Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent

A novel N-{2-amino-4-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L- glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as an a ntitumor agent. Compound 3b, the N7-benzylated analogue of 3a, was also syn thesized as an antitumor agent. The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo-[2 ,3-d]pyrimidine (10) in 5 steps from 2-acetylbutyrolactone. Protection of t he 2-amino group of 10 and regioselective iodination at the 5-position foll owed by palladium-catalyzed coupling afforded intermediate 14 which was con verted to 3a by reduction and saponification. Similar synthetic methodology was used for 3b. X-ray crystal structure of the ternary complex of 3a, DHF R, and NADPH showed that the pyrrolo[2,3-d]pyrimidine ring binds in a "2,4- diamino mode" in which the pyrrole nitrogen mimics the 4-amino moiety of 2, 4-diaminopyrimidines. This is the first example of a classical pyrrolo[2,3- d]pyrimidine antifolate shown to have this alternate mode of binding to DHF R. Compounds 3a and 3b were more inhibitory than LY231514 against TS from L actobacillus casei and Escherichia coli. Analogue 3a was also more inhibito ry against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii. Ev aluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX. Metabolite protection studies and folylpoly-gamma -glutamate synthe tase studies suggest that the antitumor activity of 3a against the growth o f tumor cells in culture is a result of dual inhibition of TS and DHFR. Com pound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED50 values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX. In contrast, compound 3b was inactive against both cell lin es. Compound 3a was evaluated in the National Cancer Institute in vitro pre clinical antitumor screening program and afforded IG(50) values in the nano molar range against a number of tumor cell lines.