Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug
© Wang et al; licensee BioMed Central Ltd. 2001
Received: 8 October 2001
Accepted: 2 November 2001
Published: 2 November 2001
Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects.
A CC-1065 analogue was conjugated to a cephalosporin affording prodrug 2. The prodrug and its corresponding free drug, 1, have IC50 values of 0.9 and 0.09 nM, respectively, against U937 leukemia cells in vitro.
For the first time, a prodrug comprised of a cephalosporin and a CC-1065 analogue has been synthesized. The preliminary in vitro studies show that the prodrug was 10-fold less toxic than the free drug. Prodrug 2 has the potential to be useful in cancer treatment using the ADEPT approach.
Antibody-directed enzyme prodrug therapy (ADEPT) [1–5] is one of the promising new approaches that selectively target tumor cells, thus reducing toxic side effects to patients. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. This strategy addresses the stoichiometry, controlled drug release and poor antibody penetration problems associated with the use of monoclonal antibody-drug conjugates [6–8]. In addition, because the process of drug release is enzymatic, a single enzyme can generate a large amount of free drug. Consequently, a small amount of antibody can be used to reduce immunogenicity.
Beta-lactamases have been widely investigated for their role in the metabolism of antibiotics including cephalosporins and penicillins. Because of the high catalytic efficiency and broad substrate specificity, β-lactamases have been extensively used in the ADEPT approach to activate prodrugs of vinca alkaloids , nitrogen mustard [28–32], doxorubicin [33–36] and others . To take advantage of the potent antitumor activity of the CC-1065 class of compounds and the ADEPT approach, we have synthesized β-lactam prodrugs. Herein, we report synthesis and preliminary cytotoxic effects of a prodrug comprised of a cephalosporin and a CC-1065 analogue (Figure 1).
Results and discussion
Cytotoxicity of compounds 1 and 2 against U937 leukemia cells in vitroa
This is the first report demonstrating synthesis of a prodrug comprised of a cephalosporin and a CC-1065 analogue. The preliminary in vitro studies show the prodrug to be less toxic than the free drug. Due to the slow non-enzymatic degradation of the cephalosporins in solution , the ratio of toxicity of cephalosporin-containing prodrugs to their corresponding free drugs is generally not very high. However, some of the prodrugs are very effective against tumors in mouse models. For example, a cephalosporin-doxorubicin prodrug was 9-fold less toxic than free doxrubicin against tumor cells in vitro, but caused tumor regression when tested in tumor xenograft models . A cephalosporin-vinca alkaloid prodrug was 5-fold less toxic than the free drug against tumor cells in vitro, but was highly effective in tumor xenograft models in vivo . When taxol was conjugated to a cephalosporin, the resulting prodrug was approximately 10-fold less toxic than free taxol against tumor cells in vitro . Thus, prodrug 2 has the potential to be useful in cancer treatment using the ADEPT approach. We will report more biological data in due course.
Materials and methods
Cephalothin sodium, 3, (2.5 g, 5.98 mmol) was suspended in dichloromethane (150 mL). Anhydrous hydrogen chloride (4 N in dioxane, 2 mL, 8 mmol) was added, and the reaction mixture was stirred for 30 min at room temperature. tert-Butyl trichloroacetimidate (3.2 mL, 17.84 mmol) was added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was washed consecutively with water (150 mL), saturated sodium hydrogen carbonate solution (150 mL) and water (150 mL). The organic solution was dried using sodium sulfate. The solvent was removed, and the product was purified by flash column chromatography eluting with a solvent consisting of dichloromethane, ethyl acetate and hexane (1/1/3, v/v) affording 1.2 g of 4 (44% yield).
Compound 4 (1 g, 2.21 mmol) was dissolved in methanol (70 mL), and solid potassium carbonate (120 mg) was added. The mixture was stirred for 2 h at room temperature, and acetic acid (200 μL) was added to quench the reaction. The solvent was removed, and the product was purified by flash column chromatography eluting with 8% acetone in dichloromethane to afford 220 mg of 5 (24% yield).
Compound 5 (280 mg, 0.68 mmol) was dissolved in anhydrous THF (40 mL), and dimethylaminopyridine (1 mg), p-nitrophenylchloroformate (0.2 g, 1 mmol) and 2, 6-lutidine (120 μL), 1 mmol) were added sequentially. The reaction mixture was stirred overnight at room temperature. The solvent was removed, and the product was purified by flash column chromatography eluting with 5% ethyl acetate in dichloromethane to afford 271 mg of 6 (69% yield).
To a solution of 6 (50 mg, 87 μmol) in dichloromethane (2 mL) cooled to 0°C was added m- chloroperoxybenzoic acid (CPBA, 26 mg, 93 μmol) in 0.5 mL of dichloromethane. The reaction mixture was stirred for 15 min at 0°C, and was then washed with 5% potassium hydrogen carbonate solution followed by brine. The solvent was removed, and the product was purified by flash column chromatography eluting with 8% ethyl acetate in dichloromethane to afford 34 mg of 7 (66% yield).
Compound 7 (15 mg, 25 μmol) was added to a solution of 1 (9 mg, 23 μmol) in DMF (0.3 mL), which was synthesized as we reported previously , and the reaction mixture was stirred overnight at room temperature. The product was purified by thin layer chromatography eluting with ethyl acetate and hexane (3/1, v/v) to afford 12 mg of 8 (62% yield). 1H NMR (DMF-d7, ppm): 10.70 (s, 1 H), 9.15 (s, 1 H), 8.63 (s, 1 H), 8.25–7.85 (m, 4 H), 7.60–7.19 (m, 7 H), 7.05–6.95 (m, 2 H), 6.05–6.01 (m, 1 H), 5.39–5.30 (d, 1 H), 5.12–4.79 (m, 5 H), 4.35–4.27 (m, 1 H), 4.19–3.75 (m, 6 H), 1.58 (s, 9 H). FAB MS m/e 866.0 (M + Na)+.
To a solution of 8 (5 mg, 5.9 μmol) in DMF (0.2 mL) and dichloromethane (1 mL) was added trifluroacetic acid (1 mL), and the solution was stirred for 2 h at room temperature. The solvent was removed, and ethyl ether was added. The solid was filtered, and washed with ether to afford prodrug 2 (3.7 mg, 79% yield). 1H NMR (DMF-d7, ppm): 11.56 (s, 1 H), 10.50 (s, 1 H), 9.65 (s, 1 H), 8.25–7.85 (m, 4 H), 7.60–7.24 (m, 7 H), 7.10–6.96 (m, 2 H), 6.10–6.01 (m, 1 H), 5.42–5.38 (d, 1 H), 5.10–4.60 (m, 5 H), 4.35–4.25 (m, 1 H), 4.20–3.75 (m, 6 H). FAB MS m/e 787.1.
We thank Jolande Murray for help with the manuscript. This work was supported in part by a grant from the National Institutes of Health (CA79357-01 to Y. W.).
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