Articles Design, Synthesis, and X-ray Crystal Structure of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate Reductase as an Antitumor Agent 1 Aleem Gangjee,* ,† Jianming Yu, † John J. McGuire, ‡ Vivian Cody, § Nikolai Galitsky, § Roy L. Kisliuk, ⊥ and Sherry F. Queener # Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282; Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263; Hauptman-Woodward Medical Research Institute, Inc., 73 High Street, Buffalo, New York 14203; Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111; and Department of Pharmacology and Toxicology, School of Medicine, Indiana University, Indianapolis, Indiana 46202 Received May 9, 2000 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 antitumor agent. Compound 3b, the N7-benzylated analogue of 3a, was also synthesized 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 the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification. Similar synthetic methodology was used for 3b. X-ray crystal structure of the ternary complex of 3a, DHFR, 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 DHFR. Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli. Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii. Evaluation 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- γ-glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR. Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED 50 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 lines. Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG 50 values in the nanomolar range against a number of tumor cell lines. Introduction Inhibitors of folate metabolism have provided impor- tant agents useful in cancer chemotherapy as a result of their inhibition of the biosynthesis of nucleic acid precursors. 2,3 The cofactor tetrahydrofolate (THF) is formed by the NADPH-dependent reduction of 7,8- dihydrofolate (7,8-DHF) by the enzyme dihydrofolate reductase (DHFR) 4 and serves as the principal compo- nent in folate metabolism as a carrier of one-carbon units in its various cofactor forms. 5 The synthesis of deoxythymidylate (dTMP) from deoxyuridylate (dUMP) is catalyzed by thymidylate synthase (TS) and utilizes 5,10-methylenetetrahydrofolate as the source of the methyl group of dTMP as well as the reductant. 6 This reaction affords 7,8-DHF which needs to be reduced by DHFR to THF. Thus TS coupled with DHFR forms a crucial link responsible for the synthesis of dTMP and hence DNA. Inhibitors of TS derived from substrate analogues such as 5-fluorouracil 7 and from folate ana- logues such as ZD1694 (Tomudex) 8 (Chart 1) have found utility as clinically important antitumor agents. Simi- larly the DHFR inhibitor methotrexate (MTX) is a main- stay in single and combination cancer chemotherapy. 9 Several TS and DHFR inhibitors which are analogues of folate have also shown antitumor activities in vitro and in vivo with some currently in clinical trials. 10,11 * To whom correspondence should be addressed. Phone: 412-396- 6070. Fax: 412-396-5593. E-mail: gangjee@duq.edu. † Duquesne University. ‡ Roswell Park Cancer Institute. § Hauptman-Woodward Medical Research Institute, Inc. ⊥ Tufts University School of Medicine. # Indiana University. 3837 J. Med. Chem. 2000, 43, 3837 10.1021/jm000200l CCC: $19.00 © 2000 American Chemical Society Published on Web 09/13/2000