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.