminated oligomeric ethylene glycol.
Further, it is possible to prepare a conjugate of a small molecule diaromatic substituted compound moiety bearing a hydroxyl group wherein the hydroxyl group-bearing small molecule diaromatic substituted compound moiety is coupled to an oligomeric ethylene glycol bearing an haloformate group [e.g., CH3(OCH2CH2)nOC(O)-halo, where halo is chloro, bromo, iodo] to result in a carbonate [—O—C(O)—O—] linked small molecule conjugate. This can be performed, for example, by combining a diaromatic substituted compound moiety and an oligomeric ethylene glycol bearing a haloformate group in the presence of a nucleophilic catalyst (such as 4-dimethylaminopyridine or “DMAP”) to thereby result in the corresponding carbonate-linked conjugate.
In another example, it is possible to prepare a conjugate of a small molecule diaromatic substituted compound bearing a ketone group by first reducing the ketone group to form the corresponding hydroxyl group. Thereafter, the small molecule diaromatic substituted compound now bearing a hydroxyl group can be coupled as described herein.
In still another instance, it is possible to prepare a conjugate of a small molecule diaromatic substituted compound bearing an amine group. In one approach, the amine group-bearing small molecule diaromatic substituted compound and an aldehyde-bearing oligomer are dissolved in a suitable buffer after which a suitable reducing agent (e.g., NaCNBH3) is added. Following reduction, the result is an amine linkage formed between the amine group of the amine group-containing small molecule diaromatic substituted compound and the carbonyl carbon of the aldehyde-bearing oligomer.
In another approach for preparing a conjugate of a small molecule diaromatic substituted compound bearing an amine group, a carboxylic acid-bearing oligomer and the amine group-bearing small molecule diaromatic substituted compound are combined, in the presence of a coupling reagent (e.g., DCC). The result is an amide linkage formed between the amine group of the amine group-containing small molecule diaromatic substituted compound and the carbonyl of the carboxylic acid-bearing oligomer.
While it is believed that the full scope of the conjugates disclosed herein behave as described, an optimally sized oligomer can be identified as follows.
First, an oligomer obtained from a monodisperse or bimodal water soluble oligomer is conjugated to the small molecule drug. Preferably, the drug is orally bioavailable, and on its own, exhibits a non-negligible blood-brain barrier crossing rate. Next, the ability of the conjugate to cross the blood-brain barrier is determined using an appropriate model and compared to that of the unmodified parent drug. If the results are favorable, that is to say, if, for example, the rate of crossing is significantly reduced, then the bioactivity of conjugate is further evaluated. Preferably, the compounds according to the invention maintain a significant degree of bioactivity relative to the parent drug, i.e., greater than about 30% of the bioactivity of the parent drug, or even more preferably, greater than about 50% of the bioactivity of the parent drug.
The above steps are repeated one or more times using oligomers of the same monomer type but having a different number of subunits and the results compared.
For each conjugate whose ability to cross the blood-brain barrier is reduced in comparison to the non-conjugated small molecule drug, its oral bioavailability is then assessed. Based upon these results, that is to say, based upon the comparison of conjugates of oligomers of varying size to a given small molecule at a given position or location within the small molecule, it is possible to determine the size of the oligomer most effective in providing a conjugate having an optimal balance between reduction in biological membrane crossing, oral bioavailability, and bioactivity. The small size of the oligomers makes such screenings feasible and allows one to effectively tailor the properties of the resulting conjugate. By making small, incremental changes in oligomer size and utilizing an experimental design approach, one can effectively identify a conjugate having a favorable balance of reduction in biological membrane crossing rate, bioactivity, and oral bioavailability. In some instances, attachment of an oligomer as described herein is effective to actually increase oral bioavailability of the drug.
For example, one of ordinary skill in the art, using routine experimentation, can determine a best suited molecular size and linkage for improving oral bioavailability by first preparing a series of oligomers with different weights and functional groups and then obtaining the necessary clearance profiles by administering the conjugates to a patient and taking periodic blood and/or urine sampling. Once a series of clearance profiles have been obtained for each tested conjugate, a suitable conjugate can be identified.
Animal models (rodents and dogs) can also be used to study oral drug transport. In addition, non-in vivo methods include rodent everted gut excised tissue and Caco-2 cell monolayer tissue-culture models. These models are useful in predicting oral drug bioavailability.
To determine whether the diaromatic substituted compound or the conjugate of a diaromatic substituted compound and a water-soluble non-peptidic polymer has activity as a diaromatic substituted compound therapeutic, it is possible to test such a compound. The diaromatic substituted compounds may be tested using available antiviral assays that are described here and are known to a person skilled in the art.
The compounds of the invention may be administered per se or in the form of a pharmaceutically acceptable salt, and any reference to the compounds of the invention herein is intended to include pharmaceutically acceptable salts. If used, a salt of a compound as described herein should be both pharmacologically and pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare the free active compound or pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention. Such pharmacologically and pharmaceutically acceptable salts can be prepared by reaction of the compound with an organic or inorganic acid, using standard methods detailed in the literature. Examples of useful salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulphonic and benzenesulphonic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium, or calcium salts of a carboxylic acid group.
The compounds of the invention may contain one or more chiral centers and for each chiral center, the invention contemplates each optical isomer as well as any combination or ratio of or an optically active form, for example, a single optically active enantiomer, or any combination or ratio of enantiomers (e.g., scalemic and racemic mixtures). In addition, the small molecule drug may possess one or more geometric isomers. With respect to geometric isomers, a composition can comprise a single geometric isomer or a mixture of two or more geometric isomers.
The present invention also includes pharmaceutical preparations comprising a conjugate as provided herein in combination with a pharmaceutical excipient. Generally, the conjugate itself will be in a solid form (e.g., a precipitate), which can be combined with a suitable pharmaceutical excipient that can be in either solid or liquid form.
Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol, myoinositol, and the like.
The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
The preparation may also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
An antioxidant can be present in the preparation as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA, zinc and other such suitable cations.
Pharmaceutically acceptable acids or bases may be present as an excipient in the preparation. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
The amount of the conjugate in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.
The amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. The optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.
Generally, however, excipients will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5%-98% by weight, more preferably from about 15-95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
These foregoing pharmaceutical excipients along with other excipients and general teachings regarding pharmaceutical compositions are described in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), the “Physician's Desk Reference”, 52nd ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.
The pharmaceutical compositions can take any number of forms and the invention is not limited in this regard. Exemplary preparations are most preferably in a form suitable for oral administration such as a tablet, caplet, capsule, gel cap, troche, dispersion, suspension, solution, elixir, syrup, lozenge, transdermal patch, spray, suppository, and powder.
Oral dosage forms are preferred for those conjugates that are orally active, and include tablets, caplets, capsules, gel caps, suspensions, solutions, elixirs, and syrups, and can also comprise a plurality of granules, beads, powders or pellets that are optionally encapsulated. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts.
Tablets and caplets, for example, can be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred when preparing tablets or caplets containing the conjugates described herein. In addition to the conjugate, the tablets and caplets will generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, flow agents, and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, promoting powder flow and preventing particle capping (i.e., particle breakage) when pressure is relieved. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums, or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Stabilizers, as well known in the art, are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.
Capsules are also preferred oral dosage forms, in which case the conjugate-containing composition can be encapsulated in the form of a liquid or gel (e.g., in the case of a gel cap) or solid (including particulates such as granules, beads, powders or pellets). Suitable capsules include hard and soft capsules, and are generally made of gelatin, starch, or a cellulosic material. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like.
Included are parenteral formulations in the substantially dry form (as a lyophilizate or precipitate, which can be in the form of a powder or cake), as well as formulations prepared for injection, which are liquid and require the step of reconstituting the dry form of parenteral formulation. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
In some cases, compositions intended for parenteral administration can take the form of nonaqueous solutions, suspensions, or emulsions, normally being sterile. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
The parenteral formulations described herein can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat.
The conjugate can also be administered through the skin using conventional transdermal patch or other transdermal delivery system, wherein the conjugate is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the conjugate is contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated structure can contain a single reservoir, or it can contain multiple reservoirs.
The conjugate can also be formulated into a suppository for rectal administration. With respect to suppositories, the conjugate is mixed with a suppository base material which is (e.g., an excipient that remains solid at room temperature but softens, melts or dissolves at body temperature) such as coca butter (theobroma oil), polyethylene glycols, glycerinated gelatin, fatty acids, and combinations thereof. Suppositories can be prepared by, for example, performing the following steps (not necessarily in the order presented): melting the suppository base material to form a melt; incorporating the conjugate (either before or after melting of the suppository base material); pouring the melt into a mold; cooling the melt (e.g., placing the melt-containing mold in a room temperature environment) to thereby form suppositories; and removing the suppositories from the mold.
In some embodiments of the invention, the compositions comprising the conjugates may further be incorporated into a suitable delivery vehicle. Such delivery vehicles may provide controlled and/or continuous release of the conjugates and may also serve as a targeting moiety. Non-limiting examples of delivery vehicles include, adjuvants, synthetic adjuvants, microcapsules, microparticles, liposomes, and yeast cell wall particles. Yeast cells walls may be variously processed to selectively remove protein component, glucan, or mannan layers, and are referred to as whole glucan particles (WGP), yeast beta-glucan mannan particles (YGMP), yeast glucan particles (YGP), Rhodotorula yeast cell particles (YCP). Yeast cells such as S. cerevisiae and Rhodotorula sp. are preferred; however, any yeast cell may be used. These yeast cells exhibit different properties in terms of hydrodynamic volume and also differ in the target organ where they may release their contents. The methods of manufacture and characterization of these particles are described in U.S. Pat. Nos. 5,741,495, 4,810,646, 4,992,540, 5,028,703 and 5,607,677, and U.S. Patent Application Publication Nos. 2005/0281781 and 2008/0044438.
The invention also provides a method for administering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with the conjugate. The method comprises administering, generally orally, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical preparation). Other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, and parenteral. As used herein, the term “parenteral” includes subcutaneous, intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections.
In instances where parenteral administration is utilized, it may be necessary to employ somewhat bigger oligomers than those described previously, with molecular weights ranging from about 500 to 30K Daltons (e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).
The method of administering may be used to treat any condition that can be remedied or prevented by administration of the particular conjugate. Those of ordinary skill in the art appreciate which conditions a specific conjugate can effectively treat. The actual dose to be administered will vary depend upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature. Generally, a therapeutically effective amount will range from about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to 750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.
The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
All articles, books, patents, patent publications and other publications referenced herein are incorporated by reference in their entireties. In the event of an inconsistency between the teachings of this specification and the art incorporated by reference, the meaning of the teachings in this specification shall prevail.
EXPERIMENTAL
It is to be understood that while the invention has been described in conjunction with certain preferred and specific embodiments, the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
All non-PEG chemical reagents referred to in the appended examples are commercially available unless otherwise indicated. The preparation of PEG-mers is described in, for example, U.S. Patent Application Publication No. 2005/0136031.
1H NMR (nuclear magnetic resonance) data was generated by an NMR spectrometer. A list of certain compounds as well as the source of the compounds is provided below.
Example 1
Synthesis of mPEGn-O-Delavirdine Conjugates




Synthesis of 2-Chloro-3-isopropylpyridine 2: Acetone (1.1 mL, 14.89 mmol) was added to a stirred solution of 3-amino-2-chloropyridine 1 (1.662 g, 12.67 mmol) in 1,2-dichloroethane at room temperature. And then acetic acid (0.7 mL, 12.22 mmol) was added. After about 10 min, NaB(OAc)3H (3.005 g, 13.47 mmol) was added. The resulting mixture was stirred at room temperature for 24 h. More of acetone (5.5 ml) and NaB(OAc)3H (356 mg) and DCE (5 mL) were added. HOAc (0.35 mL) was added. After 2 h, MeOH (5 mL) was added. After overnight at room temperature, water was added to quench the reaction. Sat. aqueous potassium carbonate was added to neutralize the acid. The organic phase was separated and the aqueous was extracted with dichloromethane (2×20 mL). The combined organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was separated by flash column chromatography on silica gel, eluting with 5-30% EtOAc/hexane to afford 1.20 g product as colorless oil, as well as 0.6326 g of starting material. 1H-NMR (CDCl3): 7.669 (dd, J=4.5-5.0 Hz and 1.5 Hz, 1 H, Ar—H), 7.075 (dd,J=8.0 Hz and 4.5-5.0 Hz, 1 H, Ar—H), 6.875 (dd, J=8.0 Hz and 1.5 Hz, 1 H, Ar—H), 4.194 (br, 1 H, NH), 3.644-3.578 (m, 1 H, CH), 1.260 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 171.1 (MH+).




Synthesis of 3-Nitro-2-(Boc-piperazinyl)pyridine 6: 1-Boc-piperazine 3 (1.135 g, 5.9 mmol) was dissolved in acetonitrile (20 mL) at room temperature, and potassium carbonate (1.02 g, 7.4 mmol) was added. The mixture was cooled to 0° C. and 2-chloro-3-nitropyridine 5 (0.950 g, 5.93 mmol) was added. The mixture was stirred at 0° C. for 20 min, at room temperature for 6 h. The mixture was concentrated to remove the solvent. The residue was mixture with water (5 mL) and brine (15 mL). The mixture was extracted with dichloromethane (3×20 mL). The combined organic solution was washed with brine ( ̃40 mL), dried over sodium sulfate, concentrated to afford a yellow residue. The residue was purified by flash column chromatography on silica gel, eluting with 5-40% EtOAc/hexane to afford 1.765 g of product in 97% yield. 1H-NMR (CDCl3): 8.342 (dd, J=4.5 Hz and 1.5 Hz, 1 H, Ar—H), 8.145 (dd, J=8.0 Hz and 1.50 Hz, 1 H, Ar—H), 6.788 (dd, J=8.0 Hz and 4.5 Hz, 1 H, Ar—H), 3.565 (m, 4 H, 2 CH2), 3.43 (br, 4 H, 2 CH2), 1.477 (s, 9 H, 3 CH3).




Synthesis of 3-Amino-2-(Boc-piperazinyl)pyridine 7: 3-Nitro-2-(Boc-piperazine)pyridine 6 (1.765 g, 5.72 mmol) was dissolved in methanol (15 mL) at room temperature. Then Pd(OH)2 (1.234 g) and cyclohexene (2.0 mL, 19.52 mmol) were added. The resulting mixture was heated at 70° C. for 2 h. The mixture was concentrated to remove the solvent. DCM was added and then it was filtered through a pad of silica gel, washed with DCM and 10% MeOH/DCM. The collected solution was concentrated and dried under high vacuum to afford a slight pink solid as the product (1.384 g, 87% yield). 1H-NMR (CDCl3): 7.794 (dd, J=5.0 Hz and 1.5 Hz, 1 H, Ar—H), 6.956 (dd, J=7.5 Hz and 1.5 Hz, 1 H, Ar—H), 6.854 (dd, J=7.5 Hz and 5.0 Hz, 1 H, Ar—H), 3.783 (br, 2 H, NH2), 3.571 (t, J=4.5-5.0 Hz, 4 H, 2 CH2), 3.059 (t, J=4.5-5.0 Hz, 4 H, 2 CH2), 1.482 (s, 9 H, 3 CH3). LC-MS: 279.2 (MH+). (ref. Romero D. L. et al. J. Med. Chem. 37, 999-1014, 1994.)




Synthesis of 2-(Boc-piperazinyl)-3-(isopropylamino)pyridine 4: 3-amino-2-(Boc-piperazine)pyridine 7 (3.813 g, about 11.16 mmol, crude) was dissolved in dichloroethane (75 mL). Acetone (1.7 mL, 23.12 mmol) was added, followed by an addition of HOAc (1.0 mL, 17.45 mmol) at room temperature. After about 10 min, NaBH(OAc)3 (7.46 g, 33.44 mmol) was added. An additional amount of dichloroethane (25 mL) was added. The resulting mixture was stirred at room temperature for 64 h. 5% aqueous NaHCO3 was added to quench the reaction. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2×50 mL). The combined organic solution was washed with brine, dried over anhydrous Na2SO4, concentrated. The residue was purified via flash column chromatography over silica gel with 5-35% ethyl acetate in hexane to afford the product as solid in quantitative yield. 1H-NMR (CDCl3): 7.673 (dd, J=5.0 Hz and 1.5 Hz, 1 H, Ar—H), 6.904 (dd, J=8.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.812 (dd, J=8.0 Hz and 1.5 Hz, 1 H, Ar—H), 4.150 (d, J=7.5 Hz, 1 H, NH), 3.569-3.517 (m, 5 H, 2CH2 and CH), 2.997 (t, J=5.0 Hz, 4 H, 2 CH2), 1.477 (s, 9 H, 3 CH3), 1.23 (d, J=6.0 Hz, 6 H, 2 CH3). (ref. Romero D. L. et al. J. Med. Chem. 37, 999-1014, 1994.)




Synthesis of 2-piperazinyl-3-(isopropylamino)pyridine 8: Trifluoroacetic acid (2.0 mL) was added to a stirred solution of 2-(Boc-piperazinyl)-3-(i -propylamino)pyridine 4 (1.426 g, 4.45 mmol) in dichloromethane (10 mL) at 0° C. The resulting mixture was stirred at 0° C. for 30 min, at room temperature for 1 h 15 min. Additional quantities of TFA (2.0 mL) were added. The mixture was stirred at room temperature for another 23 h. Sat. K2CO3 solution was slowly added to quench the reaction (gas ↑). Small amount of water was added to help the layers separation. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2×25 mL). The combined organic solution was washed with brine, dried over anhydrous Na2SO4, and concentrated to afford the product in quantitative yield. 1H-NMR (CDCl3): 7.681 (dd, J=5.0 Hz and 1.5 Hz, 1 H, Ar—H), 6.890 (dd, J=8.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.800 (dd, J=8.0 Hz and 1.5 Hz, 1 H, Ar—H), 4.170 (d, J=7.5 Hz, 1 H, NH), 3.567-3.485 (m, 1 H, CH), 3.046 (s, 8 H, 4 CH2), 1.983 (br, 1 H, NH), 1.234 (d, J=6.0 Hz, 6 H, 2 CH3).




Synthesis of Ethyl 5-O-mPEG3-indole Carboxylate 10 (n=3): A mixture of ethyl 5-hydroxyindole carboxylate 9 (264 mg, 1.29 mmol), mPEG3-OMs (632 mg, 2.61 mmol) and potassium carbonate (552 mg, 3.99 mmol) in acetonitrile (15 mL) was stirred at room temperature for 50 min, at 70° C. for 19 h. Additional quantities of mPEG3-OMs (340 mg, 1.40 mmol) were added. The mixture was stirred at 70° C. for another 23 h. The mixture was concentrated to remove the solvent. The residue was mixed with water, extracted with DCM (3×15 mL). The combined organic extractions were washed with brine, dried over anhydrous sodium sulfate, and concentrated. The residue was separated by flash column chromatography on silica gel, eluting with 30-70% EtOAc/Hexane to afford 327 mg of product (yield: 72%), as well as staring material (12.7 mg, yield: 4.7%). 1H-NMR (CDCl3): 8.762 (br, 1 H, NH), 7.300 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.128 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.084 (m, 1 H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 4.395 (q, J=7.0 Hz, 2 H, CH2), 4.165 (m, 2 H, CH2), 3.885 (m, 2 H, CH2), 3.771-3.751 (m, 2 H, CH2), 3.706-3.687 (m, 2 H, CH2), 3.673-3.654 (m, 2 H, CH2), 3.562-3.543 (m, 2 H, CH2), 3.378 (s, 3 H, CH3), 1.406 (t, J=7.0 Hz, 3 H, CH3). LC-MS: 352.2 (MH+), 374.2 (MNa+).




Synthesis of Ethyl 5-O-mPEG4-indole Carboxylate 10 (n=4): A mixture of ethyl 5-hydroxyindole carboxylate 9 (1.0734 g, 5.13 mmol), mPEG4-OMs (690 mg, 2.41 mmol) and potassium carbonate (3.0361 mg, 21.97 mmol) in acetone (40 mL) was stirred 60° C. for 7 h. Additional quantities of mPEG4-OMs (897 mg, 3.13 mmol) were added. The mixture was stirred at 60° C. for another 17 h. More mPEG4-OMs (756 mg, 2.64 mmol) was added. After 23 h at 60° C., mPEG4-OMs (438 mg, 1.48 mmol) and acetone (30 mL) were added. The mixture was stirred at 60° C. for two days. The mixture was cooled to room temperature, filtered. The solid was washed with DCM. The combined organic solution was concentrated. The residue was mixed with water, extracted with DCM (2×30 mL). The combined organic extractions were washed with brine, dried over anhydrous sodium sulfate, concentrated. The residue was separated by flash column chromatography on silica gel, eluting with 30-90% EtOAc/Hexane to afford 1.4612 g of product (yield: 72%). 1H-NMR (CDCl3): 8.803 (br, 1 H, NH), 7.300 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.123 (dd, J=2.5 Hz and 1.0 Hz, 1 H, Ar—H), 7.078 (d, J=2.5 Hz, 1 H, Ar—H), 7.023 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 4.391 (q, J=7.0 Hz, 2 H, CH2), 4.160 (m, 2 H, CH2), 3.881 (m, 2 H, CH2), 3.754-3.730 (m, 2 H, CH2), 3.702-3.624 (m, 8 H, 4 CH2), 3.545-3.527 (m, 2 H, CH2), 3.367 (s, 3 H, CH3), 1.403 (t, J=7.0 Hz, 3 H, CH3).




Synthesis of Ethyl 5-O-mPEG6-indole Carboxylate 10 (n=6): A mixture of ethyl 5-hydroxyindole carboxylate 9 (1.1334 g, 5.41 mmol), mPEG6-OMs (1.237 g, 3.3 mmol) and potassium carbonate (3.1465 mg, 3.1465 mmol) in acetone (45 mL) was stirred 60° C. for 23 h. Additional quantities of mPEG6-OMs (953 mg, 2.55 mmol) were added. The mixture was stirred at 60° C. for 24.5 h. The mixture was cooled to room temperature, filtered. The solid was washed with DCM and acetone. The combined organic solution was concentrated. The residue was separated by flash column chromatography on silica gel, eluting with 30-50% EtOAc/Hexane to afford 1.977 g of product (yield: 76%). 1H-NMR (CDCl3): 8.826 (br, 1 H, NH), 7.307 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.126 (dd, J=2.0-2.5 Hz and 1.0 Hz, 1 H, Ar—H), 7.080 (d, J=2.0 Hz, 1 H, Ar—H), 7.028 (dd, J=9.0 Hz and 2.0-2.5 Hz, 1 H, Ar—H), 4.393 (q, J=7.0 Hz, 2 H, CH2), 4.165 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.881 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.753-3.728 (m, 2 H, CH2), 3.695-3.626 (m, 16 H, 8 CH2), 3.553-3.535 (m, 2 H, CH2), 3.373 (s, 3 H, CH3), 1.406 (t, J=7.0 Hz, 3 H, CH3).
The other ethyl 5-O-mPEGn-indole carboxylate 10 (n=5, 7, 8, 9) could be synthesized employing the same procedure as the synthesis of 5-O-mPEG6-indole carboxylate.




Ethyl 5-O-mPEG5-indole Carboxylate 10 (n=5): 1H-NMR (CDCl3): 8.790 (br, 1 H, NH), 7.303 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.126 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.081 (d, J=2.5 Hz, 1 H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 4.392 (q, J=7.0 Hz, 2 H, CH2), 4.164 (d, J=4.5-5.0 Hz, 2 H, CH2), 3.881 (d, J=4.0-4.5 Hz, 2 H, CH2), 3.752-3.728 (m, 2 H, CH2), 3.701-3.623 (m, 12 H, 6 CH2), 3.549-3.350 (m, 2 H, CH2), 3.371 (s, 3 H, CH3), 1.406 (t, J=7.0 Hz, 3 H, CH3).




Ethyl 5-O-mPEG7-indole Carboxylate 10 (n=7): 1H-NMR (CDCl3): 8.851 (br, 1 H, NH), 7.310 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.126 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.080 (d, J=2.0 Hz, 1 H, Ar—H), 7.027 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 4.393 (q, J=7.0 Hz, 2 H, CH2), 4.165 (t, J=5.0 Hz, 2 H, CH2), 3.882 (t, J=5.0 Hz, 2 H, CH2), 3.752-3.728 (m, 2 H, CH2), 3.701-3.628 (m, 20 H, 10 CH2), 3.372 (s, 3 H, CH3), 1.406 (t, J=7.0 Hz, 3 H, CH3).




Ethyl 5-O-mPEG8-indole Carboxylate 10 (n=8): 1H-NMR (CDCl3): 8.892 (br, 1 H, NH), 7.311 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.123 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.077 (d, J=2.0 Hz, 1 H, Ar—H), 7.025 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 4.391 (q, J=7.0 Hz, 2 H, CH2), 4.163 (t, J=5.0 Hz, 2 H, CH2), 3.880 (t, J=5.0 Hz, 2 H, CH2), 3.751-3.726 (m, 2 H, CH2), 3.699-3.616 (m, 24 H, 12 CH2), 3.548-3.529 (m, 2 H, CH2), 3.369 (s, 3 H, CH3), 1.404 (t, J=7.0 Hz, 3 H, CH3).




Ethyl 5-O-mPEG9-indole Carboxylate 10 (n=9): 1H-NMR (CDCl3)
8.888 (br, 1 H, NH), 7.310 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.121 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.076 (d, J=2.5 Hz, 1 H, Ar—H), 7.023 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 4.388 (q, J=7.0 Hz, 2 H, CH2), 4.161 (t, J=5.0 Hz, 2 H, CH2), 3.878 (t, J=5.0 Hz, 2 H, CH2), 3.749-3.724 (m, 2 H, CH2), 3.698-3.618 (m, 28 H, 14 CH2), 3.545-3.527 (m, 2 H, CH2), 3.368 (s, 3 H, CH3), 1.402 (t, J=7.0 Hz, 3 H, CH3).




5-O-mPEG3-indole 2-Carboxylic Acid 11 (n=3): KOH (668 mg, 10.26 mmol) in water (5 mL) was added to a stirred solution of ethyl 5-O-mPEG3-indole carboxylate 10 (1.184 g, 3.37 mmol) in dioxane (15 mL). The resulting mixture was stirred at room temperature for 27.5 h. The reaction mixture was acidified with 1 N HCl to pH 4.18 and extracted with MeOH/DCM (10/90, 3×30 mL). The organic layers were combined and washed with brine, dried over Na2SO4, and concentrated under reduced pressure to afford a solid as product (1.0242 g, 94% yield). 1H-NMR (CDCl3): 8.997 (br, 1 H, NH), 7.297 (dt, J=9.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.207 (dd, J=2.0 Hz and 1.0 Hz, 1 H, Ar—H), 7.055 (d, J=2.0 Hz, 1 H, Ar—H), 7.034 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 4.157 (m, 2 H, CH2), 3.893 (m, 2 H, CH2), 3.784-3.757 (m, 2 H, CH2), 3.724-3.704 (m, 2 H, CH2), 3.690-3.671 (m, 2 H, CH2), 3.580-3.560 (m, 2 H, CH2), 3.384 (s, 3H, CH3). LC-MS: 324.2 (MH+).




5-O-mPEG4-indole 2-Carboxylic Acid 11 (n=4): KOH (872.5 mg, 13.40 mmol) in water (6 mL) was added to a stirred solution of ethyl 5-O-mPEG4-indole carboxylate 10 (1.4612 g, 3.695 mmol) in dioxane (25 mL). The resulting mixture was stirred at room temperature for 23.5 h. More KOH (794 mg, 12.20 mmol) in water (5 mL) was added. The mixture was stirred at room temperature for 16 h. The reaction mixture was acidified with 1 N HCl to pH 4.08 and extracted with MeOH/DCM (10/90, 3×30 mL). The organic layers were combined and washed with brine, dried over Na2SO4, and concentrated under reduced pressure to afford a solid as product (1.3139 g, 97% yield). 1H-NMR (CDCl3): 8.846 (br, 1 H, NH), 7.307 (d, J=9.0 Hz, 1 H, Ar—H), 7.219 (dd, J=2.0 Hz and 0.5 Hz, 1 H, Ar—H), 7.073 (d, J=2.0 Hz, 1 H, Ar—H), 7.053 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 4.165 (t, J=5.0 Hz, 2 H, CH2), 3.891 (t, J=5.0 Hz, 2 H, CH2), 3.766-3.744 (m, 2 H, CH2), 3.716-3.637 (m, 8 H, 4 CH2), 3.556-3.537 (m, 2 H, CH2), 3.373 (s, 3 H, CH3).




5-O-mPEG6-indole 2-Carboxylic Acid 11 (n=6): KOH (1.4043 g, 21.57 mmol) in water (10 mL) was added to a stirred solution of ethyl 5-O-mPEG6-indole carboxylate 10 (1.977 g, 4.09 mmol) in dioxane (15 mL). The resulting mixture was stirred at room temperature for 25 h. Water was added to dilute the mixture. The reaction mixture was acidified with 1 N HCl to pH 4.64 and extracted with DCM (3×30 mL). The pH of the aqueous solution was checked and adjusted to pH 4.37, extracted with DCM (3×25 mL). The combined organic solution was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to afford a solid as product (1.7766 g, 95% yield). 1H-NMR (CDCl3): 9.010 (br, 1 H, NH), 7.310 (d, J=9.0 Hz, 1 H, Ar—H), 7.210 (m, 1 H, Ar—H), 7.080 (br, 1 H, Ar—H), 7.042 (dd, J=9.0 Hz and 2.0-2.5 Hz, 1 H, Ar—H), 4.168 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.880 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.753-3.729 (m, 2 H, CH2), 3.694-3.678 (m, 2 H, CH2), 3.651-3.621 (m, 14 H, 7 CH2), 3.567-3.548 (m, 2 H, CH2), 3.382 (s, 3 H, CH3).
The other 5-O-mPEG6-indole 2-carboxylic acids 11 (n=5, 7, 8, 9) were synthesized from the corresponding ethyl 5-O-mPEGn-indole carboxylate by the same procedure.




5-O-mPEG5-indole 2-Carboxylic Acid 11 (n=5): 1H-NMR (CDCl3)
8.895 (br, 1 H, NH), 7.298 (d, J=9.0 Hz, 1 H, Ar—H), 7.201 (d, J=1.0 Hz, 1 H, Ar—H), 7.061 (br, 1 H, Ar—H), 7.036 (dd, J=9.0 Hz and 2.0-2.5 Hz, 1 H, Ar—H), 4.157 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.880 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.755-3.737 (m, 2 H, CH2), 3.704-3.623 (m, 12 H, 6 CH2), 3.553-3.534 (m, 2 H, CH2), 3.371 (s, 3 H, CH3).




5-O-mPEG7-indole 2-Carboxylic Acid 11 (n=7): 1H-NMR (CDCl3): 9.435 (br, 1 H, NH), 7.299 (d, J=9.0 Hz, 1 H, Ar—H), 7.172 (m, 1 H, Ar—H), 7.026 (br, 1 H, Ar—H), 6.992 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 4.125 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.858 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.740-3.722 (m, 2 H, CH2), 3.686-3.611 (m, 20 H, 10 CH2), 3.547-3.529 (m, 2 H, CH2), 3.360 (s, 3 H, CH3).




5-O-mPEG9-indole 2-Carboxylic Acid 11 (n=9): 1H-NMR (CDCl3): 9.320 (br, 1 H, NH), 7.313 (d, J=9.0 Hz, 1 H, Ar—H), 7.183 (m, 1 H, Ar—H), 7.059 (d, J=2.5 Hz, 1 H, Ar—H), 7.013 (dd, J=9.0 Hz and 2.0-2.5 Hz, 1 H, Ar—H), 4.149 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.868 (t, J=4.5-5.0 Hz, 2 H, CH2), 3.742-3.533 (m, 32 H, 16 CH2), 3.366 (s, 3 H, CH3).




Synthesis of 5-O-mPEG3-Delavirdine 12 (n=3): 5-O-mPEG3-indole 2-carboxylic acid 11 (n=3) (425.4 mg, 1.32 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (286 mg, 1.3 mmol) were dissolved in anhydrous THF (15 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.3 mL, 1.64 mmol) was added. The resulting mixture was stirred at room temperature for 5 h. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×20 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography on silica gel, eluting with EtOAc/Hexanes to result in the product (516.8 mg, yield: 76%) as white solid. 1H-NMR (CDCl3): 9.901 (br, 1H, NH), 7.689 (dd, J=5.0 Hz and 1.5 Hz, 1 H, Ar—H), 7.312 (d, J=9.0 Hz, 1 H, Ar—H), 7.043 (d, J=2.0 Hz, 1 H, Ar—H), 6.956 (dd, J=9.0 Hz and 2.0 Hz, 1 H, Ar—H), 6.928 (dd, J=8.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.842 (dd, J=8.0 Hz and 1.5 Hz, 1 H, Ar—H), 6.727 (d, J=1.5 Hz, 1 H, Ar—H), 4.187 (d, J=7.5 Hz, 1 H, NH), 4.146 (t, J=5.0 Hz, 2 H, CH2), 4.078 (br, 4 H, 2 CH2), 3.866 (t, J=5.0 Hz, 2 H, CH2), 3.755-7.737 (m, 2 H, CH2), 3.369-3.674 (m, 2 H, CH2), 3.660-3.641 (m, 2 H, CH2), 3.592-3.529 (m, 3 H, CH2 and CH), 3.361 (s, 3 H, CH3), 3.177 (t, J=5.0 Hz, 4 H, 2 CH2), 1.254 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 526.2 (MH+).




Synthesis of 5-O-mPEG4-Delavirdine 12 (n=4): 5-O-mPEG4-indole 2-carboxylic acid 11 (n=4) (680 mg, 1.85 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (406.5 mg, 1.845 mmol) were dissolved in anhydrous THF (15 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.51 mL, 2.79 mmol) was added. The resulting mixture was stirred at room temperature for 23 h. 10% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×30 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography on silica gel, eluting with 60-100% EtOAc/Hexanes and EtOAc to result in the product (779 mg, yield: 74%) as white solid. 1H-NMR (CDCl3): 9.012 (br, 1H, NH), 7.695 (dd, J=4.5 Hz and 1.5 Hz, 1 H, Ar—H), 7.315 (d, J=9.0 Hz, 1 H, Ar—H), 7.062 (d, J=2.5 Hz, 1 H, Ar—H), 6.994 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.942 (dd, J=9.0 Hz and 4.5 Hz, 1 H, Ar—H), 6.856 (dd, J=8.0 Hz and 1.0-1.5 Hz, 1 H, Ar—H), 6.741 (d, J=1.0 Hz, 1 H, Ar—H), 4.192 (s, 1 H, NH), 4.164 (t, J=5.0 Hz, 2 H, CH2), 4.062 (br, 4 H, 2 CH2), 3.885 (t, J=5.0 Hz, 2 H, CH2), 3.760-7.739 (m, 2 H, CH2), 3.710-3.634 (m, 8 H, 4 CH2), 3.591-3.532 (m, 3 H, CH2 and CH), 3.371 (s, 3 H, CH3), 3.173 (t, J=5.0 Hz, 4 H, 2 CH2), 1.267 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 570.3 (MH+).




Synthesis of 5-O-mPEG5-Delavirdine 12 (n=5): 5-O-mPEG5-indole 2-carboxylic acid 11 (n=5) (371.8 mg, 0.90 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (196 mg, 0.89 mmol) were dissolved in anhydrous THF (32 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.25 mL, 1.37 mmol) was added. The resulting mixture was stirred at room temperature for 5 h 45 min. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×20 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography to result in the product (107.5 mg, yield: 20%) as oil. 1H-NMR (CDCl3): 8.991 (br, 1 H, NH), 7.696 (dd, J=4.5-5.0 Hz and 1.0-1.5 Hz, 1 H, Ar—H), 7.307 (d, J=9.0 Hz, 1 H, Ar—H), 7.053 (d, J=2.5 Hz, 1 H, Ar—H), 6.986 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.932 (dd, J=8.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.846 (dd, J=7.0 Hz and 1.0 Hz, 1 H, Ar—H), 6.732 (d, J=1.5 Hz, 1 H, Ar—H), 4.182 (s, 1 H, NH), 4.155 (t, J=5.0 Hz, 2 H, CH2), 4.051 (br, 4 H, 2 CH2), 3.874 (t, J=5.0 Hz, 2 H, CH2), 3.748-7.736 (m, 2 H, CH2), 3.693-3.617 (m, 12 H, 6 CH2), 3.597-3.522 (m, 3 H, CH2 and CH), 3.361 (s, 3 H, CH3), 3.133 (t, J=5.0 Hz, 4 H, 2CH2), 1.258 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 614.3 (MH+).




Synthesis of 5-O-mPEG6-Delavirdine 12 (n=6): 5-O-mPEG6-indole 2-carboxylic acid 11 (n=6) (554 mg, 1.22 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (268 mg, 1.22 mmol) were dissolved in anhydrous THF (40 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.35 mL, 1.92 mmol) was added. The resulting mixture was stirred at room temperature for 47.5 h. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×20 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography to result in the product (252 mg, yield: 32%) as oil. 1H-NMR (CDCl3): 9.110 (br, 1 H, NH), 7.696 (dd, J=4.5 Hz and 1.5 Hz, 1 H, Ar—H), 7.318 (d, J=9.0 Hz, 1 H, Ar—H), 7.058 (d, J=2.0 Hz, 1 H, Ar—H), 6.990 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.945 (dd, J=8.0 Hz and 4.5 Hz, 1 H, Ar—H), 6.859 (dd, J=8.0 Hz and 1.5 Hz, 1 H, Ar—H), 6.739 (d, J=1.5 Hz, 1 H, Ar—H), 4.192 (s, 1 H, NH), 4.162 (t, J=5.0 Hz, 2 H, CH2), 4.063 (br, 4 H, 2 CH2), 3.882 (t, J=5.0 Hz, 2 H, CH2), 3.756-7.733 (m, 2 H, CH2), 3.705-3.624 (m, 16 H, 8 CH2), 3.592-3.509 (m, 3 H, CH2 and CH), 3.370 (s, 3 H, CH3), 3.178 (t, J=5.0 Hz, 4 H, 2 CH2), 1.267 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 658.4 (MH+).




Synthesis of 5-O-mPEG7-Delavirdine 12 (n=7): 5-O-mPEG7-indole 2-carboxylic acid 11 (n=7) (627.7 mg, 1.26 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (281.8 mg, 1.28 mmol) were dissolved in anhydrous THF (12 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.40 mL, 2.19 mmol) was added. The resulting mixture was stirred at room temperature for 6 h. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×25 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography on SiO2 to result in the product (574.5 mg, yield: 65%) as oil. 1H-NMR (CDCl3): 9.010 (br, 1H, NH), 7.693 (dd, J=4.5 Hz and 1.5 Hz, 1 H, Ar—H), 7.319 (d, J=9.0 Hz, 1 H, Ar—H), 7.059 (d, J=2.0 Hz, 1 H, Ar—H), 6.993 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.940 (dd, J=9.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.854 (dd, J=8.0 Hz and 1.0 Hz, 1 H, Ar—H), 6.739 (d, J=1.5 Hz, 1 H, Ar—H), 4.191 (s, 1 H, NH), 4.163 (t, J=5.0 Hz, 2 H, CH2), 4.064 (br, 4 H, 2 CH2), 3.882 (t, J=5.0 Hz, 2 H, CH2), 3.756-7.733 (m, 2 H, CH2), 3.705-3.626 (m, 20 H, 10 CH2), 3.607-3.532 (m, 3 H, CH2 and CH), 3.371 (s, 3 H, CH3), 3.171 (t, J=5.0 Hz, 4 H, 2 CH2), 1.266 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 702.4 (MH+).




Synthesis of 5-O-mPEG8-Delavirdine 12 (n=8): 5-O-mPEG8-indole 2-carboxylic acid 11 (n=8) (741 mg, 1.36 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (300 mg, 1.36 mmol) were dissolved in anhydrous THF (15 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.40 mL, 2.19 mmol) was added. The resulting mixture was stirred at room temperature for 19 h. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×25 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography on SiO2 to result in the product (623.3 mg, yield: 61%) as oil. 1H-NMR (CDCl3): 9.021 (br, 1 H, NH), 7.693 (dd, J=5.0 Hz and 1.5 Hz, 1 H, Ar—H), 7.320 (d, J=9.0 Hz, 1 H, Ar—H), 7.059 (d, J=2.0 Hz, 1 H, Ar—H), 6.983 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.940 (dd, J=9.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.857 (dd, J=8.0 Hz and 1.0 Hz, 1 H, Ar—H), 6.739 (d, J=1.5 Hz, 1 H, Ar—H), 4.191 (s, 1 H, NH), 4.163 (t, J=5.0 Hz, 2 H, CH2), 4.060 (br, 4 H, 2 CH2), 3.883 (t, J=5.0 Hz, 2 H, CH2), 3.757-7.733 (m, 2 H, CH2), 3.700-3.627 (m, 24 H, 12 CH2), 3.594-3.531 (m, 3 H, CH2 and CH), 3.371 (s, 3 H, CH3), 3.171 (t, J=5.0 Hz, 4 H, 2CH2), 1.266 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 746.5 (MH+).




Synthesis of 5-O-mPEG9-Delavirdine 12 (n=9): 5-O-mPEG9-indole 2-carboxylic acid 11 (n=9) (952 mg, 1.62 mmol) and 2-piperazinyl-3-(isopropylamino) pyridine 8 (356 mg, 1.62 mmol) were dissolved in anhydrous THF (15 mL) at room temperature. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.50 mL, 2.19 mmol) was added. The resulting mixture was stirred at room temperature for 18 h. 5% aq. NaHCO3 solution was added to quench the reaction. The mixture was extracted with DCM (4×25 mL). The organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by flash column chromatography on SiO2 to result in the product (779.2 mg, yield: 61%) as oil. 1H-NMR (CDCl3): 9.057 (br, 1 H, NH), 7.697 (dd, J=4.5 Hz and 1.5 Hz, 1 H, Ar—H), 7.325 (d, J=9.0 Hz, 1 H, Ar—H), 7.062 (d, J=2.0 Hz, 1 H, Ar—H), 6.996 (dd, J=9.0 Hz and 2.5 Hz, 1 H, Ar—H), 6.944 (dd, J=8.0 Hz and 5.0 Hz, 1 H, Ar—H), 6.858 (dd, J=8.0 Hz and 1.0 Hz, 1 H, Ar—H), 6.742 (d, J=1.5 Hz, 1 H, Ar—H), 4.196 (s, 1 H, NH), 4.167 (t, J=5.0 Hz, 2 H, CH2), 4.065 (br, 4 H, 2 CH2), 3.886 (t, J=5.0 Hz, 2 H, CH2), 3.761-7.742 (m, 2 H, CH2), 3.704-3.635 (m, 28 H, 14 CH2), 3.612-3.535 (m, 3 H, CH2 and CH), 3.376 (s, 3 H, CH3), 3.175 (t, J=5.0 Hz, 4 H, 2 CH2), 1.270 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 702.4 (MH+)
Example 2
Synthesis of mPEGn-N-Delavirdine Conjugates







Synthesis of Ethylpyruvate 4-Nitrophenylhydrazone 14: Glacial acetic acid (3.0 mL, 48.38 mmol) was added to a stirred solution of 4-nitrophenylhydrazine (from Aldrich, >30% water as stabilizer, 9.0 g, 39.9 mmol) in ethyl alcohol (350 mL) at room temperature. And ethyl pyruvate (7.2 mL, 61.68 mmol) was added. The resulting mixture was stirred at room temperature for one hour and at 60° C. for 3.5 h, cooled to room temperature. The mixture was adjusted to pH about 10.25 with aq KOH solution. The mixture was concentrated to remove organic solvents. The brown solid was collected by filtering and washing with water to afford the product (8.19 g) in 82% yield. The product was direct used for the next step without purification. 1H-NMR (CDCl3): 8.217 (d, J=9.0 Hz, 2 H, Ar—H), 7.27 (d, J=9.0 Hz, 2 H, Ar—H), 4.348 (q, J=7.0 Hz, 2 H, CH2), 2.173 (s, 3 H, CH3), 1.399 (t, J=7.0 Hz, 3 H, CH3). (From the 1H-NMR, there are about 10% of another isomer).




Synthesis of 5-Nitro-1H-indole-2-carboxylic Acid Ethyl Ester 15 Ethylpyruvate 4-nitrophenylhydrazone 14 (8.18 g, 32.56 mmol) was heated at 125° C. in polyphosphoric acid (80 g) for 5 h. The mixture was cooled to room temperature, poured into water containing some ice. The solid was filtered through a funnel, washed with water. The solid was collected and recrystallized with ethyl acetate to afford the product (5.47 g, 72% yield). 1H-NMR (DMSO-d6): 8.734 (d, J=2.0 Hz, 1 H, Ar—H), 8.131 (dd, J=2.0 Hz and 9.0-9.5 Hz, 1 H, Ar—H), 7.606 (d, J=9.0-9.5 Hz, 1 H, Ar—H), 7.442 (br, 1 H, Ar—H), 4.378 (q, J=7.0-7.5 Hz, 2 H, CH2), 1.355 (t, J=7.0-7.5 Hz, 3 H, CH3).




Synthesis of 5-Nitro-1H-indole-2-carboxylic Acid 16: 5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (2.73 g, 11.65 mmol) and KOH (2.69 g, 41.33 mmol) were stirred in dioxane/water (50 mL/10 mL) at room temperature for 41 h. Water was added to diluted the solution. The mixture was concentrated to remove the organic solvent. The remaining solution was washed with DCM (100 mL). The aqueous solution was adjusted with 1 N HCl to pH 4.16, extracted with EtOAc (3×100 mL). The combined EtOAc extraction was washed with brine, dried over sodium sulfate, concentrated to afford the product as solid (1.79 g, 74% yield).
5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (194 mg, 0.83 mmol) and KOH (342 mg, 5.25 mmol) were stirred in MeOH/water (8 mL/4 mL) at room temperature for 21.5 h. More water was added to dilute the solution. The mixture was concentrated to remove the organic solvent. The remaining solution was washed with DCM (100 mL). The aqueous solution was adjusted with 1 N HCl to pH 2.9, extracted with EtOAc (2×15 mL). The combined EtOAc extraction was washed with brine, dried over sodium sulfate, concentrated to afford the product as solid (185.5 mg, 97% yield). 1H-NMR (DMSO-d6): 8.718 (d, J=2.0-2.5 Hz, 1 H, Ar—H), 8.116 (dd, J=2.0-2.5 Hz and 9.0-9.5 Hz, 1 H, Ar—H), 7.583 (d, J=9.0-9.5 Hz, 1 H, Ar—H), 7.378 (d, J=1.5 Hz, 1 H, Ar—H). LC-MS: 207.1 (MH+).




Synthesis of 5-Nitro-Delavirdine 17: EDC (1.75 mL, 9.59 mmol) was added to a stirred solution of 5-nitro indole acid (1.65 g, 8.0 mmol) and 3-isopropylamino-2-piperazinyl-pyridine (1.951 g, 8.86 mmol) in THF (75 mL) at room temperature. The resulting mixture was stirred at room temperature for 26.5 h. 5% NaHCO3 aq solution (100 mL) was added to quench the reaction. The mixture was concentrated to remove the organic solvent. The remaining mixture was extracted with DCM (4×100 mL). The combined organic solution was concentrated to afford a residue. The residue was mixed with small amount of DCM ( ̃15 mL), filtered and washed the solid with DCM to afford a solid as product. The above aqueous solution (after extraction) was filtered through a Celite funnel and washed the solid with acetone and DCM, dried to afford another part of product. The total yield was 50%. 1H-NMR (CDCl3): 9.640 (s, 1 H, NH), 8.640 (d, J=2.5 Hz, 1 H, Ar—H), 8.184 (dd, J=2.5 Hz and 9.0-9.5 Hz, 1 H, Ar—H), 7.700 (dd, J=1.5 and 5.0 Hz, 1 H, Ar—H), 7.489 (d, J=9.0-9.5 Hz, 1 H, Ar—H), 6.988-6.947 (m, 2 H, 2 Ar—H), 6.883-6.867 (m, 1 H, Ar—H), 4.189-4.090 (m, 5 H, NH and 2 CH2), 3.618-3.554 (m, 1 H, CH), 3.204 (m, 4 H, 2 CH2), 1.275 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 409.2 (MH+).




Synthesis of 5-Amino-Delaviridine 18: A mixture of 5-nitro delavirdine 17 (1.1773 g, 2.88 mmol) and cyclohexene (2.0 mL, 19.52 mmol) in THF/MeOH (80/100 mL) in the presence of Pd(OH)2 on activated carbon (Pearlman's catalyst) (2.74 g) was stirred at 75° C. for 7.5 h. The reaction mixture was cooled to room temperature. The mixture was filtered through a pad of Celite. The Celite and the palladium were washed with MeOH (2×15 mL). The combined solution was concentrated. The residue was purified by flash column chromatography on silica gel, eluting with 50-100% EtOAc/hexane to afford 0.5974 g of 5-amino delavirdine 18. 1H-NMR (CDCl3): 8.857 (s, 1 H, NH), 7.692 (dd, J=1.5 and 5.0 Hz, 1 H, Ar—H), 7.242 (d, J=8.5-9.0 Hz, 1 H, Ar—H), 6.950-6.901 (m, 2 H, 2 Ar—H), 6.860-6.841 (m, 1 H, Ar—H), 6.774 (dd, J=2.0-2.5 and 8.5-9.0 Hz, 1 H, 1 Ar—H), 6.642 (d, J=1.5 Hz, 1 H, Ar—H), 4.181 (m, 1 H, NH), 4.069 (m, 2 CH2), 3.576 (m, 3 H, NH2 and CH), 3.164 (m, 4 H, 2 CH2), 1.264 (d, J=6.5 Hz, 6 H, 2 CH3). LC-MS: 379.2 (MH+).




Synthesis of 5-mPEG-NH-Delavirdine 19 (n=1): A reaction mixture of 5-amino delavirdine (390 mg, 1.03 mmol), mPEG1-OMs (183.8 mg, 1.19 mmol), potassium carbonate (791 mg, 5.72 mmol) and tetrabutylammonium bromide (33 mg, 0.102 mmol) in CH3CN/DMF (9/2 mL) was placed in single-mode focused microwave reactor (CEM) and irradiated at 120° C. for 150 min. The mixture was concentrated to remove the organic solvent and the residue was mixture with dichloromethane (120 mL), washed with water (2×80 mL), dried over Na2SO4, and concentrated. The residue was purified by column chromatography to afford the product pure 5-mPEG1-NH-delavirdine 18 (n=1) (103.2 mg), as well as some starting material (89.5 mg). 1H-NMR (CDCl3): 9.568 (s, 1 H, NH), 7.691 (dd, J=1.0-1.5 and 4.5-5.0 Hz, 1 H, Ar—H), 7.237 (d, J=9.0 Hz, 1 H, Ar—H), 6.941-6.916 (m, 1 H, Ar—H), 6.851-6.835 (m, 1 H, Ar—H), 6.787 (m, 1 H, Ar—H), 6.738 (dd, J=2.0 and 9.0 Hz, 1 H, 1 Ar—H), 6.662 (d, J=1.0-1.5 Hz, 1 H, Ar—H), 4.190 (d, J=7.0 Hz, 1 H, NH), 4.067 (m, 4 H, 2 CH2), 3.640 (t, J=5.0 Hz, CH2), 3.593-3.529 (m, 1 H, CH), 3.391 (s, 3 H, CH3), 3.308 (t, J=5.0 Hz, 2 H, CH2), 3.196 (t, J=5.0 Hz, 4 H, 2CH2), 1.255 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 437.3 (MH+).




Synthesis of 5-mPEG2-NH-Delavirdine 19 (n=2): A reaction mixture of 5-amino delavirdine (244.6 mg, 0.647 mmol), mPEG2-OMs (176.7 mg, 0.891 mmol), potassium carbonate (514.8 mg, 3.725 mmol) and tetrabutylammonium bromide (56 mg, 0.174 mmol) in CH3CN/DMF (3.0/1.9 mL) was placed in single-mode focused microwave reactor (CEM) and irradiated at 120° C. for 150 min. The mixture was concentrated to remove the organic solvent and the residue was mixture with water (15 mL), extracted with dichloromethane (3×40 mL). The combined organic solution was washed with brine, dried over Na2SO4, concentrated. The residue was purified by column chromatography to afford the product pure 5-mPEG2-NH-delavirdine 18 (n=2) (50.4 mg), as well as some starting material and 5-di-mPEG2-H-delavirdine. 1H-NMR (CDCl3): 9.450 (s, 1H, NH), 7.719 (dd, J=1.5 and 5.0 Hz, 1 H, Ar—H), 7.266 (d, J=8.5-9.0 Hz, 1 H, Ar—H), 6.970-6.945 (m, 1 H, Ar—H), 6.881-6.862 (m, 1 H, Ar—H), 6.813 (m, 1 H, Ar—H), 6.768 (dd, J=2.0-2.5 and 8.5-9.0 Hz, 1 H, 1 Ar—H), 6.688 (d, J=1.5 Hz, 1 H, Ar—H), 4.216 (d, J=7.0 Hz, 1 H, NH), 4.092 (m, 4 H, 2 CH2), 3.769 (t, J=5.0-5.5 Hz, CH2), 3.685-3.667 (m, 2 H, CH2), 3.611-3.562 (m, 4 H, CH, NH and CH2), 3.423 (s, 3 H, CH3), 3.356 (t, J=5.0-5.5 Hz, 2 H, CH2), 3.196 (m, 4 H, 2 CH2), 1.285 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 481.5 (MH+).




Synthesis of 5-mPEG3-NH-Delavirdine 19 (n=3): A reaction mixture of 5-amino delavirdine (100.2 mg, 0.265 mmol), mPEG3-OMs (83.8 mg, 0.346 mmol), potassium carbonate (198.4 mg, 1.435 mmol) and tetrabutylammonium bromide (15 mg, 0.174 mmol) in CH3CN (1.5 mL) was placed in single-mode focused microwave reactor (CEM) and irradiated at 120° C. for 110 min. The mixture was cooled to room temperature and filtered. The solid was washed with acetonitrile. The combined organic solution was concentrated to remove the organic solvent. The residue was purified by column chromatography to afford the product pure 5-mPEG3-NH-delavirdine 18 (n=3) (31.7 mg), as well as some starting material. 1H-NMR (CDCl3): 9.219 (s, 1 H, NH), 7.690 (dd, J=1.5 and 5.0 Hz, 1 H, Ar—H), 7.239 (d, J=8.5-9.0 Hz, 1 H, Ar—H), 6.943-6.918 (m, 1 H, Ar—H), 6.855-6.836 (m, 1 H, Ar—H), 6.782 (m, 1 H, Ar—H), 6.751 (dd, J=2.0-2.5 and 8.5-9.0 Hz, 1 H, 1 Ar—H), 6.661 (d, J=1.5 Hz, 1 H, Ar—H), 4.182 (br, 1 H, NH), 4.059 (m, 4 H, 2 CH2), 3.740 (t, J=5.0 Hz, CH2), 3.670-3.646 (m, 7 H, NH and 3 CH2), 3.565-3.547 (m, 3 H, CH and CH2), 3.379 (s, 3 H, CH3), 3.318 (t, J=5.0 Hz, 2 H, CH2), 3.166 (m, 4 H, 2 CH2), 1.259 (d, J=6.0 Hz, 6 H, 2 CH3). LC-MS: 525.3 (MH+).




Synthesis of Ethyl 5-Amino-1H-indole 2-Carboxylate 20: A mixture of 5-Nitro-1H-indole-2-carboxylic acid ethyl ester 15 (88.7 mg, 0.38 mmol) and cyclohexene (0.1 mL, 0.98 mmol) in THF/MeOH (3/8 mL) in the presence of Pd(OH)2 on activated carbon (Pearlman's catalyst) (2.74 g) was heated to reflux for 5 h. The reaction mixture was cooled to room temperature. The mixture was filtered through a pad of Celite. The Celite and the palladium were washed with EtOAc. The combined solution was concentrated. The residue was purified by flash column chromatography on silica gel, eluting with 5-45% EtOAc/hexane to afford 66.6 mg of ethyl 5-amino-1H-indole 2-carboxylate 20. 1H-NMR (CDCl3): 8.653 (Br, 1 H, NH), 7.228 (d, J=8.5-9.0 Hz, 1 H, Ar—H), 7.031 (dd, J=1.0 Hz and 2.0 Hz, 1 H, Ar—H), 6.932 (d, J=2.5 Hz, 1 H, Ar—H), 6.803 (dd, J=2.5 Hz, 1 H, Ar—H), 4.379 (q, J=7.0-7.5 Hz, 2 H, CH2), 1.396 (t, J=7.0-7.5 Hz, 3 H, CH3). LC-MS: 205.2 (MH+).




Synthesis of 5-mPEG3-amino 1H-indole 2-Carboxylic Acid 21: A reaction mixture of ethyl 5-amino-1H-indole 2-carboxylate 20 (36.3 mg, 0.18 mmol), mPEG3-OMs (65.2 mg, 0.27 mmol), and potassium carbonate (60 mg, 0.434 mmol) in water (1 mL) was placed in single-mode focused microwave reactor (CEM) and irradiated at 120° C. for 30 min. The mixture was cooled to room temperature and adjusted the pH to 4.03 with 1N HCl, washed with dichloromethane, extracted with EtOAc (3×15 mL). The combined ethyl acetate solution was washed with brine, dried over Na2SO4, concentrated to afford the product as the major one based on the results of 1H-NMR in MeOD and LC-MS.
Example 3
pKa and Log P Determination for Various Conjugates
The Sirius GLpKa instrument is used to determine the pKa and Log P. A blank standardization has to be completed first and passed in order to conduct the compound experiments. The solutions used by the instrument for the blank titration include water, a pH solution, and a surfactant (TRITON X-100). The TRITON X-100 was purchased commercially from Sirus. The 0.5% solution of TRITON X-100 was used in the experiment. In a 1 L volumetric flask 5 mls of TRITON X-100 is added and MILLI Q water to volume. The vessels used by the instrument were filled to 1⁄3 of their volume with each solution. Therefore the assay tray contains one vessel for water, pH solution, and surfactant for the blank run. The temperature of the water bath for the instrument is 25° C. The blank is calculated as an average of three “good” experiments. The “good” was denoted by a green check mark next to the assay number in the computer software. Once the blank was run successfully the instrument was ready to conduct the experiments.
The pKa has to be determined first in order to calculate the Log P. The API is used to determine the pKa needed for the Log P experiment followed by the conjugates. The base compound was first weighed into a vessel. The amount weighed for each sample was 10% of the molecular weight of the compound. The same procedure was followed for each conjugate. The vessels were placed in the assay tray of the instrument. A method had to be set up for the pKa run. The experimental parameters are entered into the computer software. These included sample weight, molecular weight, assay type (aqueous pKa), number of assays (three), and titration range (generally 3.5-12). The run was started. There are three measurements for each run that are averaged to obtain the final results from the data points collected in the calculation software. All runs were denoted as “good” by the green check mark next to the assay number. The pKa values were obtained for all compounds as listed in the table below.
An additional method has to be set up for the Log P determinations for each compound. The conjugates are weighed into vessels in the same manner as for the previous experiment, i.e., 10% of the molecular weight. The vessels are placed in the assay tray of the instrument. The experimental parameters are entered into the computer. These included sample weight, molecular weight, assay type (Partition Log P), number of assays (three), and pKa value of the parent drug from the previous experiment. The run was started. There are three measurements for each run that are averaged to obtain the final results from the data points collected in the calculation software. All runs were denoted as “good” by the green check mark next to the assay number. The Log P values were obtained for all compounds as listed in Table 1 below.







TABLE 1







pK and Log P Values of Tested Compounds












Compound
pKa1
pKa2
Log P















Delavirdine Mesylate
4.323
9.272
3.5



mPEG3-O-Delavirdine
3.792

1.981



mPEG4-O-Delavirdine
4.275

2.217



mPEG5-O-Delavirdine
4.454





mPEG6-O-Delavirdine
4.447

2.645



mPEG7-O-Delavirdine
4.683

2.733



mPEG8-O-Delavirdine
4.667

2.578



mPEG9-O-Delavirdine
4.655

2.328









Example 4
Anti-HIV-1 Cytoprotection Assay
Cell Preparation—CEM-SS cells were passaged in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 5×104 cells per ml in tissue culture medium and added to the drug containing microtiter plates in a volume of 50 μL.
Virus Preparation—The virus used for the assay was the lymphocyte-tropic virus strain HIV-IRF. The virus was obtained from the NIH AIDS Research and Reference Reagent Program and stock virus pools were produced in CEM-SS cells. A pretitred aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was re-suspended and diluted into tissue culture medium such that the amount of virus added to each well in a volume of 50 μL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection.
Plate Format—Each plate contains cell control wells (cells only), virus control wells (cells plus virus), drug toxicity wells (cells plus drug only), drug colorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus). Samples were tested in triplicate with eleven half-log dilutions per compound.
Efficacy and Toxicity XTT—Following incubation at 37° C. in a 5% CO2 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of HIV induced cell killing by anti-HIV test substances. XTT solution was prepared daily as a stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTT/PMS stock was prepared immediately before use by adding 40 μL of PMS per ml of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate was re-incubated for 4 hours at 37° C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble fosmazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader.
Example 5
HIV-1 Reverse Transcriptase Inhibition Assay
The following materials were removed from the freezer and were stored on wet ice (dGTP, 32P, purified enzyme extract, rCdG, dGTP, DNA). Test compounds were evaluated at a high test of 100 μM diluted in dH2O and if the stock solution was turbid, 1:1000 Triton-X was added to the stock solution. Thirty microliters (30 μL) of each dilution was then transferred in duplicate to the appropriate well of a 96-well plate. The positive and negative control wells each received 30 μL of dH2O instead of the compound. A 2× buffer was prepared in the following manner: 125 μL 2M Tris pH 8.0, 250 μL 3M KCl, 80 μL 1 M MgCl2, 10 μL 2 M DTT, 40 μL 25 U/mL rCdG, 100 μL 1 mM dGTP, 10 μL 800 Ci/mMol alpha 32P:dG TP, and 4.4 mL dH2O. Fifty microliters (50 μL) of 2× buffer was added to every well of the plate. BSA was diluted 1:100 and Triton-X 1:1000 in dH2O and the enzyme was added to the mixture at a 1:400 dilution. Twenty microliters (20 μL) of the enzyme solution was added to all of the wells of the plate except the negative control wells. Distilled water was used instead of enzyme in these wells. The plate was incubated at 37° C. for 30 minutes. Following incubation, 10 μL of 10 mg/mL DNA (MB-grade fish sperm) was added to each well, followed by 150 μL of cold 10% TCA in each well. The plate was incubated at room temperature for approximately 15 minutes in order to allow for precipitation of the samples. Following the incubation the contents were transferred to the appropriate filter plate. The plate was placed on the vacuum manifold and pushed firmly to induce suction. Wells were rinsed with 200 μL of cold TCA. Again, the plate was pushed down firmly and vacuum suction was applied. Once all of the liquid was aspirated from the filter plate, it was removed from the manifold and residual liquid was blotted from the bottom of the plastic base. The filtered wells were separated from the plastic base and the wells were blotted on clean, absorbent paper. The filtered wells were pushed into the cassette and a sheet of Millipore multiscreen tape was placed on the bottom of a Wallace Microbeta cassette covering all of the holes. Twenty microliters (20 μL) of Wallace Supermix Scintillant was added to each well and the top of the wells were covered with a sheet of the multiscreen tape. The plate was read on the Wallace MicroBeta counter to measure incorporated 32P.
Anti-HIV-1 Cytopathic Effects Inhibition Assay Evaluations: Eight compounds were evaluated against the RF strain of HIV-1 in CEM-SS cells using a 12 concentration dose response curve. The results of these assays are summarized in the table below. The AZT control compound was evaluated in parallel with the test materials and yielded an EC50 value of 6.0 nM, which falls within the acceptable range of activity of the control compound (1 to 10 nM).
Example 6
Anti-HIV Evaluation of Conjugates in CEM-SS Cells Against Wild Type and Drug Resistant HIV-RF 
Cells were prepared as described above. Virus Preparation—The wild type NL4-3 and drug resistant All viruses were obtained from the NIH AIDS Research and Reference Reagent Program and stock virus pools were produced in CEM-SS cells. Amino acid substitutes of L100I, Y181C and P23L were introduced by site-directed mutagenesis into pN L4-3 and stock virus pools were produced in CEM-SS cells. A pretitered aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was re-suspended and diluted into tissue culture medium such that the amount of virus added to each well in a volume of 50 μL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection.
Plate Format—Each plate contains cell control wells (cells only), virus control wells (cells plus virus), drug toxicity wells (cells plus drug only), drug colorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus). Samples were tested in triplicate with eleven half-log dilutions per compound. Efficacy and Toxicity XTT—Following incubation at 37° C. in a 5% CO2 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of HIV induced cell killing by anti-HIV test substances. XTT solution was prepared daily as a stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTTIPMS stock was prepared immediately before use by adding 40 μl, of PMS per ml of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate was re-incubated for 4 hours at 37° C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble foimazan product and the plate was read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader.
Data Analysis—Raw data was collected from the Softmax Pro 4.6 software and imported into a Excel XLFit4 spreadsheet for analysis by four parameter curve fit calculations. Both antiviral activity and toxicity are calculated. The results of these assays are summarized in the table below.












ANTI-HIV ACTIVITIES AND CYTOTOXICITIES












Wild Type RT
CEM-SS/HIV-1RF
CEM-SS
Therapeutic


Compound
IC50(μM)
EC50 (μM)
TC50 (μM)
Index





AZT

0.006
>1.0
>166.7


(Repeat test)

0.01
>1.0
>100.0


Delavirdine Mesylate
0.027
0.002
27.2
13587.0


(Repeat test)
0.001
0.006
52.43
8738.33


mPEG3-O-Delavirdine
0.05
0.054
26.9
499.0


mPEG4-O-Delavirdine
0.28
0.16
34.0
213.8


mPEG5-O-Delavirdine
0.13
0.76
14.4
19.0


mPEG6-O-Delavirdine
0.29
0.84
22.5
26.7


mPEG7-O-Delavirdine
0.70
0.47
35.5
76.1


mPEG8-O-Delavirdine
0.49
0.36
52.5
144.2


mPEG9-O-Delavirdine
0.56
0.13
40.7
323.1


5-mPEG1-NH-
0.25
0.047
50.91
1083.11


Delavirdine






5-mPEG2-NH-
0.58
0.017-~0.2*
55.53
3266.29


Delavirdine






5-mPEG3-NH-
1.0
0.16
34.88
212.67


Delavirdine










ANTI-HIV CYTOPROTECTION ASSAY USING DRUG RESISTANT VIRUS STRAINS














AZT
Nevirapine
Delavirdine

















EC50
TC50

EC50
TC50

EC50
TC50



HIV-1 Strain
(μM)
(μM)
TI
(μM)
(μM)
TI
(μM)
(μM)
TI





NL4-3
0.004
>0.5
>125.0
0.19
>10.0
>52.6
0.005
>0.5
>100.0


(repeat test)
0.01
>0.1
>10.0
0.09
>10.0
>111.0
0.01
>1.0
>100.0


NL4-3L100I
0.003
>0.5
>167.0
>10.0
>10.0
—
0.003
>0.5
>167.0


(repeat test)
0.006
>0.1
>16.7
>10.0
>10.0
—
0.24
>1.0
>4.2


NL4-3Y181C
0.002
>0.5
>250.0
>10.0
>10.0
—
>0.5
>0.5
—


(repeat test)
0.001
>0.1
>100.0
8.59
>10.0
>1.2
0.58
>1.0
>1.7


NL4-3P236L
0.007
>0.5
>71.4
0.3
>10.0
>33.3
>0.5
>0.5
—


(repeat test)
0.002
>0.1
>50.0
0.28
>10.0
>35.7
0.5
>1.0
>2.0


A17K103N/Y181C
0.003
>0.5
>167.0
>10.0
>10.0
—
>0.5
>0.5
—


(repeat test)
0.003
>0.5
>167.0
>10.0
>10.0
—
>1.0
>1.0
—














mPEG3-O-delavirdine
mPEG4-O-delavirdine
mPEG9-O-delavirdine

















EC50
TC50

EC50
TC50

EC50
TC50



HIV-1 Strain
(μM)
(μM)
TI
(μM)
(μM)
TI
(μM)
(μM)
TI





NL4-3
0.06
>5.0
>83.3
0.87
>5.0
>5.8
0.89
>5.0
>5.6


NL4-3L100I
0.03
>5.0
>167.0
0.95
>5.0
>5.3
0.94
>5.0
>5.3


NL4-3Y181C
>5.0
>5.0
—
>5.0
>5.0
—
>5.0
>5.0
—


NL4-3P236L
>5.0
>5.0
—
>5.0
>5.0
—
>5.0
>5.0
—


A17K103N/Y181C
>5.0
>5.0
—
>5.0
>5.0
—
>5.0
>5.0
—













5-mPEG1-NH-
5-mPEG2-NH-



delavirdine
delavirdine
















EC50
TC50

EC50
TC50




HIV-1 Strain
(μM)
(μM)
TI
(μM)
(μM)
TI






NL4-3
0.02
>1.0
>50.0
0.08
>1.0
>12.5



NL4-3L100I
0.56
>1.0
>1.8
0.71
>1.0
>1.4



NL4-3Y181C
0.82
>1.0
>1.2
>1.0
>1.0
—



NL4-3P236L
>1.0
>1.0
—
0.23
>1.0
>4.4



A17K103N/Y181C
>1.0
>1.0
—
>1.0
>1.0
—





*Note - variability in cytoprotection data at 0.032 and 0.102 μM made it difficult to accurately determine the EC50 value. Ignoring the variability, the EC50 was calculated as 0.017 μM.






Example 7
Caco-2 Permeability Studies
The objective of this study was to determine the bidirectional Caco-2 permeability of test compounds. Caco-2 monolayers were grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well Costar Transwell® plates. Details of the plates and their certification are shown below. The permeability assay buffer for the donor chambers was Hanks Balanced Salt Solution containing 10 mM HEPES and 15 mM glucose (HBSSg) at a pH of 7.4. The buffer in the receiver chambers also contained 1% bovine serum albumin (BSA). The dosing solution concentrations were 10 μM in the assay buffer. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37° C. with 5% CO2 in a humidified incubator. After 2 hours, aliquots were taken from the receiver and donor chambers. Each determination was performed in duplicate. The lucifer yellow flux was also measured for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC/MS/MS using electrospray ionization. Analytical conditions are outlined in Appendix A. The apparent permeability, Papp, and percent recovery were calculated as follows:

Papp=(dCr/dt)×Vr/(A×CA)  (1)

Percent Recovery=100×((Vr×Crfinal)+(Vd×Cdfinal))/(Vd×CN)  (2)

where, dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in μM s-1.

Vr is the volume of the receiver compartment in cm3.
Vd is the volume of the donor compartment in cm3.
A is the area of the cell monolayer (1.13 cm2 for 12-well Transwell®)
CN is the nominal dosing concentration of the dosing solution in μM.
CA is the average of the nominal dosing concentration and the measured 120 minute donor concentration in μM.
Crfinal is the receiver concentration in μM at the end of the incubation period.
Cdfinal is the concentration of the donor in μM at the end of the incubation period.












Description of Caco-2 Permeability Characterization (Test 1)












Passage Number
65



Age at QC (Days)
23
Acceptance Criteria


TEER Value (Ω · cm2)
563
450-650


Lucifer Yellow Papp, × 10−6 cm/s
0.18
<0.4


Atenolol Papp, × 10−6 cm/s
0.26
<0.5


Propranolol Papp, × 10−6 cm/s
20.8
15-25


Digoxin (A-to-B) Papp, × 10−6 cm/s
0.47
N/A


Digoxin (B-to-A) Papp, × 10−6 cm/s
11.0
N/A


Digoxin Efflux Ratio
23
>3.0










Recovery and Apparent Permeability (Test 1)













Average
Average





Papp,
Papp,
Efflux




A→B
B→A
Ratio



Recovery (%)
(×10−6
(×10−6 
(B→A/












Compound
A→B
B→A
cm/s)
cm/s)
A→B)





Delavirdine Mesylate
65
65
9.87
67.9
6.9


mPEG3-O-Delavirdine
66
61
38.4
33.9
0.9


mPEG4-O-Delavirdine
74
65
39.3
34.8
0.9


mPEG5-O-Delavirdine
71
65
29.9
39.1
1.3


mPEG6-O-Delavirdine
67
72
16.1
34.1
2.1


mPEG7-O-Delavirdine
74
69
14.2
36.6
2.6


mPEG8-O-Delavirdine
80
82
4.98
43.6
8.8


mPEG9-O-Delavirdine
66
72
2.22
27.7
12





















Description of Caco-2 Permeability Characterization (Test 2)













Passage Number
67
59



Age at QC (Days)
20
21
Acceptance





Criteria


TEER Value (Ω · cm2)
600
574
450-650


Lucifer Yellow Papp, × 10−6 cm/s
0.23
0.16
<0.4


Atenolol Papp, × 10−6 cm/s
0.29
0.24
<0.5


Propranolol Papp, × 10−6 cm/s
17.0
15.4
15-25


Digoxin (A-to-B) Papp, × 10−6 cm/s
0.75
0.65
N/A


Digoxin (B-to-A) Papp, × 10−6 cm/s
13.4
16.9
N/A


Digoxin Efflux Ratio
18
26
>10  










Recovery and Apparent Permeability (Test 2)













Average
Average





Papp,
Papp,
Efflux




A→B
B→A
Ratio



Recovery (%)
(×10−6
(×10−6 
(B→A/












Compound
A→B
B→A
cm/s)
cm/s)
A→B)





Delavirdine Mesylate
82
86
16.1
56.6
3.5


mPEG3-O-Delavirdine
95
101
41.4
29.1
0.7


mPEG5-O-Delavirdine
97
95
33.3
38.9
1.2


mPEG7-O-Delavirdine
94
95
23.1
39.3
1.7


mPEG9-O-Delavirdine
93
94
5.27
37.3
7.1


5-mPEG1-NH-
91
92
50.2
39.4
0.8


Delavirdine







5-mPEG2-NH-
88
88
46.2
41.1
0.9


Delavirdine







5-mPEG3-NH-
94
96
33.3
41.8
1.3


Delavirdine

## Claims

1. A compound having the following structure,




wherein n is an integer between 1 and 30, inclusive, and pharmaceutical salts thereof.

2. The compound of claim 1, wherein n is an integer between 1 and 10.

3. A composition comprising (i) a compound of claim 1, and (ii) a pharmaceutically acceptable excipient.

4. A composition of matter comprising a compound of claim 1 present in a dosage form.

5. A compound of claim 1, wherein n is 1.

6. A compound of claim 1, wherein n is 2.

7. A compound of claim 1, wherein n is 3.

8. A compound of claim 1, wherein n is 4.

9. A compound of claim 1, wherein n is 5.

10. A compound of claim 1, wherein n is 6.

11. A compound of claim 1, wherein n is 7.

12. A compound of claim 1, wherein n is 8.

13. A compound of claim 1, wherein n is 9.

14. A compound of claim 1, wherein n is 10.