Design, Synthesis, and X-ray Crystal Structure of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate Reductase as an Antitumor Agent

Abstract

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-gamma-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.

Design, Synthesis, and Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors

“…The DHFR inhibitory activity of 2-amino-4-oxopyrrolo [2,3-d]pyrimidines such as 1 can be explained by rotating the NH 2 -C2 bond by 180° such that the N7 functions as the 4-amino moiety. 47 Since analogue 4 is a pyrrolo[3,2-d]pyrimidine, we cannot explain the increased DHFR inhibitory activity on the basis of a NH 2 -C2 bond rotation of 180°. The increase in activity against human DHFR of 4 over 1 can be explained on the basis of our molecular modeling where the C7 of 4 interacts with Phe31 of human DHFR and the N7 of 1 would not have the same interaction.…”

“…The silica gel plug obtained was loaded onto a silica gel column and eluted with 9:1 ethyl acetate/nhexane The fractions containing the product (TLC) were pooled and the solvent was evaporated to afford 1.33 g (67%) of 28 as a white solid: TLC R f = 0. 47 …”

Discovery of Novel Antitumor Antimitotic Agents That Also Reverse Tumor Resistance

“…For the synthesis of 6-9 (Scheme 1), we found that the N7-benzyl group was necessary to increase the solubility of the intermediates and, more importantly, facilitated the Sonogashira coupling reaction with acetylenes. Thus, we started with the N-benzylated analog 11 40 (Scheme 1). Compound 11 was coupled using the Sonogashira reaction with appropriately substituted iodobenzenes in the presence of tetrakis(triphenylphosphine)palladium(0) (Pd-(PPh 3 ) 4 ), copper(I) iodide (CuI), and triethylamine in dichloroethane.…”

Synthesis and evaluation of a classical 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine and a 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine as antifolates

“…2,13,14 Gangjee et al 10,11 suggested a dual DHFR-TS inhibitor model and proposed two binding modes for the 2-amino-4-oxo-pyrrolo[2, 3-d]pyrimidine system as well as the 2,4-diamino-furo[2, 3-d]pyrimidine (Fig. 2), Gangjee et al 15 demonstrated that compound 10, a 4-methyl analog of compound 3, binds in the alternate mode to DHFR.…”

“…Compound 10 however showed excellent dual DHFR-TS inhibitory activities. 15 Some of these analogs were also significantly cytotoxic to the growth of tumor cells in culture (EC 50 = 1.0 × 10 −7 to 1.0 × 10 −8 M). This cytotoxicity was attributed to the efficient poly(γ-glutamylation) by the enzyme folypoly-γ-glutamate synthetase (FPGS).…”