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The glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-g lutamic acid (1b, TNP-351) and the related compound (1a), was replaced with various N(omega)-acyl-, sulfonyl-, carbamoyl- and aryl-2,omega-diaminoalkanoic acids, and the inhibitory effects of the resulting products (9, 11, 14, 18, 21, 23, 25, 30, 36) on dihydrofolate reductase (DHFR), the growth of murine fibrosarcoma Meth A cells, and methotrexate-resistant human CCRF-CEM cells, were examined. Compounds (9a-f) acylated with a hemiphthaloyl group were efficiently synthesized by coupling pyrrolo[2,3-d]pyrimidine carboxylic acids (7a,b) and N(omega)-phthaloyl 2,omega-diaminoalkanoic acid methyl esters (6a-c) and subsequent hydrolysis. The other N(omega)-acyl- and sulfonyl-ornithine analogs (21, 23, 25) were synthesized by acylation of free amino intermediates (19a,b) derived from tert-butoxycarbonyl-ornithine analogs (17a,b). A free ornithine analog (18) did not strongly inhibit Meth A cell growth, whereas all N(omega)-acyl-, sulfonyl-, carbamoyl- and aryl-ornithine analogs (9, 11, 21, 23, 25, 30, 36) exhibited much more potent inhibitory activities against both DHFR and Meth A cell growth. In particular, compounds 9c, 21k and 36a also showed remarkable growth-inhibitory activities against methotrexate-resistant CCRF-CEM cells. These results demonstrate that the potent inhibitory activities of N(omega)-masked ornithine analogs against the growth of Meth A cells and methotrexate-resistant CCRF-CEM cells, results from effective uptake via reduced folate carrier and their potent DHFR inhibition.

The glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2, 3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and related compounds was replaced with some N5-substituted glutamines. Antifolates (4A-S) were effectively prepared by coupling pyrrolo[2,3-d]pyrimidine carboxylic acids (11a, b) with some properly protected N5-substituted glutamine derivatives (10A-S), which were prepared by coupling Boc-Glu-OMe (7) with various amines (8A-S) using a suitable condensing reagent, followed by hydrolysis. The inhibitory effects of the resulting products on dihydrofolate reductase (DHFR), thymidylate synthetase (TS) and the growth of murine fibrosarcoma Meth A cells in culture were examined. All N5-substituted glutamine analogs (4A-S) inhibited DHFR much more strongly than TNP-351 and some analogs exhibited the same potent growth inhibition of Meth A cells as TNP-351. Some typical analogs (4Bb, 4Db, 4F, 4Oa) were also examined for inhibitory effects on the growth of methotrexate (MTX)-resistant human CCRF-CEM cells in culture and for in vivo antitumor activities against murine leukemia and solid tumors. MTX-resistant cells, with a defect in transport and decreased polyglutamylation activity, showed little cross resistance to the analog (4Oa) having a tetrazole moiety as a substituent of glutamine, which exhibited potent antitumor activities. These results demonstrate that the antifolate analogs (4) with N5-substituted glutamine in place of glutamic acid are novel potent DHFR inhibitors with activity against MTX-resistant tumors. The potent antitumor activity of these analogs (4) may result from their effective uptake via reduced folate carrier in combination with their potent inhibition of DHFR.

Either the alpha- or gamma-carboxyl group of the glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and its related compound (1a) was replaced with a 1H-tetrazole ring, and the inhibitory effects of the resulting compounds on dihydrofolate reductase (DHFR) and the growth of murine fibrosarcoma Meth A cells were examined. The gamma-tetrazole analogs (2) were found to be much more potent DHFR inhibitors than TNP-351, and strongly inhibited the growth of Meth A cells. On the other hand, the alpha-tetrazole analogs (3) were much less active against Meth A cells, even though their DHFR-inhibitory activity was comparable to that of TNP-351. These findings suggest that the alpha-carboxyl group plays an important role in effective uptake via the reduced folate carrier, and a novel DHFR inhibitor could be obtained by chemically modifying the gamma-carboxyl moiety while leaving the alpha-carboxyl group intact.

Novel pyrrolo[2,3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2,4-diaminopyrrolo[2,3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.