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Development and Activities of a New Melphalan Prodrug Designed for Tumor-Selective Activation David E. Kerr, Zhengong Li, Nathan O. Siemers, Peter D. Senter, and Vivekananda M. Vrudhula* Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121. Received September 12, 1997; Revised Manuscript Received January 5, 1998
The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted β-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 µM and 980 s-1, respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.
INTRODUCTION
Targeting cytotoxic agents to sites of malignancy using monoclonal antibodies (mAb)1 that bind to tumor-associated antigens has been the subject of a great deal of research. Among the cytotoxic moieties delivered using this strategy are radionuclides (1, 2), protein toxins (3), and chemotherapeutic drugs (4), all of which involve covalent attachment of the targeted molecule to the mAb. An alternative strategy is to use mAb-enzyme conjugates for the activation of anticancer prodrugs. In this two-step process, a mAb-enzyme conjugate is used to convert a prodrug substrate for the targeted enzyme to an active drug in a site-selective manner. Several reviews discussing this approach are currently available (5-7). Earlier work has demonstrated that this approach can generate higher intratumoral levels of drug (8-10) and better therapeutic effects than systemic administration of drug alone (11-15). β-Lactamases (bL) are enzymes that have been shown to be very effective and versatile for anticancer prodrug activation (8, 11, 13, 15-21). These monomeric enzymes are abundant, available in multiple forms, inexpensive, and retain their activity in vivo. Furthermore, a variety of anticancer drugs can be appended to cephalosporin so that, upon β-lactam hydrolysis, a fragmentation reaction leads to the release of active drug (Scheme 1). Several studies have demonstrated that bL can release phenylenediamine mustard (13, 17), doxorubicin (18), mitomycin C (16), platinum compounds (19), and paclitaxel (20) from * To whom correspondence should be addressed at Bristol Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492. 1 Abbreviations: bL, β-lactamase; CCM, 7-(4-carboxybutanamido)cephalosporin mustard; mAb, monoclonal antibody; CMel, cephalosporin melphalan; IC50, concentration causing 50% inhibition; L49-sFv-bL, fusion protein of the L49 sFv fragment attached to Enterobacter cloacae bL; MTD, maximum tolerated dose; PDM, phenylenediamine mustard.
cephalosporin prodrugs. We have previously shown that L6-bL in combination with a cephalosporin doxorubicin prodrug led to antitumor activities in two models of human lung carcinoma (8). Significantly better activities were obtained in melanoma models using either chemically conjugated L49-bL (13, 21) or the single-chain Fvcontaining fusion protein L49-sFv-bL (15) in combination with CCM, a prodrug of phenylenediamine mustard. Because of the pronounced activities obtained using alkylating agents in our studies (13, 15, 21) and in others (12, 14), we were prompted to develop cephalosporin prodrugs of a clinically approved nitrogen mustard. One agent that came to our attention was melphalan, an anticancer drug that alkylates DNA, mainly at the N-7 position of guanine residues (22). Since it is known that amide derivatives of melphalan are generally less cytotoxic than the parent drug (23), we reasoned that attachment of melphalan through the primary amine to a cephalosporin via a carbamate linkage would greatly reduce its cytotoxic activity. In this article, we report the synthesis and in vitro and in vivo properties of C-Mel, a cephalosporin prodrug of melphalan. MATERIALS AND METHODS
Materials. L49 is a mAb that binds to the melanotransferrin antigen expressed on most human melanomas (15). L49-sFv-bL was expressed in Escherichia coli and purified as described earlier (15). The bL used was a mutated form of the enzyme from Enterobacter cloacae (15, 24). CCM was synthesized as described earlier (17). HPLC analyses were performed on a Perkin-Elmer LC Bio 410 instrument equipped with a 5 µm C-18 column and a UV detector set at 264 nm. Acetonitrile/0.1 M sodium phosphate (1:1, pH 1.8) was used as the eluting solvent to monitor the enzymatic hydrolysis of the prodrug. Preparation of 7-(Phenylacetamido)-3-[[(1′,2′,2′,2′tetrachloroethoxycarbonyl)oxy]methyl]ceph-3-em-
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Scheme 1. Synthesis of C-Mel and bL-Mediated C-Mel Hydrolysisa
a
(a) 3-Chloroperoxybenzoic acid/CH2Cl2; (b) melphalan/THF, aqueous NaHCO3; (c) TFA-anisole/CH2Cl2; (d) β-lactamase.
4-carboxylic Acid 1-Oxide Diphenyl Methyl Ester (2). An ice cold solution of the corresponding thioether (1) (25) (7.10 g, 9.80 mmol) in methylene chloride (300 mL) was treated with 3-chloroperoxybenzoic acid (3.05 g, 80% purity). After 15 min, a cold solution containing a 10% sodium bisulfite solution (100 mL) and a 0.1 N sodium bicarbonate solution (100 mL) was added. The biphasic mixture was stirred at 0 °C for 10 min, and the layers were separated. The organic layer was washed with ice cold 0.1 N sodium bicarbonate solution (3 × 100 mL) and saturated NaCl (100 mL), dried over MgSO4, and concentrated to approximately 80 mL, at which point crystallization occurred. After cooling in ice, the crystalline material was filtered to give 5.20 g (7.01 mmol, 72%) of sulfoxide (2) as a white solid. MS (M + NH4+): 756 (found), 756 (calcd). 1H NMR (CDCl3): δ 7.60-7.24 (m, 15H, ArH), 6.92 (s, 1H, CHCl), 6.69 (d, 1H, JNH,7 ) 9.9 Hz, 7-NH), 6.62 (s, 1H, CHPh2), 6.11 (dd, 1H, J6,7 ) 4.8 Hz, H-7), 5.61 (d, 1H, JAB ) 14.1 Hz, CH2AOCO), 4.85 (d, 1H, CH2BOCO), 4.47 (d, 1H, H-6), 3.80 (d, 1H, JAB ) 18.8 Hz, H-2A), 3.67 (d, 1H, CH2APh, J ) 6.3 Hz), 3.60 (d, 1H, CH2BPh), 3.20 (d, 1H, H-2B). Anal. Calcd for C32H26N2Cl4O8S: C, H, N, S, Cl, 19.15%. Found: C, H, N, S, Cl, 19.87%. Preparation of C-Mel. To a solution of melphalan (412 mg, 1.35 mmol) in a mixture of 0.25 N sodium bicarbonate (14 mL) and THF (28 mL) was added the activated sulfoxide (2) (1.00 g, 1.35 mmol). The resulting suspension turned clear after sonicating at room temperature for 15 min. The reaction mixture was stirred for an additional 10 min at room temperature and cooled in an ice bath; 1 N HCl (5 mL) was added, and the reaction mixture was extracted with ethyl acetate (3 × 40 mL). The combined organic layer was washed with water (80 mL) and brine (80 mL), dried over MgSO4, and evaporated in vacuo. The resulting residue was suspended in methylene chloride (50 mL) and anisole (5 mL) at 0 °C, and trifluoroacetic acid (5 mL) was added over a period of 5 min. The resulting reaction mixture was stirred at 0 °C for 45 min. Volatile materials were then removed under reduced pressure. The residue was
dissolved in a minimum amount of dichloromethane, and the solution was poured into ice cold hexane (100 mL). The white precipitate formed was filtered and recrystallized from absolute ethanol/methylene chloride (1:1) to give 445 mg (0.60 mmol, 44%) of the title compound as an off-white solid. MS for C30H33Cl2N4O9S (MH+): 695 (calcd), 695 (found). 1H NMR (DMSO-d6): δ 13.80 (br s, 1H, COOH), 12.71 (br s, 1H, COOH), 8.44 (d, 1H, J ) 8.3 Hz, OCONH), 7.67 (d, 1H, JNH,7 ) 8.3 Hz, 7-NH), 7.32-7.20 (m, 5H, 7-ArH), 7.08 (d, 2H, J ) 8.6 Hz, 2Ar′H), 6.64 (d, 2H, 2Ar′H), 5.79 (dd, 1H, J6,7 ) 4.8 Hz, H-7), 5.06 (d, 1H, JA,B ) 13.3 Hz, CH2AOCONH), 4.81 (d, 1H, H-6), 4.54 (d, 1H, CH2BOCONH), 4.05-4.01 (m, 1H, NHCHCOOH), 3.78-3.21 (m, 12H, H-2A, H-2B, PhCH2CO, 2 NCH2CH2Cl), 2.91 (dd, 1H, JA,B ) 13.8 Hz, JA,X ) 4.4 Hz, CHCH2APh-N), 2.70 (dd, 1H, JB,X )10.1 Hz, CHCH2BPh-N). Anal. Calcd for C30H32Cl2N4O9S‚ 1C2H5OH‚1H2O: C, H, N, Cl, S. Enzyme Kinetics. The hydrolysis of C-Mel by bL was measured as described previously (17). The cleavage of the β-lactam ring results in a loss of absorbance at 260 nm. Initial velocities of β-lactam hydrolysis were determined at room temperature, and a Lineweaver-Burk plot of the data was used to estimate the Km and kcat values. The bL concentration was 25 ng/mL, and the C-Mel concentration was 20-100 µM. In Vitro Cytotoxicity. Type 3677 cells in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum, 60 µg/mL penicillin, and 100 µg/mL streptomycin were plated into 96-well microtiter plates at 104 cells/ mL and allowed to adhere overnight at 37 °C. For blocking experiments, the cells were incubated at 4 °C with unconjugated L49 at 2 µM (300 µg/mL) for 30 min prior to treatment with L49-sFv-bL. The cells were then treated with 2 nM L49-sFv-bL (134 ng/mL). After 30 min at 4 °C, the plates were washed four times with antibiotic-free Roswell Park Memorial Institute 1640 media with 10% fetal bovine serum, and then varying concentrations of C-Mel were added. C-Mel and melphalan were also added to cells treated with media alone. After 4 h at 37 °C, cells were washed three times with
Development of a New Melphalan Prodrug
Iscove’s modified Dulbecco’s medium and incubated for approximately 12 h at 37 °C. The cells were then pulsed for 18 h with [3H]thymidine (1 µCi/well) at 37 °C, detached by freezing at -20 °C and thawing, and harvested onto glass fiber filter mats using a 96-well harvester. Radioactivity was counted using a LKB Wallac β-plate counter. Therapy Experiments. Type 3677 tumor-bearing mice (subcutaneous implants, five animals/group, average tumor volume of 110 mm3) were injected with 1 mg/ kg L49-sFv-bL (intravenous, 8 days after tumor implant), followed 18 h later by CCM or C-Mel. Treatment with L49-sFv-bL and CCM or C-Mel was repeated 7 and 14 days later. Control mice were treated with PDM or melphalan. Animals were monitored daily for general health and one or two times per week for body weight and tumor growth. Tumor volume was estimated using the formula (longest length)[(perpendicular dimension)2/ 2]. Cures were defined as an established tumor that, after treatment, was not visually detectable for 10 tumor doubling periods (40 days in the 3677 tumor model). MTDs led to less than 10% weight loss and no treatmentrelated deaths and were within 50% of the dose where such events took place. If an animal was removed from the experiment because of tumor growth, the data set was no longer plotted, but the remaining animals were monitored for tumor size and general health. RESULTS
Synthesis of C-Mel and Enzymatic Convesion to Melphalan. C-Mel was synthesized by condensing commercially available melphalan with the activated sulfoxide (2) as shown in Scheme 1. Cephalosporin sulfoxide (2) was prepared from 1 (25) by oxidation with 3-chloroperoxybenzoic acid. HPLC analysis revealed that C-Mel was a substrate for bL and released melphalan (data not shown). Enzymatic hydrolysis of C-Mel to melphalan by bL was investigated spectophotometrically. The Km was 218 µM, and the kcat was 980 s-1. The fast rate of bL hydrolysis of the sulfoxide prodrug C-Mel is comparable to those of other similar cephalosporin sulfoxides (26) and is substantially faster than those of related thioether prodrugs (16). In Vitro Cytotoxicity. L49-sFv-bL is a recombinant fusion protein containing the sFv fragment of the anti-melanoma mAb L49 and a mutated form of the enzyme bL (24). The protein was expressed in E. coli as described earlier and was purified by affinity chromatography (15). The cytotoxic effects of L49-sFv-bL in combination with C-Mel were determined on 3677 human melanoma cells, which express the melanotransferrin (p97) antigen. The experiments were performed by treating the cells with the fusion protein, washing off unbound material, adding various concentrations of C-Mel for 4 h at 37 °C, and using [3H]thymidine incorporation as a measure of cytotoxic activity. The prodrug C-Mel (IC50 ) 53 µM) was approximately 40-fold less toxic to 3677 cells than the drug melphalan (IC50 ) 1.3 µM). As expected, L49-sFv-bL activated C-Mel, and the combination was equivalent in activity to melphalan (Figure 1). This indicates that prodrug conversion was efficient under the conditions tested. In addition, it was found that activation was immunologically specific, since a much lower level of C-Mel activation took place on 3677 cells that were saturated with unconjugated L49 prior to addition of the fusion protein. Therapeutic Activity. In vivo therapy experiments were performed in which the activities of L49-sFv-bL/
Bioconjugate Chem., Vol. 9, No. 2, 1998 257
Figure 1. Cytotoxic effects of L49-sFv-bL and C-Mel on 3677 melanoma cells as measured by [3H]thymidine incorporation. Cells were incubated with L49-sFv-bL, washed, and treated with C-Mel for 4 h. Control cells were treated with melphalan or C-Mel in the absence of L49-sFv-bL or with L49 at saturating amounts prior to L49-sFv-bL addition.
CCM and L49-sFv-bL/C-Mel were compared in nude mice with established subcutaneous 3677 tumors. This particular tumor model has previously been shown to be resistant to treatment with PDM and CCM but was responsive to treatment with the L49-sFv-bL/CCM combination (15). In the experiments reported here, conjugate treatment was initiated 8 days after tumor implant, at which time the tumors were approximately 110 mm3 in volume. CCM or C-Mel was then administered 18 h later, and the treatment protocol was repeated twice more on a weekly basis. This particular time interval was chosen on the basis of the high tumor: nontumor ratios 18 h after L49-sFv-bL administration (15). The MTD of melphalan (10 mg/kg/injection) led to a delay in tumor outgrowth of 39 days (Figure 2A). More pronounced antitumor activity was seen in mice that received C-Mel (100 mg/kg/injection) after L49-sFv-bL. Therapeutic activity was dose-dependent, as cures (three out of five mice) and complete regressions (two out of five mice) were obtained at a higher C-Mel dose (150 mg/kg/ injection). Both of these doses were well tolerated. In the same experiment, it was found that PDM at 3 mg/kg (MTD) had no effect on the growth of the 3677 tumors (Figure 2B). In contrast, animals treated with L49sFv-bL prior to CCM (100 mg/kg/injection) experienced a tumor growth delay of 63 days, with one out of five cures. L49-sFv-bL followed by CCM (150 mg/kg/ injection) resulted in cures (three out of five mice) and partial regressions (two out of five mice). These doses were nontoxic. L49-sFv-bL followed by either C-Mel or CCM at 200 mg/kg was above the MTD. Thus, CCM and C-Mel are equally effective in 3677 tumor-bearing mice that were pretreated with L49-sFv-bL. DISCUSSION
Among the many prodrugs that have been reported for tumor-selective activation by mAb-enzyme conjugates (5-7), the most pronounced activities have generally been obtained from those containing nitrogen mustards (1215). Possible reasons for this may be due to the steep dose-response curves displayed by such agents, along with the fact that nitrogen mustards tend not to be cell cycle-specific (22). The alkylating agents employed thus far have been PDM (13, 15, 21) and several benzoic acid mustard derivatives (12, 14), none of which are clinically approved. There may be some advantages in releasing clinically approved agents since their pharmacology and toxicities are well-understood. For this reason, we
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(13, 17, 21), and may be due to its high degree of instability under physiological conditions (29). This might lead to the conclusion that the portion of C-Mel that was systemically activated could contribute to therapeutic efficacy, while that of CCM would lead to greater toxicity. In conclusion, we have demonstrated that C-Mel is a prodrug of melphalan that can be activated in an immunologically specific manner by L49-sFv-bL. All of the tumors in animals treated with L49-sFv-bL and C-Mel at 150 mg/kg/injection underwent complete regressions, and three out of five were eventually cured. Although the therapeutic effects of C-Mel and CCM were equivalent in the tumor model used in these studies, we feel that C-Mel is advantageous since it releases a clinically approved anticancer drug. ACKNOWLEDGMENT
We acknowledge the contributions of Pam Gronbeck, Håkan Svensson, Steve Klohr, and David Lowe. LITERATURE CITED
Figure 2. Therapeutic effects of L49-sFv-bL in combination with (A) C-Mel or (B) CCM. Nude mice (five mice per group) were treated with L49-sFv-bL at 1 mg/kg followed 15-18 h later by prodrugs. The average tumor volumes were plotted until one or more mice were removed from the experiment due to tumor outgrowth. Melphalan and PDM at the MTDs were also injected without prior L49-sFv-bL treatment.
selected melphalan, a drug that has proven to be useful in the clinical treatment of malignant melanoma and other neoplasias (22, 27). Melphalan is well-suited for the targeting strategy described here, since acylation of its primary amino group has previously been shown to lead to a reduction in cytotoxic activity (23). Consequently, a prodrug of melphalan was prepared by attaching the drug via its side chain amine to a cephalosporin so that upon bL-catalyzed hydrolysis the parent drug was released. In the prodrug CCM, the primary amine of the phenylenediamine mustard was attached to cephalosporin through a stable carbamate linkage. We therefore decided to append melphalan to cephalosporin using a carbamate linkage. Our starting material for the synthesis of C-Mel was the tetrachloroethyl carbonate 1 (25) in which the cephalosporin carboxylic acid was protected as a diphenyl methyl ester to prevent the otherwise facile lactonization under basic conditions. In designing C-Mel, we decided to oxidize the cephalosporin thioether to a sulfoxide, since this reduces the isomerization of the ∆2 double bond under the basic conditions used in the coupling reaction (28). Cephalosporin sulfoxides have been shown to be excellent substrates for bL (26), so it was not surprising to find that the kcat value for bL-catalyzed hydrolysis of C-Mel (980 s-1) was quite high. This is substantially higher than the previously reported turnover number of 337 s-1 with CCM as the substrate (17). One of the noteworthy findings in these studies was that significant antitumor activity was obtained in animals treated with a MTD of melphalan (39 day growth delay), while the MTD of PDM was ineffective. The lack of antitumor activity of PDM has been previously noted
(1) Schubiger, P. A., Alberto, R., and Smith, A. (1996) Vehicles, chelators, and radionuclides: choosing the “building blocks” of an effective therapeutic radioimmunoconjugate. Bioconjugate Chem. 7, 165-179. (2) Knox, S. J. (1995) Overview of studies on experimental radioimmunotherapy. Cancer Res. 55, 5832s-5836s. (3) Frankel, A. E., FitzGerald, D., Siegall, C., and Press, O. W. (1996) Advances in immunotoxin biology and therapy: A summary of the fourth international symposium on immunotoxins. Cancer Res. 56, 926-932. (4) Pietersz, G. A., Rowland, A., Smyth, M. J., and McKenzie, I. F. (1994) Chemoimmunoconjugates for the treatment of cancer. Adv. Immunol. 56, 301-387. (5) Melton, R. G., and Sherwood, R. F. (1996) Antibody-enzyme conjugates for cancer therapy. J. Natl. Cancer Inst. 88, 153165. (6) Niculescu-Duvaz, I., and Springer, C. J. (1995) Antibodydirected enzyme prodrug therapy (ADEPT): A targeting strategy in cancer chemotherapy. Curr. Med. Chem. 2, 687706. (7) Senter, P. D., and Svensson, H. P. (1997) A summary of monoclonal antibody-enzyme/prodrug combinations. Adv. Drug Delivery Rev. 22, 341-349. (8) Svensson, H. P., Vrudhula, V. M., Emswiler, J. E., MacMaster, J. F., Cosand, W. L., Senter, P. D., and Wallace, P. M. (1995) In vitro and in vivo activities of a doxorubicin prodrug in combination with monoclonal antibody β-lactamase conjugates. Cancer Res. 55, 2357-2365. (9) Wallace, P. M., MacMaster, J. F., Smith, V. F., Kerr, D. E., Senter, P. D., and Cosand, W. L. (1994) Intratumoral generation of 5-fluorouracil mediated by an antibody-cytosine deaminase conjugate in combination with 5-fluorocytosine. Cancer Res. 54, 2719-2723. (10) Rogers, G. T., Burke, P. J., Sharma, S. K., Koodie, R., and Boden, J. A. (1995) Plasma clearance of an antibody-enzyme conjugate in ADEPT by monoclonal anti-enzyme: its effect on prodrug activation in vivo. Br. J. Cancer 72, 1357-1363. (11) Meyer, D. L., Jungheim, L. N., Law, K. L., Mikolajczyk, S. D., Shepherd, T. A., Mackensen, D. G., Briggs, S. L., and Starling, J. J. (1993) Site-specific prodrug activation by antibody-β-lactamase conjugates-regression and long-term growth inhibition of human colon carcinoma xenograft models. Cancer Res. 53, 3956-3963. (12) Eccles, S. A., Court, W. J., Box, G. A., Dean, C. J., Melton, R. G., and Springer, C. J. (1994) Regression of established breast carcinoma xenografts with antibody-directed enzyme prodrug therapy against c-erbB2 p185. Cancer Res. 54, 51715177. (13) Kerr, D. E., Schreiber, G. J., Vrudhula, V. M., Svensson, H. P., Hellstrom, I., Hellstrom, K. E., and Senter, P. D. (1995) Regressions and cures of melanoma xenografts following
Development of a New Melphalan Prodrug treatment with monoclonal antibody β-lactamase conjugates in combination with anticancer prodrugs. Cancer Res. 55, 3558-3563. (14) Blakey, D. C., Burke, P. J., Davies, D. H., Dowell, R. I., East, S. J., Eckersley, K. P., Fitton, J. E., McDaid, J., Melton, R. G., Niculescu-Duvaz, I. A., Pinder, P. E., Sharma, S. K., Wright, A. F., and Springer, C. J. (1996) ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts. Cancer Res. 56, 3287-3292. (15) Siemers, N. O., Kerr, D. E., Yarnold, S., Stebbins, M., Vrudhula, V. M., Hellstro¨m, I., Hellstro¨m, K. E., and Senter, P. D. (1997) Construction, expression, and activities of L49sFv-β-lactamase, a single chain antibody fusion protein for anticancer prodrug activation. Bioconjugate Chem. 8, 510519. (16) Vrudhula, V. M., Svensson, H. P., and Senter, P. D. (1997) Immunologically specific activation of a cephalosporin derivative of mitomycin C by monoclonal antibody β-lactamase conjugates. J. Med. Chem. 40, 2788-2792. (17) Vrudhula, V. M., Svensson, H. P., Kennedy, K. A., Senter, P. D., and Wallace, P. M. (1993) Antitumor activities of a cephalosporin prodrug in combination with monoclonal antibody-β-lactamase conjugates. Bioconjugate Chem. 4, 334340. (18) Vrudhula, V. M., Svensson, H. P., and Senter, P. D. (1995) Cephalosporin derivatives of doxorubicin as prodrugs for activation by monoclonal antibody-β-lactamase conjugates. J. Med. Chem. 38, 1380-1385. (19) Hanessian, S., and Wang, J. (1993) Design and synthesis of a cephalosporin-carboplatinum prodrug activatable by a β-lactamase. Can. J. Chem. 71, 896-906. (20) Rodrigues, M. L., Carter, P., Wirth, C., Mullins, S., Lee, A., and Blackburn, B. K. (1995) Synthesis and β-lactamasemediated activation of a cephalosporin-taxol prodrug. Chem. Biol. 2, 223-227. (21) Svensson, H. P., Frank, I. S., Berry, K. K., Vrudhula, V. M., and Senter, P. D. (1998) Therapeutic effects of monoclonal
Bioconjugate Chem., Vol. 9, No. 2, 1998 259 antibody-β-lactamase conjugates in combination with a nitrogen mustard anticancer prodrug in models of human renal cell carcinoma. J. Med. Chem. (in press). (22) Berger, N. A. (1993) Anticancer Drugs. In Cancer: Principles and Practice of Oncology (V. T. J. DeVita, S. Hellman, and S. A. Rosenberg, Eds.) pp 400-407, J. B. Lippincott Co., Philadelphia. (23) Vrudhula, V. M., Senter, P. D., Fischer, K. J., and Wallace, P. M. (1993) Prodrugs of doxorubicin and melphalan and their activation by a monoclonal antibody-penicillin-G amidase conjugate. J. Med. Chem. 36, 919-923. (24) Siemers, N. O., Yelton, D. E., Bajorath, J., and Senter, P. D. (1996) Modifying the specificity and activity of the Enterobacter cloacae P99 β-lactamase by mutagenesis within an M13 phage vector. Biochemistry 35, 2104-2111. (25) Hudyma, T. W., Bush, K., Colson, K. L., Firestone, R. A., and King, H. D. (1993) Synthesis and release of doxorubicin from a cephalosporin based prodrug by a β-lactamase immunoconjugate. Bioorg. Med. Chem. Lett. 3, 323-328. (26) Jungheim, L. N., Shepherd, T. A., and Meyer, D. L. (1992) Synthesis of acylhydrazido-substituted cephems. Design of cephalosporin-vinca alkaloid prodrugs: substrates for an antibody-targeted enzyme. J. Org. Chem. 57, 2334-2340. (27) Alexander, H. R., Jr., Fraker, D. L., and Bartlett, D. L. (1996) Isolated limb perfusion for malignant melanoma. Semin. Surg. Oncol. 12, 416-428. (28) Murphy, C. F., and Webber, J. L. (1972) Alteration of the dihydrothiazine ring moiety. In Cephalosporin and Penicillins: Chemistry and Biology (E. H. Flynn, Ed.) pp 134-182, Academic Press, New York. (29) Chakravarty, P. K., Carl, P. L., Weber, M. J., and Katzenellenbogen, J. A. (1983) Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard. J. Med. Chem. 26, 633-638.
BC970163L
