CA1158557A

CA1158557A - Hydrolytic enzyme-activatible pro-drugs

Info

Publication number: CA1158557A
Application number: CA000362379A
Authority: CA
Inventors: Philip L. Carl; Michael J. Weber; John A. Katzenellenbogen
Original assignee: University of Illinois Foundation
Current assignee: University of Illinois Foundation
Priority date: 1979-10-18
Filing date: 1980-10-15
Publication date: 1983-12-13
Also published as: WO1981001145A1; EP0038357A4; EP0038357A1

Abstract

ABSTRACT OF THE DISCLOSURE
Antineoplastic agents are rendered tumor-specific by derivatization with a peptide specifier so as to convert the antineoplastic agent into a pharmacologically inactive pro-drug which is selectively activatible at the tumor site. The peptide specifier has an amino acid residue sequence such that it will be selectively enzymatically cleaved from the antineoplastic agent by tumor-associated fibrinolytic and/or blood-coagulating proteases, such as plasmin and plasminogen activator, so as to effect release of the antineoplastic agent in pharmacologically active form in the vicinity of the tumor. These and other similar hydrolytic enzyme-activatible pro-drugs may be formed with their specifier moiety and their drug moiety covalently linked together through an intermediate self-immolative connector moiety having a molecular structure such that enzymatic cleavage of the bond covalently linking it to the specifier moiety will initiate spontaneous cleavage of the bond covalently linking it to the drug moiety to thereby effect re-lease or the drug in pharmacologically active form.

Description

~5~7 Description HYDROLYTIC ENZYME~ACTIVATIBLE PRO-DRUGS

BACKGROUND OF THE_INVENTION
This invention relates to hydrolytic enzyme-activatible pro-drugs and, in particular, to tumor-specific pro-drugs of antineoplastic agents which are selective substrates for drug-activating enzymatic cleavage by tumor-associated proteases.
One approach to improving the efficiency of drug action and the selectivity of drug delivery is to prepare a reversible derivative of a drug which is itself pharmacologically inactive, but which becomes activated in vivo to liberate the parent drug, typically, but not necessarily, by enzymatic attack. A drug derivative of this type, commonly known a~ a "pxo-drug" or a "latentiated drug", can be tailored to over-come certain undesirable properties of the parent drug, such as, for example, bitterness or tartness, offensive odor,gastric or intestinal upset and irritation, pain on injection, lack of absorption, slow or rapid metabolism, or lack of stability in the bulk state, the dosage form, or in vivo; or it can be designed to be activated selectively at the site of ; intended action, so that undesired effects can be lessened.
The present invention is primarily concerned with pro-drugs of this latter type, which are selectively activatible at the site of intended action, and, in particular, to pro-drugs of antineoplastic agents which are selectively activatible at the tumor site.

-2- l~S~S57 One aspect of the present invention, however, is more broadly applicable to pro-drugs in general, as will become more readily apparent hereinbelow.
- Many of the antineoplastic agents currently being used in cancer chemotherapy rely for their ef~ectiveness on being selectively cytotoxic to rapidly proliferating cells. In addition to malignant cells, however, certain normal cells are also rapidly pro-liferating, such as, for example, bone marrow and spleen cells. For this reason bone marrow and spleen toxicity are often limiting factors in the effectiveness of such antineoplastic agents in cancer chemotherapy. One approach in trying to overcome this problem is to design a pro-drug of the antineoplastic agent which will be selectively activatible at the tumor-site, for example, by being a selective substrate for drug-activating enzymatic cleavage by a tumor-associated enzyme.
In order for a pro-drug of this type to be useful in cancer chemotherapy, there are several criteria which it must meet. First of all, there must be enough of the activating enzyme in the tumor to generate cytotoxic levels of free drug in the vicinity of the tumor. Secondly, there must be means available to minimize activation of the pro-drug at sites dis-tant from the tumor, and to mitigate the effects ofsuch activation if it occurs. This criterion is clearly related to the first one, since it is the rela-tive amount of tumor-associated and extra-tumor en-zymatic activity which is critical for selectivity.
Thirdly, the pro-drug must be a suitable substrate for the tumor-associated enzyme under physiological con-ditions and a poor substrate for other enzymes.
Fourthly, the pro-drug must be considerably less toxic ;- than the activated drug, i.e., at least on the order of ten times less active and preferably on the order of a hundred or a thousand times less active. Finally, the .
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3 ~58SS7 activated species must have a reasonably short biological ' half-life so that the toxic effects of the locally activated drug are limited to the tumor and selectivity is not lost by diffusion of the drug away from the site of activation.
, A number of attempts have previously been made to develop tumor-specific pro-drugs of antineoplastic agents activatible by tumor-associated enzymes. However, such previous attempts have met with very limited success primarily due to a failure of such pro-drugs to meet one or more of the five criteria set forth above.
Another consideration in the design of hy-drolytic enzyme-activatible pro-drugs, in general, is the problem sometimes posed by the nature of the drug molecule being derivatized. If the drug molecule is ; large or has pronounced polar or apolar character, steric or electronic factors at the intended cleavage site could interfere with the enzymatic cleavage re-action and thereby prevent the pro-drug from being a suitable substrate for the target enzyme.
, It has previously been reported that many animal and human tumors exhibit elevated levels of fibrinolytic and blood-coagulating enzyme activity and, in -~ 25 particular, elevated levels of the fibrinolytic enzymes, plasmin and plasminogen activator. Both plasmin and plasminogen activator are proteases with trypsin-like specificity in the sense that they both cleave next to basic amino acids. Substantial infor-mation exists concerning the specificity of plasmin and plasminogen activator, based on the use of arti-ficial substrates as well as analysis of the peptide bonds cleaved in the natural substrate. Plasminogen activator shows considerable substrate specificity towards its natural substrate, plasminogen, in which , -:~
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~L~585S7 a single Arg-Val bond is cleaved in converting plas-minogen to plasmin. Plasmin is often regarded as a rather unspecific, trypsin-like protease. However, it cleaves a limited number of bonds in dissolving a fibrin clot. Examination of the plasmin cleavage sites in its natural substrate, fibrin, reveal that eleven of the fifteen earliest cleavages are at lysine - residues, and in fifteen of the twenty earliest cleavages (including all of the earliest nine cleavages) a hydrophobic amino acid precedes the lysine or arginine. Hence, the implication is that plasmin is selective for lysine residues preceded by a hydrophobic amino acid residue.
The elevated levels of fibrinolytic and blood-coagulating enzymes found in many tumor cells and the substrate specificity of these proteases have not previously been exploited in the design of tumor-specific pro-drugs of antineoplastic agents. While it is true that various normal cells and tissues, including the lung, kidney, squamous epithelium and activated macrophages, also exhibit elevated levels of these proteases, such normal cells by and large are not rapidly proliferating, and thus should not be highly sensitive to the cytotoxic effects of a DNA synthesis inhibitor released in their vicinity.
On the other hand, at least two major sites of high normal cell proliferation, l.e., the bone marrow and spleen, have been reported to be low in fibrinolytic and blood-coagulating enzyme activity. Hence, the specific combination of rapidly proliferating cells exhibiting high levels of fibrinolytic and blood-coagulating enzyme activity appears to be a characteristic possessed by a great many tumor cells but generally not possessed by normal cells.

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SUMMARY OF THE INVENTION
It is, accordingly, a primary object of the present invention to provide a pro-drug of an antineoplastic agent which is selectively activatible at the site of the tumor.
Another object of the invention is to pro-vide a tumor-specific pro-drug of an antineoplastic agent in accordance with the preceding object, which ; is a highly selective substrate for drug-activating enzymatic cleavage by one or more tumor-associated hydrolytic enzymes.
; A further object of the invention is to provide a tumor-specific pro-drug of an antineoplastic agent in accordance with the preceding objects, 15 wherein the activating enzyme is one which is pre-sent in the tumor in sufficient amounts to generate cytotoxic levels of free drug in the vicinity of the tumor.
Still another object of the invention is to 20 provide a tumor-specific pro-drug of an antineo-plastic agent in accordance with the preceding ob-jects, wherein the activating enzyme is one whose presence at sites distant from the tumor is insuf-ficient to generate cytotoxic levels of free drug in 25 the vicinity of such distant sites.
A still further object of the present invention - is to provide a tumor-specific pro-drug of an antineoplastic agent in accordance with the preceding objects, which is considerably less toxic than the Ç 30 activated drug.
Yet another object of the present invention is to provide a tumor-specific pro-drug of an anti-neoplastic agent in accordance with the preceding ob-jects, wheréin the activated drug has a reasonably , 35 short biological half-life so that the cytotoxic ef-', .

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-6- ~585S7 fects of the locally activated drug are limited to the tumor and selectivity is not lost by diffusion of ; the drug away from the site of activation.
A yet further object of the present invention is to provide hydrolytic enzyme-activatible pro-drugs, including those of the type set forth in the preceding objects, which include connector means for spacing the drug-activating enzymatic cleavage site sufficiently far away from the drug molecule so as to prevent steric and/or electronic interference with the enzymatic cleavage reaction, which connector means does not in itself prevent release of the free drug in pharmacologically active form following the enzymatic cleavage reaction.
The above and other objects are achieved in accordance with the present invention by derivatizing an antineoplastic agent with a peptide specifier at a reactive site appropriate for inhibiting the pharmacological activity of the antineoplastic agent, to thereby convert the antineoplastic agent into a - pharmacologically inactive peptidyl derivative pro-drug. The peptide specifier has an amino acid resi-due sequence specifically tailored so as to render the peptidyl derivative a selective substrate for drug-activating enzymatic cleavage by one or more tumor-associated fibrinolytic and/or blood-coagulating proteases, such as plasmin and plasminogen activator.
The enzymatic cleavage reaction will remove the pep-tide specifier moiety from the pro-drug and effect ,~ 30 release of the antineoplastic agent in pharmacologically active form selectively at the tumor site.
In those instances where the drug molecule is ,~ large and/or has pronounced polar or apolar character, steric and/or electronic interference of the enzymatic cleavage reaction is avoided in accordance with the present !
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- ~5~3557 .

invention by forming the peptidyl derivative pro-drug with its peptide specifier moiety and its antineoplastic agent moiety covalently linked together through an intermediate self-immolative connector moiety having a molecular structure such that enzymatic cleavage of the bond covalently linking it to the peptide specifier - moiety will initiate spontaneous cleavage of the bond covalently linking it to the antineoplastic agent moiety to thereby effect release of the antineoplastic agent in pharmacologically active form. The intermediate self-immolative connector aspect of the present invention is not limited in its application to protease-activatible pro-drugs of antineoplastic agents, but is equally applicable to a variety of other types of hydrolytic enzyme-activatible pro-drugs wherein steric and/or electronic hindrance by the drug molecule might otherwise interfere with the drug-activating enzymatic cleavage re-action. Moreover, the self-immolative connector aspect of the present invention may also be used to impart to the pro-drugs greater stability towards undesired hydrolytic processes, both enzymatic and spontaneous, and/or optimal pharmacokinetic properties without needing to chemically modify either the specifier or '~ the drug themselves.
In vitro tests thus far carried out on several protease-activatible peptidyl derivative pro-drugs of 7 antineoplastic agents in accordance with the present invention, show a five- to seven-fold improvement over the underivatized parent drug in selective cytotoxic activity against malignant cells exhibiting elevated levels of fibrinolytic enzyme activity versus well-matched (displaying similar good sensitivity to the free drug) j~ normal cells not exhibiting such levels of fibrinolytic enzyme activity. These results are indicative of the fact that the peptidyl derivative pro-drugs of the present invention are selective substrates for drug-activating 1 ' , ~

~8--enzymatic cleavage by tumor-associated fibrinolytic enzymes and are selectively activatible to release cytotoxic levels of pharmacologically active drug at - sites exhibiting elevated levels of such fibrinolytic enzyme activity. Since normal tissues exhibiting such elevated levels of fibrinolytic enzyme activity are, for the most part, limited to those having a low percentage of replicating cells, peptidyl derivative pro-drugs of antineoplastic agents which are cyto-toxic predominantly to rapidly proliferating cellsin accordance with the present invention, should be selectively cytotoxic to those malignant cells which exhibit the specific combination of properties of being rapidly proliferating and exhibiting elevated - 15 levels of fibrinolytic enzyme activity.
DESCRIPTION OF PREFERRED EMBODIMENTS
~, It will be understood that in the following de-tailed description and appended claims, the abbre-,~ viations and nomenclature employed are those which are standard in amino acid and peptide chemistry, and that all amino acids referred to are in the L-form unless otherwise specified.
The hydrolytic enzyme-activatible pro-drugs in accordance with the present invention may be broadly ~' ~ 25 described as having a molecular structure comprised of ',' a drug moiety and a specifier moiety. The specifier moiety, by means of its chemical structure, targets the pro-drug to one or more species of hydrolytic enzymes, and renders the pro-drug a selective substrate for drug-activating enzymatic cleavage by the target hydrolytic enzyme. The drug moiety and the specifier moiety are covalently linked together either directly to form a bipartate molecular structure, or through an intermediate self-immolative connector moiety to form a tripartate ', molecular structure. In either case, the covalent ., f,4?.~ --,,~
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~5~557 g linkage between the moieties will be such that the drug moiety is rendered pharmacologically inactive, the site of the drug-activating enzymatic cleavage will be at the bond covalently linking the specifier moiety to its immediately adjacent moiety, and the drug-activating enzymatic cleavage will effect release of the drug moiety in pharmacologically active form. The intermediate self-immolative connector moiety, when employed in the pro-drug molecule, has a molecular structure such that the drug-activating enzymatic cleavage of the bond covalently linking it to the specifier moiety will initiate spontaneous cleavage of the bond covalently linking it to the drug moiety, to thereby effect release of the drug moiety in pharmacologically active form.
The peptidyl derivative pro-drugs of antineoplastic agents in accordance with the present invention have an antineoplastic agent as their drug moiety and a peptide as their specifier moiety,and are specifically designed to be selective substrates for drug-activating enzymatic cleavage by one or more tumor-associated proteases selected from the group consisting of fib-rinolytic enzymes and blood-coagulating enzymes.
' Blood-coagulating enzymes are those which are involved in the intrinsic or extrinsic system of fibrin clot formation, and include, but are not necessarily limited to, thrombin, thromboplastin, Factor Va, Factor VIIa, Factor VIIIa, Factor IXa, Factor Xa, Factor XIa, and Factor XIIa. Fibrinolytic enzymes are those which are involved in the physiological mechanism for dissolving fibrin clots, and include plasmin and plasminogen activator.
Recent evidence suggests that all of these proteases are associated with a great many tumors, and that plasmin and plasminogen activator, in particular,are present in these . 35 tumors at elevated levels sufficient for pro-drug activation.

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~58557 In order to be suitable for conversion into a pro-drug in accordance with the present invention, the anti-neoplastic agent should be one having an unhindered chem-ically reactive site whose derivatization will inhibit the pharmacological activity of the antineoplastic agent.
Such reactive site will typically be a free amino group or a free hydroxyl group, since these groups are most readily derivatizable with peptides. However, where an intermediate self-immolative connector is employed in forming the pro-drug, the reactive site for derivatiza-tion of the antineoplastic agent may also be a free sulf-hydryl group. A number of known antineoplastic agents meet the above requirements, including, for example, cytosine arabinoside, adriamycin,daunomycin,6-thioguanine, fluorodeoxyuridine, bis-(2-chloroethyl) amine, phenylene-diamine mustard, 3'-aminothymidine, L-alanosine, 2-amino-thiodiazole, 1,4-dihydroxy-5,8-bis(2-aminoethylamino)-9, 10-anthracenedione, O NH2 l C1 -CH-COOH (AT-125) and HO-C-CH-CH2CH2-C-CH=N--N (DON).
The peptide specifier employed for derivatizing the antineoplastic agent so as to convert it into a tumor-specific pro-drug in accordance with the present invention, has an amino acid residue sequence specifically tailored so that it will be selectively enzymatically cleaved from the resulting peptidyl derivative pro-drug by one or more of the tumor-associated fibrinolytic and/or blood-coagulating proteases. Examination of the cleavage sites in the natural substrates for these proteases provides a basis for choosing appropriate amino acid residue sequences for the peptide specifier. Since at least most of the fibrinolytic and blood-coagulating proteases appear to have in common a relatively high degree of specificity toward cleavage sites in their natural substrates which have a ` ~

~ ~58557 ~11-basic amino acid residue on the carboxyl side thereof, it is preferred to form the peptide specifier with a basic amino acid residue in its C-terminal position, and to carry out the derivatization of the antineoplastic agent with the C-terminus of the peptide specifier.
Suitable basic amino acid residues for use as the C-terminal amino acid residue of the peptide specifier include lysine, arginine, histidine, ornithine, and citrulline, with lysine and arginine being particularly preferxed.
The amino acid residue in the position immediately adjacent to the C-terminal amino acia residue also appears to play a significant role in imparting the desired protease-specificity to the peptide specifier. Such penultimate amino acid residue is preferably a hydrophobic amino acid residue or glycine. Suitable hydrophobic amino acid residues include alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline. Alanine, leucine and glycine are particularly pref~rred amino acid residues for use in such penultimate position of the peptide specifier.
For facilitating preparation of the pro-drug and enhancing its stability against undesired hydrolytic processes, the N-terminal amino acid residue of the peptide specifier is preferably a D-amino acid residue, a protected L-amino acid residue, or protected glycine.
Suitable protecting groups are well known in the art of peptide chemistry, and include,for example carbobenzoxy (CBZ), t-butoxycarbonyl (Boc), p-toluene sulfonyl, and benzoyl, with CBZ being particularly preferred. Most preferably, the N-terminal amino acid residue is a D-amino acid residue, such as, for example, D-valine or D-isoleucine, since this provides the peptide specifier with better solubility properties than with the protected L-amino acid residue or protected glycine.

-12- ~ ~58557 The amino acid residue chain length of the peptide specifier preferably ranges from that of a tripeptide to that of a pentadecapeptide. It will be understood, however, that peptide specifiers as short as dipeptides and longer than pentadecapeptides may also suitably be employed.
With the foregoing basic considerations serving as a general guideline, numerous specific peptide specifier molecules suitable for use in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular one of the tumor-associated fibrinolytic and blood-coagulating proteases. Based upon the information which is presently available in regard to the cleavage site specificities and the tumor-associated concentrations of these proteases, the presently preferred peptide specifiers for use in the present invention are those which are optimized toward the fibrinolytic proteases, plasmin and plasminogen activator. Its high degree of cleavage site specificity makes plasminogen activator a particularly attractive target protease from the standpoint of designing pro-drugs with optimal selectivity.
On the other hand, since one plasminogen activator molecule is capable of converting numerous molecules of plasminogen to plasmin, plasmin will generally have a substantially greater tumor-associated concentration than plasminogen activator and, notwithstanding its lower degree of cleavage site specificity, may be more likely to provide a target large enough to generate pharmacologically significant concentrations of the antineoplastic agent from the pro-drug. In any event, both plasminogen activator, due to its high degree of cleavage site specificity, and plasmin, due to its high tumor-associated concentration, appear to be the target proteases of choice in determining optimal amino acid residue sequences for the :

~58SS7 peptide specifier under the aforementioned general guidelines.
In the peptide specifiers optimized toward plasmin as the target protease, the C-terminal amino acid residue is preferably lysine, the amino acid residue in the position immediately adjacent to the C-terminal amino acid residue is preferably leucine, and the N-terminal amino acid residue is preferably D-valine or D-isoleucine.
Specific examples of this preferred embodiment of peptide specifiers include the tripeptides D-Val-Leu-Lys and D-Ile-Leu-Lys, and the tetrapeptides D-Val-Ser-Leu-Lys and D-Ile-Ser-Leu-Lys.
In the peptide specifiers optimized toward plasminogen activator as the target protease, the amino acid residue sequence preferably substantially mimics the amino acid residue sequence on the carboxyl side of the Arg-Val bond in plasminogen which serves as the site of cleavage of plasminogen by plasminogen activator, with the C-terminal amino acid residue preferably being arginine, and the amino acid residue in the position immediately adjacent to the C-terminal amino acid residue being glycine. Specific examples of this preferred em-bodiment of peptide specifiers include the tripeptide CBZ-Pro-Gly-Arg, the tetrapeptide CBZ-Cys-Pro-Gly-Arg, R

the pentapeptide CBZ-Lys-Cys-Pro-Gly-Arg, and the hexapeptide cBz-Lys-Lys-cys-pro-Gly-Arg. In addition, R
CBZ-Gly-Gly-Arg is a suitable preferred specifier.

.
Optimization of the peptide specifier toward one or more of the blood-coagulating enzymes as the target tumor-associated protease may similarly be accomplished by choosing an amino acid residue sequence in accordance with the aforementioned general guidelines, but which substantially mimics the amino acid residue sequence on the carboxyl side of the cleavage site in the appropriate natural or known artificial substrates for the particular enzyme. Examples of such substrates i 10 are disclosed by Claeson, et al, "Substrate Structure and Activation Relationship"j appearing in New Methods For the Analysis of Coagulation Using Chromogenic Sub-strates, Ed. I. Witt, ~alter de Gruyter, Berlin, New York, Pa~es 37-54 (1977).
Representative peptide specifiers within the scope of the present invention and optimized 'oward thrombin as the target protease, include the tripeptides p-toluene sulfonyl-Gly-Pro-Arg and benzoyl-Phe-Val-Arg.
A representative peptide specifier in accordance with the present invention and optimized toward Factor Xa as the target protease is the tetrapeptide benzoyl-Ile-Glu-Gly-Arg.
In the preferred procedure for synthesizing the peptidyl derivative pro-drugs in accordance with the present invention, the peptide specifier is first separately prepared with its C-terminus in the free acid form, and with all of its other reactive groups suitably blocked. Synthesis of the peptide specifier may be carried out by standard peptide synthesis techniques well known in the art, including either solution-phase or solid-phase methods. Particularly where the peptide being synthesized is of relatively short chain length, the solution-phase methods offer certain advantages in that the peptide is directly prepared in the blocked form needed for the subsequent derivatization of the drug, and the intermediates in the synthesis can be purified, .
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-15- ~58557 insuring product peptide purity. If solid-phase methods are employed, various known techniques may be used for the removal of the blocked peptide from the resin, for example, by using either photocleavable attachment linkages, or by acyl transfer with 2-dimethylaminoethanol followed by hydrolysis.
If the antineoplastic agent being converted into a pro-drug contains more than one reactive site on its molecule, those reactive sites other than the one being derivatized may be suitably protected prior to the derivatization reaction. Any of the conventional protecting groups well known in the art may suitably be used for this purpose. For example, in derivatizing the 5'-hydroxyl group of cytosine arabinoside, the

4-amino group of the base may suitably be protected as the Schiffs base using dimethylformamide dimethyl ketal or diisopropylformamide dimethyl ketal, with the latter providing more favorable organic solubility characteristics important in attaining good product recoveries. Deprotection following the derivatization reaction may be effected with trifluoroacetic acid.
Another instance where protecting groups might be used to achieve more favorable organic solubility char-acteristics would be in the derivatization of the 2-amino group of 6-thioguanine, wherein benzylation of the 6-thio group and at the N4 position will increase the solubility of the parent drug. Deprotection of S and N benzyl protecting groups following the de-rivatization reaction can be accomplished by treatment with sodium in liquid ammonia.
In preparing the bipartate peptidyl derivative pro-drugs in accordance with the present invention, the peptide specifier, with its C-terminus in the free acid form, and with all of its other reactive groups suitably blocked, is directly reacted with a carboxyl-reactive site of the antineoplastic agent whose derivatization inhibits pharmacological activity, to thereby form a direct covalent linkage between the C-terminus of the peptide specifier and said carboxyl-reactive site of the antineoplastic agent. Such covalent linkage will either be an amide linkage, i.e., when the carboxyl-reactive site is a free amino group; or an ester linkage, i.e., when the carboxyl-reactive site is a free hydroxyl group. Where a choice is available between synthesizing the pro-drug with either an amide linkage or an ester linkage, amide linkages are generally preferred in view of the fact that ester-linked pro-drugs tend to lose at least some selectivity due to hydrolysis by non-specific esterases.
Standard ester-forming and amide-forming techniques well known in the art may be used for carrying out the derivatization reaction. For example, the amide-linked derivatives may suitably be prepared via a mixed anhydride reaction with the aid of isobutyl chloroformate and either triethylamine or N-methyl morpholine in a suitable solvent such as dimethylformamide or dioxane/tetrahydrofuran. Following the derivatization reaction, the protecting groups are removed, for example, by treatment with trifluoroacetic acid in methylene chloride, to yield the desired peptidyl derivative pro-drug.
The tripartate pro-drugs in accordance with the present invention employ an intermediate self-immolative connector moiety which spaces and covalently links together the drug moiety and the specifier moiety.
Since the self-immolative connector aspect of the present invention is believed to be a novel concept in the design of hydrolytic enzyme-activatible pro-drugs in general, it will be described, first of all, in terms of its broader applications, and thereafter, !

~5~35S7 as it more specifically relates to the peptidyl deriva-tive pro-drugs of antineoplastic agents in accordance with the present invention.
In its broadest sense, a self-immolative connector may be defined as a bifunctional chemical moiety which is capable of (1) covalently linking together two spaced chemical moieties into a normally stable tripartate molecule; (2) releasing one of said spaced chemical moieties from the tripartate molecule by means of an enzymatic cleavage; and (3) following said enzymatic cleavage, spontaneously (i.e., non-enzymatically) cleaving from the remainder of the molecule to release the other of said spaced chemical moieties. As applied to the design of hydrolytic enzyme-activatible pro-drugs in accordance with the present invention, the self-immolative connector is covalently linked at one of its ends to the specifier moiety and covalently linked at its other end to the reactive site of the drug moiety whose derivatization inhibits pharmacological activity, so as to space and covalently link together the specifier moiety and the drug moiety into a tripartate molecule which is stable and pharmacologically inactive in the absence of the target hydrolytic enzyme, but which is enzymatically cleavable by such target hydrolytic enzyme at the bond covalently linking the connector moiety to the specifier moiety to thereby effect release of the specifier moiety from the tripartate molecule. Such enzymatic cleavage, in turn, will activate the self-immolating character of the connector moiety and initiate spontaneous cleavage of the bond covalently linking the connector moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.

~58557 A self-immolative connector offers several potential advantages in hydrolytic enzyme-activatible pro-drug design. First of all, in those instances where the drug molecule being derivatized is large and/or has pronounced polar or apolar character, a bipartate pro-drug formed by a direct linkage of the specifier moiety to the drug moiety may not be a suitable substrate for the target hydrolytic enzyme due to steric and/or electronic hindrance at the intended enzymatic cleavage site caused by the close proximity of such site to the drug molecule.
By inserting an intermediate self-immolative connector moiety between the specifier moiety and the drug moiety, the drug-activating enzymatic cleavage site may be spaced sufficiently far away from the drug molecule so as to prevent steric and/or electronic interference with the enzymatic cleavage reaction, and without the connector itself preventing release of the free drug in pharmacologically active form following the enzymatic cleavage reaction. This should allow construction of many more classes and types of hydrolytic enzyme-activatible pro-drugs. Secondly, by varying the functionality of the drug-derivatizing end of the self-immolative connector from that of the specifier, the self-immolative connector may pro-vide greater versatility in the type of linkage used for derivatizing the drug, and may enable linkages which are more stable towards undesired hydrolytic processes (both enzymatic and spontaneous) than are the direct specifier-drug linkages. Thirdly, by providing the self-immolative connector with numerous sites for chemical substitution, it should be possible to design tripartate pro-drugs with optimal pharmacokinetic properties without needing to chemically modify either the specifier or the drug -9 ~ ~51~5S~

themselves. This should allow the specifier and drug to be individually optimized for their own particular tasks, e.g., the specifier might be optimized as a substrate for the desired hydrolytic enzyme and made relatively resistant to hydrolysis by undesired hydrolytic enzymes, and the drug might be optimized for specific inhibition of some target enzyme.
A connector moiety which has been found to have all of the above-described characteristics rendering it particularly suitable for use as a self-immolative connector, and the manner in which it is employed in the design of hydrolytic enzyme-activatible tri-partate pro-drugs in accordance with the present invention, may be represented by the following general formula:

Specifier-~4- ~ -C-O-C-Drug (I) wherein Rl is hydrogen or one or more substituent groups which are either electron-donating groups or electron-withdrawing groups; R2 and R3 may be the same or different and are each selected from the group consisting of hydrogen, alkyl, phenyl, and phenyl substituted with either electron-donating groups or electron-withdrawing groups; R4 is NH or O; when R4 is NH, the specifier moiety is selected from the group consisting of a peptide, an amino acid, a carboxylic acid, and phosphoric acid; when R4 is O, the specifier moiety is selected from the group consisting of a carboxylic acid, phosphoric acid, and sulfuric acid;
and the drug moiety is a normally pharmacologically active agent having a reactive site whose derivatization inhibits pharmacological activity, said reactive site being selected from the group consisting of a ., , ~ .
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~58557 -free amino group, a free hydroxyl group and a free sulfhydryl group, the covalent linkage between said drug moiety and its adjacent carbonyl group being at said reactive site so as to inhibit the pharmacological activity of said drug moiety.
In the above definitions of Rl, R2 and R3, any of the common electron-donating and electron-withdrawing groups well known in the art may suitably be employed. By way of example suitable electron-'~10 donating groups include -NH2, -OH, -OCH3, -NHCOCH3, -C6H5, and -CH3; and suitable electron-withdrawing groups include -NH (CH3)3, -NO2, -CN, -SO3H, -COOH, -CHO, -COR, -Cl, -Br, -I, and -F. In the above definitions of R2 and R3, the alkyl group will generally be a lower alkyl group but may, if desired, be of longer chain length, for example, up to about 15 carbon atoms. Preferred embodiments of the connector moiety are those in which Rl is hydrogen, R2 is hydrogen or methyl, and R3 is hydrogen or methyl.
In Formula I, above, the specifier moiety and '; the R4 group together constitute a substrate recognition site for a particular class of target hydrolytic enzymes, which is dependent upon the specific combination of specifier moiety and R4 group selected. The various classes of target hydrolytic enzymes for each specific combination of specifier moiety and R4 group within the Formula I definitions set forth above, are listed in Table I, below.
Table I
30 Specifier Moiety R Target Hydrolytic Enzyme Peptide or amino acid NH Peptide hydrolase (protease) Carboxylic acid NH Amidase Phosphoric acid NH Phosphoramidase Carboxylic acid O Carboxylic ester hydrolase 35 Phosphoric acid O Phosphoric monoesterase or diesterase Sulfuric acid O Sulfuric ester hydrolase ,,~ .
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"

." , ~ -~ ' ~8557 The carbonate end of the connector moiety enables . derivatization of either a free amino group (by forming acarbamate linkage), a free hydroxyl group (by forming a mixed carbonate linkage), or a free sulfhydryl group (by forming a mono-thio mixed carbonate linkage) on a drug molecule. A wide variety of different types of pharmacologically active agents having one or more of such reactive groups on their molecule will have their pharmacological activity inhibited by derivatization of such reactive groups, and hence would be suitable for use as the drug moiety - of Formula I. Representative pharmacologically active agents falling in this category, in addition to the various antineoplastic agents previously listed, are set forth, together with their respective types of pharmacological activity and derivatizable sites for inhibition thereof, in Table 2, below.
Table 2 Pharmacological Derivatizable Drug Activity ~ite Fluocinonide Anti-psoriasas cll_oH
- Betamethasone Anti-rheumatiod OH
arthritis Amantadine Antiviral 2 25 Aminocaproic acid Hemostatic NH2 Oxytocin Labor-inducing NH2 Isoniazid Antibacterial, NH2 antitubercular Cycloserine Antibacterial NH2 30 Methyl dopa Antihypertensive NH2 Methimazole Antithyroid hormone SII
Anileridine Analgetic NH2 Mestranol Estrogen Cl7 Phenelzine Antidepressant NH2 35 Phenylprop- Adrenergic stimulant NH2 anolamine , , ;~
., s .
,:
:, . . .:

;
;,,, , , , . ~ ~:
.,. : .

-22- ~ 5~5~7 The theory underlying the mechanism of the self-immolative connector in accordance with the present invention, i5 explained by the following reaction - scheme:
R2 Enzymatic Specifier~R4- ~ -Cl-O-C-DrUg Hydrolysis >
R

HR4- ~ -C-O-C-Drug Spontaneous >

+ O C D~ug Spontaneous ~ /
C2 + Drug The specifier moiety and R4 together act as a group with poor electron-donating capacity. However, enzymatic cleavage of the bond between the specifier moiety and R4 by the target hydrolytic enzyme converts R4 into a strongly electron-donating group, i.e., either NH2 if R4 is NH,or OH if R4 is O. This electron donating effect greatly labilizes the benzylic bond to oxygen, which spontaneously ionizes. Spontaneous decarboxylation of the carbonate anion will then release the drug moiety in pharmacologically active form.

, .
.

~.

~58557 The overall release of the drug moiety from the tripartate pro-drug is determined by two processes, namely, (1) the rate of enzymatic hydrolysis of the bond linking the specifier moiety to R4, and (2) the rate of ionization of the bond to the - benzylic center. If Rl in Formula I is an electron-donating group, this would tend to decrease process (l) and increase process (2). On the other hand, if Rl is an electron-withdrawing group, this would tend to increase process (l) and decrease process (2).
The net effect of Rl on the rate of release of the drug moiety from the tripartate pro-drug may thus be relatively independent of whether Rl releases or withdraws electrons. The system is thus at least partially buffered against the electronic nature of Rl, at least over a certain range. This means that Rl can be chosen to be either relatively polar, for example, -NH2 or -NH (CH3)3, or relatively , non-polar, for example, -C6H5 or -CH3, in order to alter the pharmocokinetic properties of the molecule : as desired. The resulting tripartate molecules should not vary greatly in the rate of drug release once they equilibrate with the compartment where the target hydrolytic enzyme acts. Rl may thus be chosen to optimize, for example, log P(l-octanol-water partition coefficient).
! In regard to R2 and R3 in Formula I, the pre-dominant effect of these groups will be on the rate , of ionization of the bond to the benzylic center.
Electron-donating substituents on these groups-will tend to increase the ionization rate, while electron-withdrawing substituents on these groups will tend to decrease the ionization rate.

, .

~, , ,' , ., ' ' , -24- ~S85S7 The hydrolytic enzyme-activatible tripartate pro-drugs in accordance with the present invention, may be readily synthesized by, first of all, reacting the specifier with a p-substituted benzyl alcohol reactant having the general formula H-R4- ~ - C - OH ( II ) Rl ;~ wherein Rl, R2, R3 and R4 have the same meanings ' as defined above, to obtain a specifier-benzyl alcohol intermediate derivative having the general 10 formula Specifier-R4- ~ -C-OH (III) Rl R3 The specifier-benzyl alcohol intermediate derivative is then reacted with either phosgene or a chloro-formate reagent, such as pentafluorophenyl chloro-formate, pentachlorophenyl chloroformate, orp-nitrophenyl chloroformate, to form a second inter-mediate derivative. This second intermediate derivative will be either a specifier-benzyl - chloroformate intermediate derivative, if the reactant is phosgene, or a specifier-benzyl mixed carbonate intermediate derivative, if the reactant is a chloroformate reagent. In either case, such second intermediate derivative is then reacted with the reactive site of the drug whose derivatization in-- 25 hibits pharmacological activity (i.e., either a - free amino group, a free hydroxyl group, or a free sulfhy~ryl group), to obtain the pro-drug of Formula I.

, ,~ .

':

, ~58~S7 Applying the above-described general procedure to the synthesis of tripartate peptidyl derivative pro-drugs of antineoplastic agents in accordance with the present invention, the peptide specifier, as described in detail hereinabove,with its C-terminus in the free acid form and with all of its other reactive groups suitably blocked with protecting groups, is reacted at its C-terminus with the free amino group of a p-amino benzyl alcohol reactant having the general formula ~ R3 (IV~

wherein Rl, R2, R3 and R4 have the same meanings as defined above, to obtain a peptidyl benzyl alcohol having the general formula Peptide-NH- ~ _¦_OH (V) ,; Rl This reaction is preferably carried out using a suitable condensing reagent, such as for example, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ) in dimethylformamide, to avoid the necessity for protecting the benzylic alcohol function. The peptidyl benzyl alcohol is then reacted with either phosgene or a chloroformate reagent, such as pentafluorophenyl . chloroformate, pentachlorophenyl chloroformate, or p-nitrophenyl chloroformate,to convert the peptidyl benzyl alcohol into either a peptidyl benzyl chloroformate (if phosgene is used as the reactant) or a peptidyl ; benzyl mixed carbonate (if a chloroformate reagent is used as the reactant). The peptidyl benzyl chloroformate or peptidyl benzyl mixed carbonate is ; , " - . . :

-26~ 5~7 then reacted with a reactive site (a free amino group, a free hydroxyl group, or a free sulfhydryl group) of the antineoplastic agent whose derivatization inhibits pharmacological activity, to obtain a derivatization reaction product from which the pro-tecting groups are then removed, such derivatization reaction product having the general formula Peptide-NH- ~ -C-0-3-Antineoplastic Agent (VI) Rl - In preparing the peptidyl derivative pro-drugs of antineoplastic agents in accordance with the present inVentiGn~ the tripartate molecular structure of Formula VI, consisting of the peptide specifier moiety, intermediate self-immolative connective moiety, and the antineoplastic agent moiety, is particularly advantageous when the antineoplastic agent moiety is adriamycin, daunomycin, or bis-(2-chloroethyl) amine, since the molecules of these drugs are such as to tend to cause steric or electronic hindrance problems if the intended drug-activating enzymatic cleavage site is in close proximity to the drug molecule.
These problems are minimized by the spacing provided ' by the self-immolative connector moiety.
The hydrolytic enzyme-activatible pro-drugs of the present invention, whether of the bipartate or tripartate molecular structure, will generally be administered in the same manner as the parent drug, i.e., orally or parenterally, with parenteral administration, e.g., intravenous, intramuscular or intraarterial, being generally preferred in order to minimize the possibility of premature activation of the pro-drug by non-specific hydrolytic enzymes.
The dose levels of the pro-drug should be such ~85X7 as to provide the requisite dose of the free drug.
This will generally require that the pro-drug be administered in somewhat larger doses than the parent drug sufficien~ to allow for the possibility of incomplete activation of the pro-drug into the free drug.
The invention will be further illustrated by way of the following examples.
Example 1 . .
(A) Synthesis of Peptide Specifier ~oc-D-Val was condensed with Leu-OMe ester using dicyclohexylcarbodiimide (DCC) in dimethylformamide/
méthylene chloride solution. The resulting di-peptide was converted to the free acid by hydrolyzing the ester with NaOH to yield Boc-D-Val-Leu-OH.
N -CBZ-N -Boc-Lys was converted to the methyl , ester via treatment with CH2N2 and the amino group freed by hydrogenolysis over Pd/C to yield N -Boc-Lys-OMe. This latter compound was condensed with the Boc-D-Val-Leu-OH (prepared as above) via a mixed anhydride reaction with isobutylchloroformate in dimethylformamide to yield Boc-D-Val-Leu-N -Boc-Lys-OMe. The crude tripeptide was purified by column chromatography on silica gel, and the methyl ester freed by alkaline hydrolysis to yield Boc-D-Val-Leu-N -Boc-Lys-OH.
(B) Synthesis of Bipartate Pro-Drug The antineoplastic agent AT-125 having the formula ~0~ INM2,, , ,N l-CH-COOH
~1 . Cl ,, -28- ~1 ~ 8 Ss7 was derivatized at its free amino group by con-densing it with the protected peptide specifier Boc-D-Val-Leu-NE-Boc-Lys-OH (prepared as above) via a mixed anhydride reaction with isobutyl-chloroformate in dioxane/tetrahydrofuran. The - resulting product was deprotected with trifluoro-acetic acid (TFA) in methylene chloride to yield the desired pro-drug D-Val-Leu-Lys-AT-125.
A portion of this peptidyl derivative pro-drug was treated with trypsin in Tris buffer.
Two products were obtained which showed Rf on TLC silica plates corresponding to the tri-peptide D-Val-Leu-Lys-OH and the free drug AT-125.
This demonstrates that the pro-drug is a sub-strate for trypsin and, presumably, other trypsin-like proteases such as plasmin and plasminogen activator, which generally show a specificity similar to that of trypsin. A similar result was obtained with plasmin.

Example 2 , The peptidyl derivative pro-drug prepared in accordance with Example 1, was tested for its tumor-specific cytotoxic activity by means of an in vitro test procedure utilizing a cell culture system with well-matched normal and malignant cells which differed substantially in fibrinolytic enzyme (l.e., plasmin and plasminogen activator) levels but displayed similar good sensitivity to the free drug. The cell culture system employed was chick embryo fibroblasts, both normal and transformed with Rous Sarcoma virus. The transformed cells exhibit a substantially higher level of fibrinolytic enzyme ; activity than the normal cells. The test procedure was carried out as follows.

-29~ llS8SS7 Chick embryo fibroblasts, either normal ~N) or transformed with Rous Sarcoma virus (SR) were plated in 35 mm plastic dishes at an initial titer of 1.5 x - 105 cells per dish (N) or 3 x 105 cells per dish

5 ~SR) . Allowing for the difference in plating efficiencies between normal and transformed cells, this leads to similar numbers of cells per plate.
' The medium used was Dulbecco's Modified Minimal s Eagle''s Medium (DME) supplemented with 10% Tryptose-, 10 Phosphate broth', 4% calf serum, and 1~ chick serum.
Cells grow exponentially in this medium with a ~;~ doubling time of 18 to 20 hours. After 24 hours the cells were changed to DME medium containing ' 5% chick serum and lacking Tryptose-Phosphate broth and calf serum. After a further 18 hours, the ' test drugs ~which included both the peptidyl de-rivative pro-drug and, for purposes of comparison, the underivatized parent drug) were added at various concentrations for a further 5 hours. Finally, in order to measure DNA synthesis 3H-thymidine at a final concentration of 1 microcurie/ml was added to each dish for 30 minutes. The medium was removed ~' and the cells were incubated with cold 5% trichloroacetic acid for a further 15 minutes. After 3 further washes with cold trichloroacetic acid, the DNA was hydrolyzed with 10% trichloroacetic acid at 70C for two hours.
~he solubili~ed material was counted in an Omnifluor-Triton-X-100 scintillation fluid. Control experiments have demonstrated that this measurement of thymidine incorporation into DNA corresponds to cell numbers as , determined in a Coulter counter.
By means of plotting the residual 3H-thymidine incorporation as a funct.ion of drug concentration com-pared to untreated control, the ED50 (i.e., the drug concentration at which incorporation of thymidine *
Trade mark.
. ,~
" ,3~' .. . . . .. ..
':, ' . ::

''-. .

_30~ 8S57 is reduced to 50% of the untreated control) is de-termined for each of the two test drugs (i.e., the peptidyl derivative pro-drug and the corresponding underivatized parent drug) against both the normal -cells and the transformed cells. The therapeutic index of each drug (i.e., the ratio of its pharm-acological activity to its toxicity) is then determined as the ratio of its ED50 against the normal cells to its ED50 against the transformed cells. Following - 10 this procedure, the therapeutic index for the peptidyl derivative pro-drug in accordance with the present invention was determined to be 5.3, in comparison to a therapeutic index of 1.2 for the corresponding underivatized parent drug. Thus, the ,' 15 peptidyl derivative pro-drug in accordance with the present invention exhibits an approximately 5-fold improvement in therapeutic index over the corresponding underivatized parent drug.
The above-described test results are indicative of the fact that the peptidyl derivative pro-drugs , of the present invention are selective substrates for drug-activating enzymatic cleavage by tumor-associated fibrinolytic enzymes,and are selectively , 25 activatible to release cytotoxic levels of pharm-acologically active drug at sites exhibiting elevated levels of such fibrinolytic enzyme activity.

Example 3 '~ Employing a synthesis procedure similar to that described in Example 1, above, the antineoplastic agent, phenylenediamine mustard, was derivatized at its free amino group with D-Val-Leu-Lys peptide ; specifier to convert it into the peptidyl derivative pro-drug D-Val-Leu-Lys-phenylenediamine mustar~.

-31- ~58557 When tested by means of the in vitro assay described in Example 2, above, the D-Val-Leu-Lys-phenylenediamine mustard pro-drug showed a 7-fold improvement in its therapeutic index in comparison with the underivatized phenylenediamine mustard parent drug.
., .
Example 4 Employing a synthesis procedure similar to that described in Example 1, above, the antineoplastic agent, adriamycin, was derivatized at its free amino group on daunosamine with the D-Val-Leu-Lys peptide specifier to convert it into D-Val-Leu-Lys-adriamycin pro-drug. When tested by means of the in vitro assay described in Example 2, above,the D-Val-Leu-Lys-adriamycin pro-drug showed a 6-fold improvement in its therapeutic index, in comparison with the underivatized adriamycin parent drug.

Example 5 ; The feasibility of the self-immolative connector aspect of the present invention in the design of hydrolytic enzyme-activatible tripartate pro-drugs was demonstrated by means of the following model study which utilized p-nitroaniline as the "drug" because of the ease of colorimetric detection.
~, Synthesis of the tripartate pro-drug model was carried out as follows. N -t-Boc-N -TFA lysine was coupled to p-aminobenzyl alcohol using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in dimethyl-formamide. The product was purified by crystallization from ethyl acetate:ether. Next this material was reacted with p-nitrophenylisocyanate in dry pyridine to yield NC''-Boc-N -TFA-Lys-N-~_cH2o-c-N- ~-N2 -32- ~158557 which was purïfied by preparative TLC on silica.
Finally, the TFA group was removed with tetramethyl-guanidine in acetonitrile:water l:l to yield the tripartate pro-drug model having the formula N -Boc-Lys-N- ~ I ~ NO2 The final structure was characterized by NMR and IR
spectroscopy.
In order to demonstrate the possibility of self-immolation with this tripartate pro-dru~ model, it was dissolved in l ml 50 mM Bis-Tris-Cl buffer pH 6.7 ;~ at 1 mM and treated with 2 ~g of trypsin. There was an instantaneous release of p-nitroaniline as measured by the increase of optical density at 405 nm.
The rate of release of p-nitroaniline was comparable to the rate of release expected for cleavage of the Lys-anilide bond, and no lag was noted. TLC analysis . showed the expected products N~-Boc-Lys-OH, p-aminobenzyl i alcohol, and p-nitroaniline. The release of p-nitro-, aniline was blocked by prior treatment of trypsin ; 20 with tosyl-lysyl chloromethyl ketone (TLCK).
f Two similar tripartate pro-drug models were . similarly synthesized, one with one of the benzylic i hydrogens replaced by a methyl group, and the other with p-nitroaniline replaced by aniline. In both 25 cases, TLC analysis again showed that trypsin hydrolysis released the expected products-,including p-nitroaniline or aniline. This was confirmed spectrophotometrically in the case of the p-nitroaniline derivative.
The above-described model studies demonstrate 30 the feasibility under physiological conditions of the self-immolation of the intermediate connector moiety in the hydrolytic enzyme-activatible tripartate pro-drugs in accordance with the present invention.

, ~5~S57 While the fibrinolytic and blood-coagulating enzyme-activatible peptidyl derivative pro-drugs in accordance with the present invention have been described with particular reference to the preferred embodiments thereof 5 wherein the drug moiety is an antineoplastic agent, it s will be understood that the drug moiety, in either the bipartate or tripartate structure, could be any normally pharmacologically active compound which is suitably con-vertible into a pro-drug by derivatization with the r 10 peptide specifiers described above and whose site of intended action is known to exhibit elevated levels of 'i fibrinolytic and/or blood-coagulating enzyme activity.
By way of example, elevated levels of fibrinolytic and/
or blood-coagulating enzymes are normally exhibited in 15 the skin, which is the site of intended action of anti-psoriasis agents, such as fluocinonide; in the joint, which is the site of intended action of anti-arthritic agents, such as betamethasone; and in the uterus, which is the site of intended action of antifertility or 20 implantation agents such as estrogenic and progestational steroids. Any of these drugs could be suitably deriva-tized with the peptide specifiers as described above to convert them into peptidyl pro-drugs, of either the bipartate or tripartate structure, which are selective 25 substrates for fibrinolytic and/or blood-coagulating proteases so as to be selectively activatible at their site of intended action.

Claims

1. A method of rendering an antineoplastic agent tumor-specific which comprises derivatizing said antineoplastic agent either directly or through an intermediate self-immolative connector with a peptide specifier at a reactive site appropriate for inhibiting the pharmacological activity of said antineoplastic agent, said peptide specifier having an amino acid residue sequence such that it will be selectively enzymatically cleaved from said antineoplastic agent by one or more tumor-associated proteases selected from the group consisting of fibrinolytic enzymes and blood-coagulating enzymes so as to effect release of said anti-neoplastic agent in pharmacologically active form.

2. The method of Cliam 1, wherein said tumor-associated protease is a fibrinolytic enzyme selected from the group consisting of plasmin and plasminogen activator.

3. The method of Claim 1, wherein said reactive site is a free amino group, a free hydroxyl group, or a free sulfhydryl group.

4. The method of Claim 3, wherein said antineoplastic agent is selected from the group con-sisting of cytosine arabinoside, adriamycin, daunomycin, 6-thioguanine, fluorodeoxyuridine, bis-(2-chloroethyl) amine, phenylenediamine mustard, 3'-aminothymidine, L-alanosine, 2-aminothiodiazole, 1,4-dihydroxy-5,8-bis (2-aminoethylamino)-9,10-anthracenedione,

5. The method of Claim 1, wherein said derivatization is carried out either directly or through an intermediate self-immolative connector with the C-terminus of said peptide specifier, and the C-terminal amino acid residue of said peptide specifier is a basic amino acid residue.

6. The method of Claim 5, wherein said peptide specifier has an amino acid residue chain length ranging from that of a tripeptide to that of a pentadecapeptide, and its N-terminal amino acid residue is a D-amino acid residue, a protected L-amino acid residue or protected glycine.

7. The method of Claim 6, wherein said peptide specifier contains a hydrophobic amino acid residue or glycine in the position immediately adjacent to said C-terminal amino acid residue.

8. The method of Claim 7, wherein said C-terminal amino acid residue is lysine, said amino acid residue in the position immediately adjacent to said C-terminal amino acid residue is leucine, and said N-terminal amino acid residue is D-valine or D-isoleucine.

9. The method of Claim 7, wherein the amino acid residue sequence in said peptide speci-fier substantially mimics the amino acid residue sequence on the carboxyl side of the Arg-Val bond in plasminogen which serves as the site of cleavage of plasminogen by plasminogen activator, with said C-terminal amino acid residue being arginine, and the amino acid residue in the position immediately adjacent to said C-terminal amino acid residue being glycine.

10. The method of Claim 1, wherein the peptide specifier reactant in the derivatization reaction has its C-terminus in the free acid form and all of its other reactive groups suitably blocked with protecting groups, said reactive site is a free amino group or a free hydroxyl group of said antineoplastic agent, said derivatization reaction is carried out directly between said C-terminus of said peptide specifier reactant and said reactive site of said antineoplastic agent, and subsequent to said derivatization reaction said protecting groups are removed from the reaction product.

11. The method of Claim 1, wherein the peptide specifier reactant in the derivatization reaction has its C-terminus in the free acid form and all of its other reactive groups suitably blocked with protecting groups; said reactive site is a free amino group, a free hydroxyl group or a free sulfhydryl group of said antineoplastic agent; said derivatization reaction is carried out in a multi-step procedure comprising:
(a) reacting said C-terminus of said peptide specifier reactant with the free amino group of a p-aminobenzyl alcohol reactant having the general formula wherein R1 is hydrogen or one or more substituent groups which are either electron-donating groups or electron-withdrawing groups, and R2 and R3 may be the same or different and are each selected from the group consisting of hydrogen, alkyl, phenyl, and phenyl substituted with either electron-donating groups or electron-withdrawing groups, to obtain a peptidyl benzyl alcohol having the general formula (b) reacting said peptidyl benzyl alcohol with either phosgene or a chloroformate reagent to convert said peptidyl benzyl alcohol into, respectively, either a peptidyl benzyl chloroformate or a peptidyl benzyl mixed carbonate, and (c) reacting said peptidyl benzyl chloroformate or peptidyl benzyl mixed carbonate with said reactive site of said antineoplastic agent to obtain a derivatization re-action product having the general formula and subsequent to said derivatization reaction said protecting groups are removed from said derivatization reaction product.

12. The method of Claim 11, wherein R1 is hydrogen, R2 is hydrogen or methyl, and R3 is hydrogen or methyl.

13. The method of Claim 11, wherein said chloroformate reagent is selected from the group consisting of pentafluorophenyl chloroformate, pentachlorophenyl chloroformate, and p-nitrophenyl chloroformate.

14. In a method of converting a pharmacologically active drug into a hydrolytic enzyme-activatible pro-drug by derivatizing said drug at a reactive site thereof appropriate for inhibiting its pharmacological activity, with a specifier designed to be selectively enzymatically cleaved from the resulting pro-drug by a target hydrolytic enzyme so as to effect release of said drug in pharmacologically active form, the improvement consisting of spacing the specifier moiety from the drug moiety by means of an intermediate self-immolative connector moiety covalently linked at its one end to said specifier moiety and covalently linked at its other end to said reactive site of said drug moiety, said intermediate self-immolative connector moiety having a molecular structure such that the drug-activating enzymatic cleavage of the bond covalently linking it to said specifier moiety will initiate spontaneous cleavage of the bond covalently linking it to said drug moiety to thereby effect release of said drug in pharmacologically active form.

15. The method of claim 14, wherein said reactive site of said drug is selected from the group consisting of a free amino group, a free hydroxyl group, and a free sulfhydryl group,and said specifier is selected from the group consisting of a peptide, an amino acid, a carboxylic acid, phosphoric acid and sulfuric acid.

16. The method of Claim 15, wherein said resulting pro-drug has the general formula wherein R1 is hydrogen or one or more substituent groups which are either electron-donating groups or electron-withdrawing groups; R2 and R3 may be the same or different and are each selected from the group consisting of hydrogen, alkyl, phenyl, and phenyl substituted with either electron-donating groups or electron-withdrawing groups; R4 is NH or O; when R4 is NH, the specifier moiety is selected from the group consisting of a peptide, an amino acid, a carboxylic acid, and phosphoric acid; and when R4 is 0, the specifier moiety is selected from the group consisting of a carboxylic acid, phosphoric acid, and sulfuric acid.

17. The method of Claim 16, wherein the preparation of said pro-drug comprises the steps of:
(a) reacting said specifier with a p-substituted benzyl alcohol reactant having the general formula wherein R1 R2,R3 and R4 have the same meanings as defined above, to obtain a specifier-benzyl alcohol intermediate derivative having the general formula (b) reacting said specifier-benzyl alcohol intermediate derivative with either phosgene or a chloroformate reagent to convert said specifier-benzyl alcohol intermediate derivative into, re-spectively, either a specifier-benzyl chloroformate intermediate derivative or a specifier-benzyl mixed carbonate intermediate derivative; and (c) reacting said specifier-benzyl chloro-formate intermediate derivative or specifier-benzyl mixed carbonate intermediate derivative with said re-active site of said drug to obtain said pro-drug.

18. The method of Claim 17, wherein said chloroformate reagent is selected from the group consisting of pentafluorophenyl chloroformate, penta-chlorophenyl chloroformate, and p-nitrophenyl chloroformate.

19. The method of Claim 17, wherein R1 is hydrogen, R2 is hydrogen or methyl, and R3 is hydrogen or methyl.

20. The method of Claim 17, wherein said drug is an antineoplastic agent.

21. The method of Claim 17, wherein R4 is NH and the specifier is a peptide.

22. The method of Claim 19, wherein said drug is an antineoplastic agent, R4 is NH, and the specifier is a peptide.

23. The method of Claim 16, wherein R1 is hydrogen, R2 is hydrogen or methyl, and R3 is hydrogen or methyl.

24. The method of Claim 16, wherein said drug moiety is an antineoplastic agent,

25. The method of Claim 16, wherein R4 is NH
and the specifier moiety is a peptide.

26. The method of Claim 16, wherein said drug moiety is an antineoplastic agent, R4 is NH and the specifier moiety is a peptide.

27. The method of Claim 1, wherein said tumor-associated protease is a blood coagulating enzyme selected from the group consisting of thrombin, thromboplastin, Factor Va, Factor VIIa, Factor VIIIa, Factor IXa, Factor Xa, Factor XIa, and Factor XIIa.

28. A tumor-specific pro-drug of an antineo-plastic agent comprising a peptidyl derivative of said antineoplastic agent, said peptidyl derivative being a selective substrate for drug-activating proteases selected from the group consisting of fibrinolytic enzymes and blood-coagulating enzymes, whenever prepared by the process of Claim 1 or by an obvious chemical equivalent thereof.

29. The pro-drug of Claim 28, wherein said tumor-associated protease is a fibrinolytic enzyme selected from the group consisting of plasmin and plasminogen activator, whenever prepared by the process of Claim 2 or by an obvious chemical equivalent thereof.

30. The pro-drug of Claim 28, wherein said tumor-associated protease is a blood-coagulating enzyme selected from the group consisting of thrombin, thromboplastin, Factor Va, Factor VIIa, Factor VIIIa, Factor IXa, Factor Xa, Factor XIa, and Factor XIIa, whenever prepared by the process of Claim 27 or by an obvious chemical equivalent thereof.

31. The pro-drug of Claim 28, wherein the mole-cular structure of said peptidyl derivative is comprised of a peptide specifier moiety and an antineoplastic agent moiety, said two moieties being covalently linked together either directly or through an intermediate self-immolative connector moiety in a manner such that said antineoplastic agent moiety is rendered pharmacologically inactive, the site of said drug-activating enzymatic cleavage will be at the bond covalently linking said peptide specifier moiety to its immediately adjacent moiety, and said drug-activating enzymatic cleavage will effect release of said antineoplastic agent moiety in pharmacologically active form, whenever prepared by the process of Claim 5, or by an obvious chemical equivalent thereof.

32. The pro-drug of Claim 28, wherein said re-active site of said antineoplastic agent moiety is a free amino group, a free hydroxyl group, or a free sulfhydryl group, whenever prepared by the process of Claim 3, or by an obvious chemical equivalent thereof.

33. The pro-drug of Claim 28, wherein said re-active site of said antineoplastic agent moiety is a free amino group, a free hydroxyl group, or a free sulfhydryl group, and wherein said antineoplastic agent moiety is selected from the group consisting of cytosine arabinoside, adriamycin, daunomycin, 6-thioguanine, fluorodeoxyuridine, bis-(2-chloroethyl) amine, phenylenediamine mustard, 3'-aminothymidine, L-alanosine, 2-aminothiodiazole, 1,4-dihydroxy-5,8-bis(2-aminoethylamino)-9,10-anthracenedione, whenever prepared by the process of Claim 4, or by an obvious chemical equivalent thereof.

34. A hydrolytic enzyme-activatible pro-drug having the general formula wherein R1 is hydrogen or one or more substituent groups which are either electron-donating groups or electron-withdrawing groups; R2 and R3 may be the same or dif-ferent and are each selected from the group consisting of hydrogen, alkyl, phenyl, and phenyl substituted with either electron-donating groups or electron-withdrawing groups; R4 is NH or O; when R4 is NH, the specifier moiety is selected from the group consisting of a peptide, an amino acid, a carboxylic acid, and phosphoric acid; when R4 is 0, the specifier moiety is selected from the group consisting of a carboxylic acid, phosphoric acid, and sulfuric acid; and the drug moiety is a normally pharmacologically active agent having a reactive site whose derivatization inhibits pharmacological activity, said reactive site being selected from the group consisting of a free amino group, a free hydroxyl group and a free sulfhydryl group, the covalent linkage between said drug moiety and its adjacent carbonyl group being at said reactive site so as to inhibit the pharmacological activity of said drug moiety, whenever pre-pared by the process of Claim 16, or by an obvious chemical equivalent thereof.

35. The pro-drug of Claim 34, wherein R1 is hydrogen, R2 is hydrogen or methyl, and R3 is hydrogen or methyl, whenever prepared by the process of Claim 23 or by an obvious chemical equivalent thereof.

36. The pro-drug of Claim 34, wherein said drug moiety is an antineoplastic agent, whenever prepared by the process of Claim 24, or by an obvious chemical equivalent thereof.

37. The pro-drug of Claim 34, wherein R4 is NH
and the specifier moiety is a peptide, whenever prepared by the process of Claim 25, or by an obvious chemical equivalent thereof.

38. The pro-drug of Claim 34, wherein said drug moiety is an antineoplastic agent, R4 is NH and the speci-fier moiety is a peptide, whenever prepared by the process of Claim 26, or by an obvious chemical equivalent thereof.