Para-hydroxyphenylhydrazines as in situ precursors of iminoquinones and quinones

Iminoquinone precursors having the formula ##STR1## wherein R.sub.1 represents hydrogen or an enzymatically hydrolyzable group; R.sub.2 and R.sub.3 independently represent hydrogen, a non-electron-withdrawing organic substituent or an enzymatically hydrolyzable group except that when R.sub.1 is hydrogen, R.sub.2 must be an enzymatically hydrolyzable group and R.sub.3 must not inhibit the hydrolysis of R.sub.2 ; R.sub.4 represents hydrogen or an electron-withdrawing or non-electron-withdrawing organic substituent, except that if R.sub.1 is hydrogen, R.sub.4 may not inhibit the hydrolysis of R.sub.2 ; W, X, Y and Z independently represent hydrogen or an electron-withdrawing or non-electron-withdrawing organic substituent, or any one of X and W, Y and Z, or X and R.sub.4 taken together represents a cyclic or heterocyclic ring having 5 to 6 ring atoms; and wherein said enzymatically hydrolyzable group is selected so that a hydrolysis product is biologically benign for the biological system in which said precursor is present is produced by hydrolysis of the hydrolyzable group. Also disclosed are methods of synthesis and methods of use of these compounds.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to precursors which will undergo elimination 
reactions to produce iminoquinones and quinones in situ after cleavage of 
a blocking group, and more particularly to para-hydroxyphenylhydrazine and 
its derivatives and their use as in situ precursors of iminoquinones and 
quinones. 
2. Description of the Prior Art 
Quinones and iminoquinones have many uses in biological systems as a result 
of their toxicity. The simplest quinone, p-benzoquinone, for example, was 
shown to be a potent bactericidal substance against Salmonella typhosa as 
early as 1911. Many other quinones and iminoquinones are also known to 
have biological activity. For example, chloranil 
(2,3,5,6-tetrachloro-1,4-benzoquinone) has marked antifungal properties, 
while blue-green algae are inhibited by 2,3-dichloro-1,4-naphthoquinone. 
When used in this application, the term "quinones" will refer generically 
to all the quinones whatever the number of rings on the substituents and 
the term "iminoquinones" will similarly refer to all iminoquinones, unless 
otherwise specified. 
Nitrogen analogues of the quinones (i.e., iminoquinones) have not been as 
extensively studied as the quinones themselves, perhaps because 
iminoquinones hydrolyze rapidly in aqueous solutions to the corresponding 
quinones. Thus, they have been difficult to study in biological systems 
because of the uncertainty as to what the reactive species is, a quinone 
or an iminoquinone. This may not be important in biological systems since 
iminoquinones appear to exhibit roughly similar reactivity to quinones in 
the typical reactions that both undergo (both act as oxidizing agents and 
electrophiles). Iminoquinones have been prepared and tested for biological 
activity, for example in Hodnett et al, J. Med. Chem. 21, 11-16 (1978) 
which is hereby incorporated by reference, and have been shown to exhibit 
cytotoxic activity. 
The toxicity of quinones and iminoquinones, and hence their biological 
activity, is caused by their high degree of reactivity, particularly with 
nucleophiles, such as the --SH group of cysteine and other biological 
thiols. However, this very reactivity works against them since they often 
react with other nucleophiles before reaching the site in the biological 
system where their activity is desired. 
Accordingly, there have been attempts to use precursors of quinones and 
iminoquinones in order to produce the reactive compounds in situ. For 
example, p-benzohydroquinone has been used to generate p-benzoquinone in 
situ to prevent phage (viral) infections from proliferating in tissue 
cultures. However, this oxidation reaction to form the quinone is itself 
not very selective for biological systems, since oxidation can occur on 
standing in air. More desirable would be a precursor which forms the 
reactive quinone or iminoquinone selectively at a site of biological 
activity, e.g., an enzyme active site. In such a case the quinone would be 
generated only at sites of biological activity where reaction was desired. 
Although quinones and some iminoquinones have been extensively studied and 
reviewed, for example in J. L. Webb, "Enzyme and Metabolic Inhibitors", 
V.III, Academic Press, New York and London (1966), pp. 421-594, which is 
hereby incorporated by reference, no such precursors of general utility 
have previously been known. 
There is however, a known reaction in which an iminoquinone is generated in 
situ in a biological system, although it represents only a minor side 
reaction rather than a general case. Researchers investigating the 
toxicity of N-(4-hydroxyphenyl)acetamide, also known as acetaminophen, 
discovered that about 2% of the acetaminophen was oxidized in liver cells 
(hepatocytes) to an N-hydroxylated compound that could lose a water 
molecule to form N-acetyl-p-iminobenzoquinone. 
##STR2## 
This iminoquinone was shown to be responsible for the toxic effects in the 
liver (1. Gillette, J. R. et. al.: "Biochemical Mechanism of Drug 
Toxicity" in Ann. Rev. of Pharmacology. 14:271 (1974) 2. Mitchell, J. R. 
et. al.: Handb. Exp. Pharmacol. 28/3 (1975) p. 383. 3. Potter, W. J. et 
al.: Pharmacology 12:129 (1974)). 
Other investigators showed that by replacing the hydrogen of the phenolic 
hydroxyl group with a sulfate that could later be enzymatically cleaved, 
the intermediate N-hydroxylated amide could be isolated and studied 
(Gemborys, M. W. et al.: J. Med. Chem. 21:649-652 (1978)). 
However, this reaction sequence does not provide a general method of 
producing quinones or iminoquinones in situ in other biological systems 
since it requires an initial oxidation by hepatocytes. 
SUMMARY OF THE INVENTION 
It is therefore an object of this invention to provide a method of 
introducing quinones or iminoquinones into a biological system without 
their being subjected to reactions before they reach the desired site of 
reactivity. 
It is a further object of this invention to provide a method of generating 
quinones and iminoquinones in situ in biological systems. 
It is yet another object of this invention to provide a precursor of 
quinones and iminoquinones that can be selectively de-blocked by the 
enzymatic systems of a biological organism to release a compound that will 
spontaneously form a toxic quinone or iminoquinone within the cell. 
It is a still further object of this invention to provide blocking groups 
and precursors having structures that facilitate transport into the cells 
of the target organism. 
These and other objects of this invention which will hereinafter become 
more readily apparent have been attained by providing precursors of 
iminoquinones having the formula 
##STR3## 
wherein R.sub.1 represents hydrogen or an enzymatically hydrolyzable 
group; R.sub.2 and R.sub.3 independently represent hydrogen, a 
non-electron-withdrawing organic substituent, or an enzymatically 
hydrolyzable group except that when R.sub.1 is hydrogen, R.sub.2 must be 
an enzymatically hydrolyzable group and R.sub.3 must not inhibit the 
hydrolysis of R.sub.2 ; 
R.sub.4 represents hydrogen or an electron-withdrawing or 
non-electron-withdrawing organic substituent except that if R.sub.1 is 
hydrogen, R.sub.4 must not inhibit the hydrolysis of R.sub.2 ; 
W, X, Y and Z independantly represent hydrogen, or an electron-withdrawing 
or non-electron-withdrawing organic substituent, or any one of X and W, Y 
and Z, or X and R.sub.4 taken together represents a cyclic or heterocyclic 
ring having 5 to 6 ring atoms; and O represents Oxygen and --NH--; and 
wherein said enzymatically hydrolyzable groups are selected so that 
hydrolysis products that are biologically benign for the biological system 
in which said precursor is present are produced by hydrolysis. 
By a group that inhibits the hydrolysis of R.sub.2 is meant any substituent 
that prevents the enzymatic hydrolysis of R.sub.2 from occurring. An 
example, not intended to be limiting, is where R.sub.1 .dbd.H, R.sub.2 
.dbd.Leucyl, and R.sub.3 .dbd.--CH.sub.3. The methyl group inhibits the 
hydrolysis of the Leucyl-hydrazide bond. Other examples are well known in 
the art of enzyme biochemistry and can easily be determined using 
well-known methods for the study of enzymatic reactions. 
By electron-withdrawing organic substituent is meant an organic substituent 
having a bond between the substituent and the atom to which it is attached 
in which the electrons are polarized toward the substituent more than they 
would be if the substituent was replaced with a hydrogen. A 
non-electron-withdrawing organic substituent is one in which either the 
electrons are polarized away from the substituent relative to the 
polarization that exists when a hydrogen is present or no difference in 
polarization exists. By biologically benign is meant not only products 
that do no harm to the individual cell in which hydrolysis takes place, 
but also those products that harm the cells in which the hydrolysis takes 
place but do not do major harm to other cells in the biological system to 
which the iminoquinone precursor has been administered. 
Compounds having formula I have at least one blocking group that is 
hydrolyzable by an enzyme in a living organism. While the blocking group 
is present, no rearrangement or elimination reactions to form 
iminoquinones, and later to form quinones, is possible. However, once the 
blocking groups are removed, a spontaneous elimination of ammonia takes 
place to give an iminoquinone. This reaction can be catalyzed by either 
acid or base and is shown in Scheme II, where R.sub.2, R.sub.3 and R.sub.4 
have the same meanings given above where R.sub.1 .dbd.H, and W, X, Y and Z 
have the same meanings given above: 
##STR4## 
As can be seen in Scheme II, further reaction to give a quinone may also 
take place. 
As indicated above, these rearrangements are prevented from occurring 
prematurely by providing a blocking group on the .beta.-nitrogen or 
phenolic oxygen. By chosing a blocking group that is hydrolyzable by an 
enzymatic system, release of the compound capable of ammonia elimination 
does not occur until the precursor is present in the desired biological 
system. In fact, the precursor can be easily designed for hydrolysis by a 
particular enzyme or class of enzymes. 
Any blocking group that is hydrolyzable by an enzyme is suitable for the 
compounds of the present invention, within the limitations discussed 
herein. Examples of classes of enzymes present in biological systems 
include carboxylic acid hydrolases, phosphoric monoester hydrolases, 
phosphoric diester hydrolases, sulfuric ester hydrolases, glycoside 
hydrolases, N-glycosyl hydrolases, peptide hydrolases, 
.alpha.-amino-acyl-peptide hydrolases, peptidyl-amino-acid hydrolases, 
dipeptide hydrolases, peptidyl-peptide hydrolases, and any other 
hydrolytic enzyme present in biological systems. 
The suitability of a particular blocking group for hydrolysis by a specific 
enzyme system can be easily determined by simple experimentation since the 
blocked precursor compound, being derivative of phenylhydrazine, will have 
a greatly different visible/ultraviolet absorption spectra from the 
iminoquinone and quinones that will be produced by spontaneous reactions 
upon deblocking in aqueous solutions, as is well known in the art. 
Blocking groups suitable for the phenolic oxygen include phosphate, 
pyrophosphate, glycosyl, ribosyl, deoxyribosyl, acyl, arylacyl, aminoacyl, 
peptidyl and sulfate. Each of these groups is cleavable by a particular 
class of hydrolase, e.g., phosphate by phosphatases, pyrophosphate by 
pyrophasphatases, glycosyl by glycoside hydrolases, ribosyl by riboside 
hydrolases, deoxyribosyl by deoxyriboside hydrolases, acyl and arylacyl by 
carboxylic ester hydrolases, aminoacyl and peptidyl by peptide hydrolases, 
and sulfate by sulfuric ester hydrolases. There is no particular limit to 
the nature of the individual types of blocking groups, except that the 
corresponding hydrolysis reaction should not be impaired severely. This is 
easily determined by simple experimentation, as has been discussed above. 
Preferred glycosyl blocking groups are prepared from C.sub.5 or C.sub.6 
naturally occurring glycosides. Preferred aminoacyl groups are 
.alpha.-amino acids, while preferred acyl or arylacyl groups have 2-30 
carbons. Most preferred are compounds in which R.sub.1 .dbd.H and R.sub.2 
acts as the blocking group. 
Substituents R.sub.2 and R.sub.3 are not required to be enzymatically 
hydrolyzable if R.sub.1 is an enzymatically hydrolyzable group. When 
R.sub.1 is hydrolyzable, R.sub.2 may be hydrogen, alkyl, aryl, or 
arylalkyl and R.sub.3 may be hydrogen or alkyl. R.sub.2 may at any time be 
a hydrolyzable group such as aminoacyl, peptidyl, glycosyl, ribosyl, 
deoxyribosyl, acyl, arylacyl, or phosphate. When R.sub.2 is one of the 
hydrolyzable groups listed herein, R.sub.3 must not inhibit the hydrolysis 
of R.sub.2. 
Substituent R.sub.3 may be hydrogen or an alkyl group except that when 
R.sub.2 is a hydrolyzable group, R.sub.3 must not inhibit the hydrolysis 
of R.sub.2. When R.sub.3 is an alkyl group, 1-5 carbons are preferred and 
method is most preferred. 
Substituent R.sub.4 may be hydrogen, alkyl, aryl, acyl, arylacyl, 
aminoacyl, peptidyl, glycosyl, ribosyl, deoxyribosyl, phosphate, or 
halogen except that when R.sub.2 is a hydrolyzable group, R.sub.4 may not 
inhibit the hydrolysis of R.sub.2. When R.sub.4 is hydrolyzable, it is 
subject to the same conditions and limitations as discussed for R.sub.1. 
Preferred alkyl groups are carboxyalkyl groups. Most preferred are (alkyl) 
carboxymethyl groups having an alkyl substitutent on the methyl carbon 
selected from the group consisting of side chains present in naturally 
occurring amino acids. Preferred aryl groups are those based on benzene 
and most preferred is phenyl. Preferred halogens are chlorine and bromine 
with chlorine most preferred. 
W, X, Y and Z may independently represent hydrogen, halogen, hydroxyl, 
amine, alkoxy, alkyl, alkenyl, carboxyl, formyl, aryloxy, alkyl or dialkyl 
amine, thioalkyl, cyano, acyl amine, aryl, or arylalkyl. Preferred 
halogens are chlorine and bromine and most preferred is chlorine. 
Preferred alkoxy groups are those having 1-30 carbon atoms and most 
preferred are those having 1-12 carbon atoms. Preferred alkyl groups are 
those having 1-30 carbon atoms and most preferred are those having 1-12 
carbon atoms. Preferred alkenyl groups are those having 1-30 carbon atoms 
and 1-10 double bonds while most preferred are those having 1-20 carbon 
atoms and 1-5 double bonds. Preferred aryloxy groups are those based on 
benzene and most preferred is phenyl. Preferred alkyl and dialkyl amines 
are those having 1-30 carbon atoms per alkyl group and most preferred are 
those having 1-12 carbon atoms per alkyl group. Preferred thioalkyl groups 
are those having the sulfur attached to the ring and 1-30 carbon atoms 
with 1-12 carbon atoms being most preferred. Preferred acylamines are 
those containing 1-30 carbon atoms or formed from a naturally occurring 
.alpha. aminoacid or peptide while most preferred are those containing 
1-12 carbons. Preferred aryl groups are those based on benzene and most 
preferred is phenyl. Preferred arylalkyl groups are those having 1-20 
carbons in an alkyl chain and an aryl group based on benzene while most 
preferred is benzyl. 
Alternately, any one of X and W, Y and Z, or X and R.sub.4 taken together 
may represent a cyclic or heterocyclic ring having 5 to 6 atoms when 
considered together with the atoms of the phenylhydrazine to which they 
are connected. Suitable heteroatoms include nitrogen, oxygen, and sulfur. 
The ring may additionally contain unsaturation in the form of 
carbon-carbon or carbon-nitrogen double bonds. Preferred for X and W or Y 
and Z are 6-membered aromatic rings such that the resulting structure is a 
naphthalene or anthracene derivative. Most preferred are naphthalene 
derivatives. Preferred for X and R.sub.4 are 5- or 6-membered rings. Most 
preferred are 5- or 6-membered rings having the .alpha.nitrogen of the 
hydrazine functional group present as the only hetero atom. 
The names of different types of substituents disclosed herein refer to the 
linkage of these substituents to the phenylhydrazine and are not intended 
to limit the occurrence of other functional groups within the substituent. 
The compounds of the present invention may easily be synthesized by known 
methods of organic chemistry. Many synthetic methods are known for 
producing phenylhydrazines, the basic structural class to which the 
compounds of the present invention belong, for example, as disclosed in: 
Edgar Enders: Methoden Zur Herstellung und Umwandlung von Arylhydrazinen 
und Arylhydrazonen. pp 169-691 in Methoden der Organischen Chemie 
(Houben-Weyl), Eugen Muller (Ed.), Band X12, Stickstoff-Verbindungen I, 
Teil 2, Rudolf Stroh(Ed.). Georg Thieme Verlag (1967) Stuttgart. One easy 
method that exists for converting quinones having known toxicity and end 
uses into the compounds of the present invention is shown in Scheme III. 
##STR5## 
In this reaction, an .beta.N-(amino acyl) phenyl hydrazine is prepared by 
reacting an aminoacyl chloride or anhydride having the .alpha.nitrogen and 
any other reactive groups on the side chain protected by a suitable 
blocking group, e.g., tert-butoxycarbonyl, with hydrazine. In these 
reactions, W, X, Y and Z have the meanings previously assigned, A is a 
protective group typically used in peptide synthesis (e.g. 
tert-butoxycarbonyl, and R.sub.5 is selected from the group consisting of 
side chains present in naturally occurring amino acids. The resulting 
hydrazide is reacted with a quinone to give an iminoquinone that can be 
reduced with ascorbic acid or other mild reducing agents to the desired 
phenylhydrazine derivative. If desired, the protecting groups on the amino 
acid moiety may now be removed. 
The .beta.N-(aminoacyl)phenylhydrazine compounds produced in these 
reactions are compounds of the present invention. The aminoacyl blocking 
group is cleavable by aminopeptidases to give the phenylhydrazine 
derivative that is the immediate precursor of the iminoquinone (and 
quinone), as was shown in Reaction Scheme II. The quinone that is released 
as the final product of these reactions is the same quinone that was used 
in the synthesis of the .beta.--N-(aminoacyl)phenylhydrazine. Accordingly, 
this synthetic pathway provides a simple way of converting quinones having 
a known end use as a toxic substance, e.g., fungicide, bactericide, etc., 
into a more stable precursor for ease of administration. 
Many examples of such biologically active quinones, which could be used to 
synthesize the iminoquinone precursors of the present invention, are 
known, including .rho.-Benzoquinone, Toluquinone, .rho.Xyloquinone, 
Cumoquinone, Duroquinone, Thymoquinone, 5-Methoxy-toluquinone, 
Tetrachloroquinone (chloranil), 1,4-Naphthoquinone, Menadione, Phthiocol, 
Lawsone, Juglone, Naphthazarin, 2,3-Dichloro-1,4-naphthoquinone, Lapachol, 
Lomatiol, and 9,10-Anthraquinone. 
One preferred form of the compounds of the invention are blocked 
.alpha.N-(carboxymethyl)-p-hydroxyphenylhydrazines or derivatives thereof. 
Such a compound is an aminoacid analogue per se and in its more preferred 
form uses a naturally occurring amino acid or another module of itself as 
a blocking group on the .beta.nitrogen. The resulting compound is a 
peptide analogue, and thus suitable for any biological system containing 
peptidase enzymes. Compounds having the general formula 
##STR6## 
are compounds of this preferred type, where W, X, Y and Z have the 
meanings previously described, R.sub.5 is selected form the group 
consisting of side chains present in naturally occurring amino acids, and 
R.sub.6 is hydroxy, alkoxy, --O--M.sup.t where M.sup.t represents a singly 
charged metal ion, amino acid, or 
##STR7## 
and AA is an .alpha.aminoacid, acyl, arylacyl, or phosphate. These 
compounds can be synthesized by known methods of organic synthesis, 
including the method outlined in Scheme IV below. 
##STR8## 
In this method a .rho.hydroxyaniline having a protective group G.sub.1, 
(typically benzyl) and having substituents W, X, Y and Z as previously 
defined, is reacted with a compound having the formula 
##STR9## 
where Cl represents chlorine or bromine, R.sub.5 is as previously defined, 
and G.sub.2 is a protective group, typically a lower alkyl group such as 
methyl or ethyl. The resulting secondary amine is reacted in typical 
fashion with sodium nitrite in aqueous HCl or with nitrous acid generated 
in some other fashion to form a N-nitrosamine. The nitrosamine is reduced 
by any suitable method, typically with an aluminum amalgam, to form the 
hydrazine. The hydrazine is reacted with an aminoacid having a protecting 
group G.sub.3 on the .alpha.nitrogen and on any other basic nitrogens that 
are present. A typical protective group is Boc (tert-butoxycarbonyl). If 
the various protecting groups have been chosen so that each may be removed 
separately, as illustrted by the typical examples of protecting groups 
G.sub.1 -G.sub.3 given above, individual protecting groups may be removed 
and additional reactions may occur at the de-protected positions to give 
further products of the invention, as illustrated in part by Reaction 
Scheme IV. These and the other embodiments of the invention are within the 
normal scope of manipulations commonly practiced by those skilled in the 
art to which this section of the invention pertains, organic chemistry, 
and particularly the chemistry of peptide synthesis. 
Compounds of the present invention may be used as replacements for quinones 
and iminoquinones in any biological use that relies on the toxicity of 
these compounds. They have an advantage in that quinones and iminoquinones 
are generated spontaneously from them in situ after enzymatic cleavage of 
the blocking group. Thus, degradation or reaction cannot occur except in a 
biological system to which they are administered. 
Compounds having a carboxymethylphenylhydrazine structure can be easily 
attached to a protein or polypeptide by known methods of attaching 
acyl-containing compounds to proteins. Preferred are naturally occurring 
or synthetic proteins and polypeptides have 1 to 500 functional groups 
that can be acylated. Coupling of pro-drugs to proteins and peptides is a 
method known to enhance cellular ingestion by pinocytosis and to reduce 
the toxic effects of the pro-drug prior to ingestion and release of the 
drug form itself. Bovine serum albumin is a typical naturally occurring 
protein that may be used in this fashion. 
In order to better reach a location in which iminoquinone formation is 
desired, compounds of the invention may be administered in a 
pharmaceutical carrier chosen for the type of organism being treated. The 
resulting mixture may be a solution, suspension, cream salve, powder, or 
other physical form intended for use by injection, ingestion, or contact. 
Suitable carriers depend on the type of organism being treated and the 
desired physical characteristics of the resulting mixture and generally 
include water, alcohol, oils, dimethylsulfoxide, and other non-toxic 
solvents as well as talc and inert ingredients. In certain applications, 
e.g. killing algae in non-potable water, the restriction to non-toxic 
solvents may be removed. If desired other active ingredients may be 
combined with the compounds of this invention for ease of administration. 
The compounds of this invention provide a convenient means by which 
iminoquinones can be formed in situ in a biological system. By biological 
system is meant either intracellularly in a single- or multi-cellular 
organism or in a fluid containing biological products of such cells that 
is in contact with the cells. By proper choice of blocking groups so that 
the compounds are prepared for a specific enzyme or transport system, 
selective formation of the iminoquinone, and hence the quinone, can occur 
in one cell or tissue in the presence of a second cell or tissue. By 
selective formation is meant formation either more rapidly or in a higher 
concentration for whatever reason. Selective formation is likely to be 
caused by differences in the amount of targeted enzyme in different cell 
or tissue types. 
An additional advantage of the present invention over direct use of the 
quinones themselves is that the enzymatically hydrolyzable blocking groups 
of the present invention also should participate in transport systems that 
move substrates into cells. Mediated and active transport of many 
biological substrates, such as glycosides, ribosides, and peptides, is 
well documented. Since the compounds of the present invention are all 
substrates for an enzyme reaction that can occur in the interior of a 
cell, they should all be transportable, to at least some extent, by the 
same transport systems that convey the normal substrates for the enzymes. 
It is anticipated that the compounds of the present invention will find use 
as in multicelluluar organisms. They are most active in cells having the 
highest hydrolytic activity for the enzymatically hydrolyzable blocking 
group present on a particular compound. By routine design and experimental 
choices of a blocking group or groups, it will be possible to produce 
quinones and iminoquinones in selected cells or tissues of a multicellular 
organism. Compounds of the invention have been shown to be substrates of 
leucine aminopeptidase (LAP), an enzyme known to be present in leukemia 
cells, and to inhibit the synthesis of DNA of these cells. The inhibition 
of DNA synthesis by quinones is known. In vitro hydrolysis of 
.beta.N-(L-leucyl)-.alpha.N-(carboxymethyl)-p-hydroxy-phenylhydrazine 
(Leu-CHP) and several other compounds of the invention by LAP has been 
demonstrated. The ultimate elimination products of iminoquinones and 
p-benzoquinone have been demonstrated in this in vitro experiment and in 
an in vivo experiment in which the quinone and iminoquinones were 
generated in situ in L1210 murine leukemia cells. Thus, the original 
hypothesis of in situ quinone production has been proven. However, an 
attempt to prolong the life of mice infected with leukemia using compounds 
of the invention was not effective, apparently due to a poor choice of 
blocking group resulting in low solubility and rapid clearing by the 
kidneys. Nevertheless, choice of proper blocking groups and quinoidal 
structures is well within the routine experimentation commonly practical 
in pharmacology, now that the general principles and specific examples of 
the present invention have been disclosed. 
The above disclosure generally describes the present invention. A more 
complete understanding can be obtained by reference to the following 
specific examples, which are provided herein for purposes of illustration 
only and are not intended to be limiting unless otherwise specified.

EXAMPLE 1 (See FIG. 1) 
The free base of p-benzyloxyaniline (I) was obtained by extraction of the 
hydrochloride from 1N NaOH into diethyl ether (Et.sub.2 O). The Et.sub.2 O 
solution was separated, dried over Na.sub.2 SO.sub.4, filtered and 
rotoevaporated down to a beige prismatic solid. I (79.0 gm, 0.397 mole), 
ethylchloroacetate (57.5 gm, 0.47 mole), and dry triethylamine (TEA) (47.5 
gm, 0.47 mole) were added to absolute ethanol (EtOH)(300 ml) and refluxed 
under nitrogen (N.sub.2) in a 1000 ml, three-necked, round bottom flask 
(3N-RBF) fitted with an overhead mechanical stirrer (OMS) and 
water-jacketed condenser. After 48 hours, the reaction was cooled to room 
temperature and to it was then added Et.sub.2 O (700 ml) and the resulting 
TEA-HCl precipitate was filtered off. The solution was extracted with 0.2N 
HCl, 0.2N NaOH and finally H.sub.2 O and the organic layer was separated, 
dried over Na.sub.2 SO.sub.4, filtered and rotoevaporated down to a light 
brown prismatic solid (II). Yield: 91 gm (80 % theoretical). 
In an ice bath cooled 1000 ml 3N-RBF fitted with an OMS, II (35 gm, 0.122 
mole) was added to a solution of concentrated HCl (20 ml) and ice (70 ml). 
To this a solution of NaNO.sub.2 (9.5 gm) in H.sub.2 O (30 ml) was added 
dropwise and allowed to react at 4.degree. C. for 2-3 hours. Et.sub.2 O 
(500 ml) was then added directly to the reaction mixture and the product 
was extracted into the Et.sub.2 O. This was then extracted with 0.2N NaOH, 
then H.sub.2 O, dried over Na.sub.2 SO.sub.4, filtered and rotoevaporated 
to yield a white solid. This was washed with petroleum ether and filter 
dried. Yield: 26.4 gm (68.5%). 
In order to provide N(p-hydroxyphenyl)glycine ethyl ester for later 
comparisons, II (2.85 gm, 0.01 mole) was dissolved into 15-20 ml of 
dioxane, palladium on carbon catalyst (Pd/C)(150 mg) was added while 
N.sub.2 was bubbled through the solution and the suspension was then 
catalytically hydrogenated (45-50 psi H.sub.2) for about 2 hours. The 
catalyst was filtered off and the solvent rotoevaporated to a light yellow 
liquid which was dried on the high vacuum pump for a few hours and allowed 
to stand at room temperature overnight. The next day the oil had 
crystallized into yellow needles which were washed with petroleum ether 
and filter dried. The structure was confirmed by NMR. Yield: 1.85 gm 
(95%). 
Synthesis of .alpha.-N-(carboxymethyl ethyl 
ester)-p-benzyloxphenylhydrazine (CHP(OBz)OEt)(IV). 
In an ice bath cooled 1000 ml 3N-RBF fitted with an OMS, ethyl acetate 
(EtOAc)(300 ml), EtOH (50 ml) and H.sub.2 O (25 ml) were mixed. Freshly 
prepared aluminum amalgam (Al(Hg)) was added by taking cut up aluminum (6 
gm) and exposing it to 0.1N NaOH until gas evolution was rapid. The water 
was decanted, the foil was washed briefly with tap water, decanted again 
and enough 2% HgCl.sub.2 solution added to cover the Al. After about 2 
minutes, the gray solution was decanted and the Al(Hg) was washed 
successively with H.sub.2 O, EtOH and EtOAc. After the EtOAc was decanted, 
the Al(Hg) was added directly to the solution containing III. A second 
batch of Al(Hg) was prepared in an identical fashion and also added to the 
reaction vessel. The reduction was complete after 2 hours as judged by 
silica gel thin layer chromatography (SG-TLC). The major product was the 
desired IV while the minor product was regenerated II. The unreacted 
Al(Hg) was first filtered off and washed with EtOAc using a Buchner funnel 
without filter paper. The filtrate was then refiltered and the clear 
filtrate was dried over Na.sub.2 SO.sub.4. This was filtered off and the 
filtrate was rotoevaporated to yield a white prismatic solid which was 
washed with petroleum ether and filter dried. Yield: 17.4 gm. To remove II 
(as the impurity), the solid was taken up in 100 ml of hot EtOH and 
recrystallized. After at least 8 hours, the solid IV was filtered off, 
washed with petroleum ether and filter dried. Yield: 14.6 gm (71%). 
EXAMPLE 2 (See FIG. 1) 
Synthesis of .alpha.-N-(carboxymethyl ethyl ester)-p-hydroxyphenylhydrazine 
CHP(OEt) (IVa) 
All preparations of IVa were done the same day experiments were to be 
performed using it. IV (2.0 gm), (0.0067 mole) was dissolved in dry THF 
and hydrogenated for 2 hours. (45-50 psi H.sub.2) in the presence of Pd/C. 
The catalyst was filtered off and the THF was rotoevaporated off leaving a 
clear light yellow oil behind. (The analysis reveals a mixture of IIa and 
IVa.) Et.sub.2 O (20-30 ml) was added to this and the solution (10 ml) was 
preparatively chromatographed on thick SG-TLC plates with fluorescent 
indicator using an anhydrous Et.sub.2 O/Pet ether (2:1) solvent system. 
Each plate was developed twice. The desired product (IVa) (R.sub.f =0.05) 
was obtained after scraping off the silica gel, stirring thoroughly in 
anhydrous Et.sub.2 O, filtering off the silica gel and rotoevaporating off 
the Et.sub.2 O. The light yellow oil was dried for 2 hours with a high 
vacuum pump down to a colorless oil which was further dried on the high 
vacuum pump overnight. This product was used in subsequent in vitro and in 
vivo experiments. 
EXAMPLE 3 (See FIG. 3) 
Synthesis of 
.beta.-N-(N-tert-butoxycarbonyl-L-Leucyl)-.alpha.-N-(carboxymethyl ethyl 
ester)-p-benzyloxy phenylhydrazine (Boc-Leu-CHP(OBz)OEt) (V) 
To CH.sub.2 Cl.sub.2 (30 cc) were added IV (9.0 gm, 0.03 mole) and 
N-tert-butoxycarbonyl-L-leucine (Boc-Leu, 8.4 gm, 0.036 mole) with 
stirring. After cooling to 4.degree. C. under N.sub.2 
dicyclohexylcarbodiimide (DCCI, 7.4 gm, 0.036 mole) was added and stirring 
continued for 18 hours, then EtOAc (300 cc) was added and dicyclohexylurea 
(DCCU) ws filtered off. The solvent was rotoevaporated leaving a yellow 
oil. Et.sub.2 O was added and the solution was extracted sequentially with 
1N HCl, 0.1N NaOH, H.sub.2 O, then dried over Na.sub.2 SO.sub.4, filtered 
and rotoevaporated down to a white solid (V). This was washed with pet 
ether and filter dried. Yield: 15 gm (97%). 
EXAMPLE 4 
Synthesis of 
.beta.-N-(N-tert-butoxycarbonyl-L-phenylalanyl)-.alpha.-N-(carboxymethyl 
ethyl ester)-p-benzyloxyphenylhydrazine(BOC-Phe-CHP(OBz)OEt)(VI) 
To dry THF (20 cc) cooled in an ice bath and under N.sub.2, 
N-tert-butoxycarbonyl-L-phenylalanine (Boc-Phe, 3.0 gm, 0.011 mole) and IV 
(3.0 gm, 0.01 mole) was added and stirred until completely dissolved. DCCI 
(2.4 gm. 0.012 mole) was added with stirring and reacted overnight. EtOAc 
(200 ml) was added the DCCU filtered off and solvent removed by 
rotoevaporation. The resulting oil was dissolved in Et.sub.2 O and 
extracted sequentially with 1NHCl, 0.1N NaOH and H.sub.2 O. The organic 
fraction was separated, then dried over Na.sub.2 SO.sub.4, filtered and 
rotoevaporated leaving a white solid. This solid was washed with pet ether 
and filter dried. Yield: 5.0 gm (91.4%). 
EXAMPLE 5 
Synthesis of 
.beta.-N-(N-tert-butoxycarbonyl-L-alanyl-.alpha.-N-(carboxymethyl ethyl 
ester)-p-benzyloxy-phenylhydrazine (Boc-Ala-CHP(OBz)OEt)(VII) 
N-Tert-butoxycarbonyl-L-alanine (Boc-Ala, 2.4 gm, 0.013 mole) and IV (3.0 
mole) were dissolved in THF (15 cc) and cooled in an ice bath under a 
N.sub.2 atmosphere. DCCI (2.4 gm, 0.012 mole) was added with stirring and 
allowed to react overnight. EtOAc (200 cc) added and the DCCU was filtered 
off. 
The solvent was rotoevaporated and the remaining oil was taken up in 
Et.sub.2 O and extracted successively with 1N HCl, 0.1N NaOH, H.sub.2 O. 
The organic layer was separated, dried over Na.sub.2 SO.sub.4, filtered 
and rotoevaporated leaving an off-white solid. This was dried with the 
high vacuum pump overnight. Yield: 4.3 gm (91%). The product (VII) could 
be recrystallized by dissolving the compound in the minimum quantity of 
Et.sub.2 O and then pouring it into a 30-50 fold volume of pet ether and 
allowing it to stand at room temperature overnight, yielding white 
crystals. 
EXAMPLE 6 
Synthesis of .beta.-N-(N,N.sup.1 
-ditert-butoxycarbonyl-L-lysinyl)-.alpha.-N-(carboxymethyl ethyl 
ester)-p-benzyloxyphenylhydrazine (Boc-Lys-CHP(OBz)OEt)(VIII) 
IV (3.0 gm, 0.01 mole) and N,N.sup.1 
-di-tert-butoxycarbonyl-L-lysine(.alpha.-.epsilon.-di-Boc-Lys, 4.2 gm, 
0.012 mole) were dissolved in THF (20 cc) under N.sub.2 and cooled in an 
ice bath. DCCI (2.4 gm, 0.012 mole) was added and the reaction stirred 
overnight. EtOAc (200 cc) was added and DCCU was filtered off. The solvent 
was rotoevaporated and the remaining oil taken up in Et.sub.2 O, extracted 
sequentially with 1N HCl, 0.1N NaOH, H.sub.2 O and dried over Na.sub.2 
SO.sub.4, filtered and rotoevaporated again to leave a thick yellow oil. 
This was dried on a high vacuum pump for 24 hours. Yield: 6.2 gm (98.5%). 
The light beige product could be precipitated from Et.sub.2 O-Pet ether 
similar to VII. 
EXAMPLE 7 
Synthesis of .beta.-N-(N-tert-butoxycarbonyl-L-glutamyl .gamma.-methyl 
ester)-.alpha.-N-(carboxymethyl ethyl 
ester)-p-benzyloxyphenylhydrazine(BOC-.gamma.-OMe-Glu CHP(OBz)OEt)(IX) 
N-tert-butoxycarbonyl-L-glutamyl-.gamma.-methyl ester(Boc-OMe-Glu, 5.0 gm, 
0.02 mole) and IV (5.5 gm, 0.018 mole) were dissolved in THF (25 ml) under 
N.sub.2 and cooled in an ice bath. DCCI (4.4 gm, 0.021 mole) was added and 
allowed to react overnight. EtOAc (300 ml) was added and DCCU filtered 
off. The solvent was rotoevaporated off leaving a yellow oil. Et.sub.2 O 
(150 ml) was added and the solution extracted with 1N HCl, 0.1N NaOH, 
H.sub.2 O and dried over Na.sub.2 SO.sub.4, filtered and rotoevaporated 
down to an off-white solid. Yield: 9.4 gm (96%). 
EXAMPLE 8 (See FIG. 3) 
Synthesis of .beta.-L-Amino 
Acid-(.alpha.-N-carboxymethyl)-p-hydroxyphenylhydrazines(AA-CHPs) (X, XI, 
XII, XIII, XIV) 
The respective BOC-AA-CHP(OBz)OEt compound (1 ester equivalent) (i.e., V, 
VI, VII, VIII, IX) was dissolved in a stirred solution of EtOH and THF 
(1:1) at room temperature so that the concentration was about 1M. To this 
was added 1N NaOH (2.1 ester equivalents) and the de-esterification was 
allowed to proceed overnight. When the reaction was complete, as judged by 
SG-TLC the reaction solution was extracted with Et.sub.2 O, the aqueous 
layer separated, acidified with 1N HCl and extracted into Et.sub.2 O 
followed by EtoAc and then the organic fractions were combined, dried over 
Na.sub.2 SO.sub.4, filtered and the solvents rotoevaporated off. The 
products were sufficiently pure to use in the next reaction. Yields: 
71-93% 
R.sub.f (SG-TLC, EtOAc/EtOH(3:1)); 
BOC-Leu-CHP(OBz)=0.42 Boc-Glu-CHP(OBz)-0.34 
BOC-Phe-CHP(OBz)=0.38 di-Boc-Lys-CHP(OBz)=0.40 
Boc-Ala-CHP(OBz)=0.27. 
Removal of protective benyzl group by hydrogenation 
The respective Boc-AA-CHP(OBz) compounds (2-6 gm) were dissolved in dioxane 
(20-50 ml) and glacial acetic acid (HAc)(10-15 ml) and the solution was 
partially deoxygenated by bubbling N.sub.2 gas through it for several 
minutes before the palladium on carbon catalyst (Pd/C)(200-300 mgm) was 
added and the compound was then hydrogenated for 24-48 hours (40-50 psi 
H.sub.2). After hydrogenation was complete, as judged by SG-TLC, the 
catalyst was filtered off and the filtrate was rotoevaporated down to 
clear to light brown oils. These were dried overnight using a high vacuum 
pump and the resulting solid or oil was washed with Et.sub.2 O. In the 
case of the Leu, Phe and Ala derivatives, the solid was then filter dried 
and weighed. For the lysyl and glutamyl derivatives, the resulting oils 
were dissolved in Et.sub.2 O and, upon standing, a light beige solid 
precipitated out. This was filtered off, washed with Et.sub.2 O again and 
filter dried. Yields: 84-98%. 
R.sub.f (SG-TLC, EtoAc/EtOH(3:1)); 
BOC-Leu-CHP 0.27 di-Boc-Lys-CHP=0.04 
BOC-Phe-CHP 0.27 Boc-Glu-CHP=0.28 
BOC-Ala-CHP 0.11. 
Removal of the Boc Protecting Group 
The respective Boc-AA-CHP was then deblocked by adding it to distilled 
trifluoroacetic acid (TFA, 15 ml/gm Boc-AA-CHP) under N.sub.2 and cooled 
in an ice bath. The reaction was stirred for 1/2 hour and then poured 
directly into anhydrous Et.sub.2 O (100 cc Et.sub.2 O/10 cc TFA). The 
desired product precipitated out immediately as a white solid (TFA salt). 
After continued stirring for 1/2 hour, the solid was filtered off and, if 
hygroscopic, redissolved in the minimum quantity of EtOH which was then 
rotoevaporated off and the remaining solid dried with a high vacuum pump 
overnight. Once dry, all the compounds (as the TFA salt) are white solids, 
stable at room temperature. 
For analytical purposes, the Leu-CHP (X) and Phe-CHP (XI) derivatives were 
repurified by dissolving in water (pH 11) and acidifying to neutrality 
with 1N HCl. The precipitate (off-white) was filter dried and washed with 
EtOAc. The Ala-CHP (XII) derivative was recrystallized from boiling water. 
The Lys-CHP (XIII) and Glu-CHP (XIV) derivatives were left in their TFA 
salt form. Yield: 70-80%. 
EXAMPLE 9 (See FIG. 4) 
Synthesis of .beta.-N-(L-leucyl)-.alpha.-N-(carboxymethyl ethyl 
ester)-p-hydroxyphenylhydrazine (Leu-CHP-OEt) (XV) 
Boc-Leu-CHP(OBz)OEt (3.0 gm, 0.0058 mole) was dissolved in dioxane (40 ml) 
and HAc (10 ml) and catalytically hydrogenated (Pd/C) (45-50 psi H.sub.2) 
for 24 hours. The catalyst was filtered off and the solvent rotoevaporated 
down to an oil which was further dried with the high vacuum pump 
overnight. Yield: 2.2 gm (Boc-Leu-CHP-OEt, 89%) 
This product (1.0 gm, 0.0024 mole) was then deblocked with distilled TFA (6 
cc) at 0.degree. C. under N.sub.2 for 1/2 hour. The reaction solution was 
poured directly into dry Et.sub.2 O (200 ml) and pet ether (50 ml) and the 
white product precipitated out within one hour. Yield: 0.9 gm (3/2 
TFA.Leu-CHP-OEt) Yield=77%. 
EXAMPLE 10 (See FIG. 5) 
Coupling of Leu-CHP to bovine serum albumin 
Boc-Leu-CHP (3.5 gm, 0.0089 mole) and N-hydroxysuccinimide (NHSu) (2.0 gm, 
0.07 mole) were added to CH.sub.2 Cl.sub.2 (20-30 ml) with stirring. The 
reaction solution was cooled in an ice bath and placed under N.sub.2. DCCI 
(3.0 gm, 0.0146 mole) was added and the reaction was allowed to proceed 
for 18 hours. EtOAc was added and the DCCU was filtered off. After 
extracting with saturated NaHCO.sub.3, the organic fraction was separated, 
dried over Na.sub.2 SO.sub.4, filtered and rotoevaporated down to a light 
yellow oil which solidified after drying overnight with the high vacuum 
pump. This product was used directly in the coupling reaction. Yield: 3.6 
gm (83%). 
In a 200 ml 3N-RBF fitted with an overhead mechanical stirrer bovine serum 
albumin (BSA, 2.76 gm) was dissolved with stirring into saturated 
NaHCO.sub.3 (10 ml). To this was added in 2 ml increments a solution of 
Boc-Leu-CHP-NHS.mu..(3.2 gm, 0.0065 mole) in EtOH (10 ml). Enough 1N NaOH 
was added to raise the pH to 8.0 and the reaction was stirred. After 1 
hour the reaction solution became viscous and more saturated NaHCO.sub.3 
(20 ml) and EtOH (5 ml) were added. This was stirred at room temperature 
overnight. The protein forms a viscous orange gel overnight and more 
H.sub.2 O (20 ml) was added. After 48 hours of coupling time, the reaction 
solution had changed from orange to brown. The reaction mix was poured 
into dialysis tubing (30.times.3 cm) and dialyzed against 20 volumes of 
demineralized H.sub.2 O which was changed every day for 6 days. The 
modified protein product within the dialysis tubing was dark brown. After 
6 days, the dialysis tubing was opened and filtered twice: first through 
#41 Whatman filter paper (coarse), then #42 Whatman filter paper 
(ultrafine). The clear brown filtrate was then gradually acidified with 1N 
HCl to pH 4.0. The modified protein product precipitated out immediately 
as a beige solid. This was allowed to stand overnight at 4.degree. C. and 
then centrifuged at 10000 rpm.times.20 min. The clear supernatant was 
discarded and the protein precipitate was shell-frozen in a dry 
ice-acetone bath and vacuum dried with a high vacuum pump. The product was 
a brown solid. Yield: 3.0 gm (80.6% ) (based on 100% coupling to 61 Lysyl 
residues per alubmin molecule) 
Boc-Leu-CHP-BSA (XVI) (1.0 gm) was added to distilled TFA (50 ml) under 
N.sub.2 at 4.degree. C. for 1/2 hour with stirring. The reaction 
suspension was then added to dry Et.sub.2 O (200 ml) and the product 
filter dried and pulverized with a mortar and pestle. The product XVI is 
brown in color. Yield: 1 gm (99%). 
TABLE 1 
__________________________________________________________________________ 
Analytical Data 
Com- 
Mol. Calc. Obs. 
pound 
Wt. % Yield 
Mp(.degree.C.) 
C H N C H N R.sub.f 
[.alpha.].sub.D.sup.20 
__________________________________________________________________________ 
II 285.3 
80 64-65 71.56 
6.71 
4.91 
71.88 
6.66 
4.65 
.59.sup.a,.27.sup.b 
III 314.3 
68.5 67.5-69 
64.96 
5.77 
8.91 
64.84 
5.73 
8.95 
.57.sup.a,.41.sup.b 
IV 300.36 
71 95-96 67.96 
6.72 
9.33 
67.86 
6.76 
9.12 
.44.sup.a,.065.sup.b 
V 513.64 
97 114-115.5 
65.48 
7.65 
8.18 
65.48 
7.65 
8.37 
.83.sup.a 
VI 547.65 
91.4 133-135 
67.99 
6.81 
7.67 
67.99 
7.02 
7.59 
.81.sup.a 
VII 471.55 
91 88-90 63.68 
7.05 
8.91 
63.69 
7.13 
8.95 
.76.sup.a 
VIII 
628.77 
98.5 59-61 63.04 
7.70 
8.91 
62.80 
7.89 
8.95 
.59.sup.a 
IX 543.62 
96 92-94 61.86 
6.86 
7.73 
61.64 
6.77 
7.74 
.65.sup.a 
X 295.34 
75 222-224 
56.92 
7.17 
14.23 
56.76 
7.24 
14.15 
.82.sup.c 
+23.1.sup.k 
XI 338.36.sup.d 
70 215-216 
60.35 
5.96 
12.42 
60.41 
5.94 
12.20 
.79.sup.c 
+27.9.sup.g 
XII 266.77.sup.l 
80 227-229 
49.53 
6.23 
15.75 
49.46 
6.30 
15.67 
.44.sup.c 
+24.1.sup.m 
XIII 
556.41.sup.e 
80 198-199 
38.86 
4.71 
10.07 
38.58 
4.85 
10.31 
.16.sup.c 
+20.5.sup.h 
XIV 377.3.sup.f 
80 200-201 
44.57 
4.94 
11.13 
44.17 
5.00 
11.14 
.29.sup.c 
+17.2.sup.i 
XV 494.4.sup.j 
77 190-191 
48.92 
5.72 
9.01 
48.70 
5.66 
9.32 
.98.sup.c 
__________________________________________________________________________ 
.sup.a = Et.sub.2 O/Pet Ether (1:1), .sup.b = CCl.sub.4 /CH.sub.2 Cl.sub. 
(2:1) Both on SG--TLC. .sup.c = Cellulose TLC; Butanol (20): Pyridine 
(13.3): H.sub.2 O (16): Acetic Acid (4) .sup.d = 1/2 mole H.sub.2 O 
present; .sup.e = 2Tfa.1H.sub.2 O salt; .sup.f = 1/2 Tfa.1/2 H.sub.2 O 
salt; .sup.g:c = 1.5 of Phe--CHP.1/2 H.sub.2 O; .sup.h:c = 1.5 of 
Lysyl--Tfa.1H.sub.2 O; .sup. i:c = 1.5 of Glu--CHP.1/2 Tfa.1/2 H.sub.2 O; 
.sup.k:c = 1.5; .sup.j = 3/2 Tfa salt; .sup.l = 3/4 H.sub.2 O present; 
.sup.m:c = 1.5 of Ala--CHP.3/4 H.sub.2 O. 
EXAMPLE 11 (See FIG. 7) 
Leucine Aminopeptidase In Vitro Hydrolysis 
Leucine Aminopeptidase (LAP), type III-CP (Sigma) was stored at 
0.degree.-4.degree. C. in 0.75M saturated ammonium persulfate, 0.1M Tris 
and 0.005M MgCl.sub.2 (pH 8) and contained 210 U/ml. Prior to the 
hydrolysis experiment, LAP (50.lambda.) was activated by incubating it in 
0.025M MnCl.sub.2 (0.2 ml), 0.5M Tris.HCl buffer (pH 9.0, 0.2 ml) and 
demineralized and ultra-filtered H.sub.2 O (0.6 ml) for at least 2 hours 
at 37.degree. C. To each sample test tube was added AA-CHP (1.5 mg) in 
0.02M Tris HCl buffer (pH 9.0, 0.5 ml) so that the final concentration was 
1 mg/ml AA-CHP. The hydrolysis reaction was allowed to proceed for up to 
12 hours and then TLC's were performed by spotting the reaction solution 
(2-3 .mu.l) on cellulose plates and developing them in Butanol (20 
ml)/HAc(4ml)/Pyridine(13.3 ml)/H.sub.2 O (16 ml) solvent system. They were 
dried and sprayed with 0.2% Ninhydrin in EtOH and the color was developed 
at 85.degree. C. for 5 minutes. The in vitro hydrolysis of Leu-CHP by LAP 
was also concomitantly studied by scanning spectrophotometry at different 
times after the start of incubation with the enzyme. A blank reference 
cell containing only enzyme and buffer was used. 
The enzyme was able to cleave Leu-CHP, Phe-CHP, Ala-CHP, Leu-CHP-OEt and 
Leu-CHP-BSA but not Lys-CHP, Glu-CHP, or Boc-Leu-CHP. 
In order to characterize the products of hydrolysis of AA-CHP's, the 
hydrolysis of Leu-CHP by LAP was studied by absorption spectrophotometry. 
As shown in FIG. 7, during the hydrolysis of Leu-CHP by LAP at pH 8.5 the 
original maximum at 300 nm disappeared and another more intense maximum at 
350 nm appeared. This spectral change was mimicked by free CHP-OEt alone 
at pH 9.0 as shown in FIG. 8A. FIG. 8A shows that the absorption maximum 
of CHP-OEt was at 300 nm and that up to 17.5 minutes after addition of 
CHP-OEt (0.2 mg) in DMSO (0.02 ml) to 0.01M Tris buffer at pH9.0 (3.0 ml) 
the absorbance at 300 nm continued to increase. This was also true for the 
new maximum at 350 nm and for a second new "maximum" which appeared as a 
low, broad "peak" merging with 350 peak and centered at about 400 nm. 
However, by 22 minutes (not shown) the absorbance at 300 and 400 nm had 
diminished in intensity while the 350 maximum continued to increase, 
albeit at a slower rate. At pH 7.0 (FIG. 8B), the absorbance at 300 nm 
continued to slowly increase in intensity even up to 90 minutes after 
dissolving CHP-OEt (0.2 mg) in DMSO (0.02 ml) into 0.05M Phosphate buffer 
(3 ml). Furthermore, a second maximum at 350 nm developed but it remaind 
smaller in intensity than the 300 nm peak. 
Because of the obvious instability of CHP-OEt in aqueous solution and 
because of the apparent multiplicity of absorbing species appearing during 
its rearrangement and elimination reactions to form quinones and 
iminoguinones, this process was further characterized by including in the 
reaction mixture a reducing agent (NaBH.sub.4) a nucleophile which was not 
also a reducing agent (Lysine) or 3 mercaptoproprionic acid (3-MPA) which 
could function both as a nucleophile and as a reducing agent. 
As shown in FIG. 9A, a ten-fold excess of NaBH.sub.4, a reducing agent, 
completely abolished formation of the 350 nm peak at pH 9.0. Once all the 
NaBH.sub.4 had decomposed (after 50 minutes), the peak at 350 nm could be 
seen. In FIG. 9B, a ten-fold excess of Lysine, a nucleophile but not a 
reducing agent, had little if any effect on the development of the 350 nm 
peak. On the other hand, a ten-fold excess of 3-MPA, both a nucleophile 
and a reducing agent, completely abolished any spectral change over the 
time span of several hours. (FIG. 9C). 
Table 2 summarizes the results of several separate experiments involving 
the reactions of CHP-OEt, CHA-OEt, pIQ-OEt and pBQ (0.5-1.0 mM) with 
various nucleophiles (lysine, 3-MPA) reducing agents (NaBH.sub.4, 3-MPA) 
and oxidizing agents (NaIO.sub.4, O.sub.2) in ten-fold molar excess in 
Tris-HCl buffer (0.05M, pH 9.0). 
In these studies, CHP-OEt, CHA-OEt and pBQ* were all pure compounds, thus, 
their absorption could be unequivocably assigned, pIQ-OEt was generated by 
the autoxidation of CHA-OEt in solution and its presence was noted by the 
appearance of one absorbance maximum at 400 nm and the development of a 
distinct yellow color to a previously clear solution of CHA-OEt. As shown 
in Table 2, there was a striking similarity in the absorption maxima that 
resulted when CHP-OEt was reacted with Lysine (350 nm) and 3-MPA (300 nm) 
with those that occur when pBQ was reacted with these same compounds. On 
the other hand, there was a dissimilarity between the spectra of CHP-OEt 
and CHA-OEt. CHA-OEt was oxidized by O.sub.2 or NaIO.sub.4 to the pIQ-OEt 
with a .lambda..sub.max =400 nm whereas NaIO.sub.4 had no effect on the 
rate or nature of spectral change that occurred when . . . 
TABLE 2 
______________________________________ 
Summary of Spectral Study Experiments 
With CHP--OEt, (IVa) CHA--OEt, (IIa), and 
p-Benzoquinone (pBQ) at pH 8-9 
Initial Compound Final 
Compound Maximum (nm) Added Maximum (nm) 
______________________________________ 
CHP--OEt 300 none 350 
CHP--OEt 300 Lysine 350 
CHP--OEt 300 3-MPA 300 
CHP--OEt 300 NaBH.sub.4 
300 (350.sup.a) 
CHP--OEt 300 NaIO.sub.4 
350.sup.b 
CHA--OEt 300 none 300, 400.sup.c 
CHA--OEt NaIO.sub.4 
300, 400.sup.d 
pIQ--OEt + 
300, 400 Lysine 300 
CHA--OEt 
pIO--OEt + 
300, 400 3-MPA 300 
CHA--OEt 
pBQ 240 Lysine 350 
pBQ 240 3-MPA 300 
______________________________________ 
.sup.a Appears after NaBH.sub.4 has decomposed. 
.sup.c Formation of piminoquinone (pIQ--OEt) via autoxidation. 
.sup.b No change in rate of rearrangement. 
.sup.d Formation of pIQ--OEt accelerated when CHP--OEt was dissolved in 
alkaline H.sub.2 O. 
When CHA-OEt was allowed to autoxidize to pIQ-OEt and then reacted with 
Lysine, the 400 nm peak disappeared and only the 300 nm peak remained, 
whereas lysine had no effect on the spectral changes that occurred when 
CHP-OEt was dissolved in alkaline H.sub.2 O. Both CHP-OEt and CHA-OEt, 
when reacted with 3-MPA, showed stable maxima at 300 nm for several hours. 
The proposed explanation for these data is given in FIG. 10. This scheme 
postulates a central role for the p-iminoquinone (pIQ-OEt) the product of 
reaction A or H. In the case of reaction A, pIQ-OEt is generated by the 
spontaneous rearrangement of CHP-OEt to give the iminoquinione by losing 
ammonia. This rearrangement can be acid or base catalyzed (see FIG. 2). 
The present data does not allow differentiation between these mechanisms. 
It is important to note that from FIG. 10, the maxima at 350 nm are 
assigned to disubstituted aminophenols, the products of the reaction 
sequence ABCD. Thus, even though the development of the peak at 350 nm was 
severaly retarded when CHP-OEt was dissolved in water (pH 7.0) (FIG. 8B). 
This is only a reflection of the decreased rate of formation of these 
final products (BCD) at this more acidic pH and not a measure of the 
generation of pIQ-OEt. The fact that at pH 7.0 a peak at 400 nm did not 
develop when CHP-OEt was incubated in aqueous solution may be explained by 
the very low .epsilon..sub.400 for pBQ. Thus, when pIQ-OEt underwent 
disassociation at this pH giving pBQ and glycine ethyl ester (E), pBQ 
would have made an insignificant contribution to the absorbance at 400 nm. 
At alkaline pH, CHP-OEt itself can attack pIQ-OEt as a nucleophile (B) as 
can lysine. The oxidation of that product (.lambda..sub.max =300 nm) by 
either O.sub.2 or pIQ-OEt (C) gives a second quinone which can undergo a 
second nucleophilic attack at one of two sites (D). Thus lysine would not 
be expected to have an effect on the development of the peak at 350 nm. 
That this is the case was borne out in the experiment reported in FIG. 9B. 
In the presence of NaBH.sub.4, any pIQ-OEt that was formed was immediately 
reduced to CHA-OEt (G). Once the NaBH.sub.4 had decomposed, any pIQ-OEt 
generated was able to follow pathway BCD (FIG. 9A). On the other hand, 
3-MPA could react with pIQ-OEt (F) or reduce it (N) and reduce any 
dissolved oxygen by forming a disulfide (I,O). This would effectively 
quench any reoxidation of the pIQ-OEt-3-MPA adduct or CHA-OEt, thereby 
inhibiting the formation of a disubstituted aminophenol and the 
development of the 350 nm peak. In reaction H, the generation of pIQ-OEt 
from CHA-OEt depends on an alkaline pH and the presence of a suitable 
oxidizing agent (e.g., O.sub.2, NaIO.sub.4) and in turn, reduces that 
agent (H). Once oxidized to pIQ-OEt, the quinone was fairly stable because 
CHA-OEt is a poor nucleophile (unlike CHP-OEt). If lysine was added, 
however, to a solution of pIQ-OEt, only the mono-adduct was formed because 
the supply of an oxidizing agent (e.g., O.sub.2, had been depleted in the 
formation of pIQ-OEt. This contrasted strongly with CHP-OEt, which can 
spontaneously rearrange to pIQ-OEt and hence did not require oxygen for 
reaction A or an external oxidizing agent for reaction C, since the 
continued production of pIQ-OEt (A) ensured the re-oxidation of the 
mono-adduct (C) and formation of the disubstituted products (D). When pBQ 
was reacted with 3-MPA and Lysine, the spectral results obtained were very 
similar to those achieved when these two compounds were reacted with 
CHP-OEt. When a ten-fold excess 3-MPA was added to pBQ at pH 9.0 a peak at 
300 nm developed rapidly, but the peak at 350 nm appeared only after 
several hours. (J). On the other hand, when a ten-fold excess of lysine 
was reacted with pBQ at pH 9.0, an initial peak at 300 nm (K) was observed 
which rapidly diminished while a strong peak at 350 nm appeared (M). This 
350 nm peak appeared at a faster rate than the 350 nm peak observed when 
CHP-OEt was dissolved in alkaline water. 
From these data. one must infer that CHP-OEt acts both as a quinone and 
nucleophile, leading to the conclusion that CHP-OEt (in a protic solvent, 
such as H.sub.2 O) undergoes a rearrangement resulting in the formation of 
pIQ-OEt which can then undergo subsequent nucleophilic attack, 
reoxidation, and nucleophilic attack again depending on the pH and the 
presence or absence of appropriate nucleophiles and reducing and oxidizing 
agents. Furthermore, this spontaneous rearrangement of CHP-OEt to pIQ-OEt 
would not be dependant on the presence or absence of an oxidizing agent 
(e.g., oxygen) and therefore should be as likely to occur in an hypoxic 
environment. Since it is well known that quinones are cytotoxic and having 
established that various AA-CHP's were capable of being enzymatically 
cleaved by LAP in vitro, and that upon enzymatic cleavage, CHP is released 
and can undergo a rearrangement to give an iminoquinone, these compounds 
were next tested for toxicity to leukemia cells. 
EXAMPLE 12 
In vitro Inhibition of .sup.3 H-Thymidine Incorporation into DNA of L1210 
Murine Leukemia Cells 
An L1210 murine leukemia cell line was maintained in mice (female DBA/2J 
strain from the Jackson Laboratory, Bar Harbor, Maine) by transferring 
every seven days via intraperitoneal (i.p.) injection of 10.sup.5 cells. 
L1210 cells were also stored at liquid nitrogen temperature in 2 ml 
aliquots containing 10.sup.6 cells/ml in a storage medium consisting of 
72% (v/v) RPMI Medium 1640 (Grand Island Biochemicals), 18% (v/v) fetal 
calf serum, 10% (v/v) dimethylsulfoxide (Me.sub.2 SO) and 1% Glutamine. 
The frozen cells maintained their viability for over two years. For all in 
vitro and in vivo experiments only freshly harvested intraperitoneally 
grown L1210 cells were used. 
A DBA/2J mouse bearing L1210 cells for 6 days after i.p. inoculation of 
1.times.10.sup.5 cells was sacrificed by cervical dislocation and, using 
sterile technique, the abdominal skin was incised and pinned back. Using 
two 10 ml syringes with 21 gauge needles, one being filled with cold 
phosphate buffer saline (PBS) (9 ml, pH 7.4) and in 3 ml increments, the 
solution was injected into the peritoneal cavity through one side and 
withdrawn with the other syringe through the other side. The light pink 
cell suspension was then centrifuged at 2000 rpm for 5 minutes and the 
supernatant was decanted. The cells were resuspended in cold 80% RPMI 
Medium 1640/20% heat inactivated fetal calf serum (10 ml). After having 
counted the cells with a hemocytometer and stained them for viability with 
Trypan blue (usually 99% viable), they were diluted to a final 
concentration of 1.times.10.sup.6 viable cells/ml (unless otherwise 
noted.) Each test tube then received 100 .mu.l of the cell suspension and 
25 .mu.l of the test compound or control solution (PBS) and was then 
incubated at 37.degree. C. under a 95% O.sub.2 /5% CO.sub. 2 atmosphere. 
After 0, 2, 4, 5 or 6 hours preincubation, 3H-Thymidine (1 .mu.l; 1 .mu.C) 
was added and then the incubation was continued for another 1/2 hour. The 
cells were then killed and their proteins precipitated with 5% TCA. This 
was then filtered through Whatman GF/C filter discs, washed three times 
with 1% TCA, then 95% EtOH and then oven dried for 5 minutes (100.degree. 
C.). The filter discs were then digested with NCS tissue solubilizer (0.5 
ml) and PPO/POPOP in toluene scintillation cocktail (2.0 ml) for 1/2 hour. 
To each vial was then added more PPO/POPOP/Toluene cocktail (1.5 ml) and 
the samples were counted. All determinations were made in triplicate. The 
results of these experiments are shown in FIGS. 11, 12 and 13. These 
experiments compared the various compounds at given concentrations (FIG. 
11 and FIG. 13) and describe the dependance of the degree of inhibition on 
both the incubation time and the concentration of a representative AA-CHP 
(FIG. 12). All of the AA-CHP were roughly equivalent with respect to their 
ability to inhibit 3H-Thymidine incorporation, and this inhibition 
increased for each compound as the concentration was increased (FIG. 11). 
As shown in FIG. 12, no inhibition of .sup.3 H-thymidine into DNA was 
achieved by Leu-CHP if the L1210 cells were exposed to .sup.3 H-thymidine 
and Leu-CHP at the same time. As the time of pre-incubation with Leu-CHP 
or its dose was increased, the degree of inhibition of .sup.3 H-thymidine 
incorporation also increased suggesting that either uptake or hydrolysis 
of Leu-CHP was rate limiting. 
Whereas the AA-CHP's required pre-incubation with L1210 cells in order to 
achieve inhibition of DNA synthesis, the MPD's achieved inhibition of 
.sup.3 H-thymidine incorporation in the absence of pre-incubation (i.e., 
during the half-hour the L1210 cells were exposed to both MPD's and 
3H-Thymidine (FIG. 13). While Leu-CHP-BSA was more effective during the 
first 4 hours of incubation, after 6 hours, there were no differences in 
inhibition (83%) between Boc-Leu-CHP-BSA (XVI) and Leu-CHP-BSA (XVII) at 
the concentrations shown. 
The observation that these seven structurally different compounds (5 
AA-CHP's, 2 MPD's) all inhibited 3H-Thymidine incorporation into DNA 
combined with the fact that all of these compounds are dipeptide or 
protein derivatives and not structural analogs of DNA constituents or DNA 
polymerase substrates indicated that they shared a common mechanism of 
inhibition which was quite different from such analogs. The common 
mechanism proposed here is that, upon enzymatic cleavage of the common 
hydrazide bond, toxic quinones are generated which react with protein 
(e.g., DNA polymerase-.alpha.) and non-protein bound (e.g., glutathione) 
sulfhydryl groups resulting in inhibition of sulfhydryl dependant enzymes 
and depletion of the intracellular sulfhydryl concentration. 
EXAMPLE 13 
In Vivo Toxicity and Cysteine "Rescue" Experiments 
Experiments were performed which were designed to establish the LD.sub.50 
of the compounds tested and to see whether the toxicity of these compounds 
could be reduced by exogenously administered cysteine which would increase 
the intracellular concentrations of reduced sulfhydryl groups to react 
with quinones that might be generated and hence protect the animal from 
the toxicity of the AA-CHP's. 
Female DBA/2J mice, 8 weeks old, were injected intraperitoneally with test 
compound solutions (X, XI, XII, XIII, XIV) made up by dissolving the 
compound in 1N NaOH (1 ml), adding 1N HCl (approximately 1 ml) to pH 7-8 
and diluting with PBS (pH 7.4) to give a final concentration of 30 mg/ml. 
In the case of compounds XVI and XVII, they were first dissolved in 1N 
NaOH and acidified gradually with 1N HCl to pH 7-8 so that the final 
maximum concentration was 30 mg/ml. Compound IVa, just prior to injection, 
was dissolved in DMSO and then added to PBS so that the final 
concentration was 20 mg/ml in 7% DMSO. The yellow solution was injected 
immediately because the compound decomposes rapidly in aqueous media. 
Cysteine hydrochloride was neutralized with 1N NaOH and diluted with PBS so 
that the final concentration was 30 mg/ml. In those groups that received 
only the test compounds, a single dose (up to 1000 mg/kg) was given i.p. 
and any toxic deaths were autopsied and hematoxylin and eosin sections 
obtained. Surviving mice were observed for thirty days. In those groups 
receiving both test compound and cysteine, cysteine (1000 mg/kg) was given 
i.p. both 1 hour prior to and 2 or 3 hours after i.p. injection of the 
test compound. In Table 3 are the results of these experiments. The data 
indicated that the most toxic AA-CHP was Phe-CHP, followed by Leu-CHP and 
that Ala-CHP, Glu-CHP and Lys-CHP were non-toxic up to 1000 mg/kg. 
Furthermore, CHP-OEt had an LD.sub.50 between 400-550 mg/kg and that 
Boc-Leu-CHP-BSA and Leu-CHP-BSA were non-toxic up to 2000 mg/kg. 
TABLE 3 
__________________________________________________________________________ 
Toxicity of AA--CHP's, CHP--OEt, and MPD's 
Dose Histologic Findings 
Compound 
##STR10## and EdemaCongestionPulmonary 
Tubular NecrosisAcute 
__________________________________________________________________________ 
Renal 
Leu--CHP 
600(5/5),750(3/3),1000(0/9) 
750-1000 
++-+++ 
+-0 
Phe--CHP 
400(3/5),600(1/7) 400 0-+ +++ 
Ala--CHP 
600(5/5),1000(6/6) 
&gt;1000 
Lys--CHP 
600(5/5),1000(6/6) 
&gt;1000 
Glu--CHP 
600(5/5),1000(6/6) 
&gt;1000 
CHP--OEt 
400(3/3),550(0/4) 400-550 
+++ 0 
XVI 2000(6/6) &gt;2000 
XVII 2000(6/6) &gt;2000 
__________________________________________________________________________ 
0 = absent 
+ = mild 
++ = moderate 
+++ = severe 
Phe-CHP was the most toxic of all the compounds tested and 6 of 7 animals 
receiving an i.p. dose of 600 mg/kg died within 36-44 hours after 
injection. Histologic examination of these animals revealed very 
consistent findings from animal to animal. Phe-CHP resulted in severe 
acute renal tubular coagulative necrosis (ATN). Pulmonary congestion and 
edema, as seen with Leu-CHP and CHP-OEt were minimal. Both Leu-CHP and 
CHP-OEt showed a similar histologic pattern of toxicity with each other 
but a different pattern from that of Phe-CHP. Nine of nine animals 
receiving an i.p. injection of Leu-CHP (1000 mg/kg) and four of four 
animals receiving an i.p. injection of CHP-OEt in 7% DMSO (550 mg/kg) died 
within 8 hours of treatment. All animals showed moderate to severe 
pulmonary congestion and edema with an eosinophilic protein transudate 
present in some alveoli. While these histologic changes could be due to 
left heart failure caused by myocardial toxicity of the administered 
compounds careful light microscopic examination of the heart revealed no 
evidence of a toxic insult. In mice treated with Leu-CHP, one could also 
see early signs of ATN but these changes were not nearly as striking as in 
the case of Phe-CHP. None of the other AA-CHP's caused even a transient 
illness in any of the mice after intraperitoneal injection. 
The MPD's (XVI and XVII) were non-toxic at doses up to 2000 mg/kg. This 
dose had to be injected in two doses of 1000 mg/kg each three hours apart 
since the maximum solubility of the MPD's in H.sub.2 O at pH 7-8 was 30 
mg/ml. Since these compounds are macromolecules and since it was expected 
that the cells of the reticulo endothelial system of the liver and spleen 
would ingest these compounds by pinocytosis, evidence of splenic toxicity 
was sought in 5 mice receiving 2000 mg/kg of Leu-CHP-BSA (XVII) i.p. and 
compared with that in 5 mice receiving 2000 mg/kg BSA i.p. The mice were 
sacrificed two days after injection and their spleens removed and weighed. 
There was no statistically significant difference between the two groups. 
When 3 mice given a 600 mg/kg i.p. dose of Phe-CHP (LD.sub.100) were 
pretreated 1 hour before with a 1000 mg/kg i.p. dose of cysteine and 
post-treated 3 hours after with 1000 mg/kg i.p. dose of cysteine, none of 
the cysteine-treated mice died while 6 of 7 given Phe-CHP died within 44 
hours. When the same experiment was performed using Leu-CHP (1000 mg/kg 
i.p., LD.sub.100) only one of three treated mice died and that was on Day 
3, not within 8 hours as was typical of all 9 control mice. Histologic 
sections of the cysteine and Leu-CHP treated mouse that died on Day 3 
showed no evidence of pulmonary congestion or edema. 
From these data, summarized in Table 4, it is clear that cysteine was 
effective in protecting mice from the lethal toxicity of both Phe-CHP and 
Leu-CHP attesting to the sulfhydryl reactivity of the hydrolytic products 
of these two compounds. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein. 
TABLE 4 
______________________________________ 
Effect of Cysteine on Toxicity of Phe--CHP and Leu--CHP 
Compound 
ineCyste- 
Dose 
##STR11## DeathTime of 
______________________________________ 
Leu--CHP 
No 1000 mg/kg 
9/9 &lt;8 hours 
Leu--CHP 
Yes.sup.a 
1000 mg/kg 
1/3 Day 3 .COPYRGT. 
Phe--CHP 
No 600 mg/kg 
6/7 36-44 hours 
Phe--CHP 
Yes.sup.b 
600 mg/kg 
0/3 
______________________________________ 
.sup.a 1 injection (1000 mg/kg) given 1 hour prior to treatment with 
Leu--CHP, 1 injection (1000 mg/kg) given 2 hours after treatment. All 
injections were given i.p. 
.sup.b 1 injection (1000 mg/kg) given 1 hour prior to treatment with 
Phe--CHP, 1 injection (1000 mg/kg) given 3 hours after treatment. All 
injections were given i.p. 
.COPYRGT. Autopsy H & E Sections showed no evidence of pulmonary 
congestion and edema.