Communications to the Editor A Novel Connector Linkage Applicable in Prodrug Design Scheme I a. Bipartate Prodrug Sir:We wish to report a chemical linkage that may be useful in solving certain problems in prodrug design.1 Consider the bipartate prodrug illustrated in Scheme I consisting of a carrier or specifier moiety A and a drug moiety B. Moiety A may serve to target the drug to a particular site (e.g., by making it a specific substrate for a particular

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We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.

Many types of malignant cells and human tumors display increased concentrations of the protease plasminogen activator that converts plasminogen to the highly active protease, plasmin. Because plasmin rapidly cleaves various low molecular weight compounds coupled to appropriate peptide specifiers, we hypothesized that coupling of such peptide specifiers to anticancer drugs might create "prodrugs" which would be locally activated by tumor-associated plasmin and consequently would be less toxic to normal cells. To provide an initial test ofthis concept we have synthesized peptidyl prodrugs of the structure D-Val-Leu-Lys-X in which the peptidyl portion has been designed to allow the prodrug to serve as an excellent plasmin substrate and X is an anticancer drug-either the glutamine analog (aS,5S) a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) or the alkylating agent NN-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard).Treatment of these prodrugs with plasmin generated the free peptide and the free drug, demonstrating that these prodrugs are plasmin substrates. The prodrugs and free drugs were tested in an in vitro system against either normal chicken embryo fibroblasts, which display a low level of plasminogen activator, or their virally transformed counterparts, which produce high levels of plasminogen activator. In each case the peptidyI prodrugs displayed at least a 5-fold increase in selectivity for the transformed cells compared to the free drug. The greater selectivity of action of the peptidyl prodrugs against transformed cell cultures suggests that these or similar prodrugs that are substrates for tumor-associated proteases may show increased therapeutic effectiveness in the treatment of tumors that produce sufficiently increased amounts of plasminogen activator.A persistent problem in cancer chemotherapy is the low therapeutic index of most anticancer drugs-i.e., the concentrations needed to kill tumor cells are close to those that produce severe toxicity to the host. One often-proposed approach to overcoming this problem has been to design anticancer "prodrugs" which are inactive until locally activated by some tumor-associated enzyme (1-5). Although this approach has heretofore shown little success, this may be due to incomplete characterization of tumor-associated enzymes and their substrate specificity as well as to the admittedly severe difficulty that no enzyme unique to tumor cells is known, so that the prodrugs are probably activated by some normal cells as well as tumor cells.One characteristic of many cancer cells-including cells transformed in vitro by viral (6-9), chemical (10), or physical (11) agents and cell lines established from various human neoplasms (12-16)-is an increased production of the serine protease plasminogen activator. This enzyme cleaves the serum zymogen plasminogen to generate the serine protease plasmin. The plasmin production associated with this increased plasminogen activator activity of many tumors offers an attractive possibility for...