5-DEAZA-7-DESMETHYLENE ANALOGS OF 5,10-METHYLENE-5,6,7,8-TETRAHYDROFOLIC ACID AND RELATED-COMPOUNDS - SYNTHESIS AND IN-VITRO BIOLOGICAL-ACTIVITY .49.

-[4-(tert-Butyloxycarbonyl)phenyl]-3-pyrrolidinone and 1-[3-(tert-buty loxycarbonyl)phenyl]-4-piperidinone were condensed with ethyl cyanoace tate or malononitrile to form ylidene derivatives, which were then sub jected sequentially to (i) catalytic or chemical reduction, (ii) conde nsation with guanidine, and (iii) gentle trifluoroacetic acid treatmen t to obtain -oxopyrimidin-5-yl)-1-(4-carboxyphenyl)pyrrolidine (27), 5 H)-oxopyrimidin-5-yl)-1-(carboxyphenyl)piperidine (35), and iaminopyri midin-5-yl)-1-(carboxyphenyl)pyrrolidine (40). Condensation of 27, 35, and 40 with diethyl or di-tert-butyl L-glutamate followed by removal of the ester groups yielded -oxopyrimidin-5-yl)pyrrolidino]benzoyl]-L- glutamic acid (13), )-oxopyrimidin-5-yl)piperidino]benzoyl]-L-glutamic acid (14), and minopyrimidin-5-yl)pyrrolidino]benzoyl]-L-glutamic aci d (15). Compounds 13 and 14 may be viewed as 5-deaza-7-desmethylene an alogues of 5,10-methylene-5,6,7,8-tetrahydrofolic and 5,10-ethylene-5, 6,7,8-tetrahydrofolic acid, respectively. Compounds 13 and 15 were goo d substrates for mouse liver folylpolyglutamate synthetase, with K(m) values of 20 and 18 muM and a relative first-order rate constant V(max )/K(m) of 2.2 (aminopterin = 1.0). In contrast, 14 was a very poor sub strate, with a K(m) of 490 muM and a relative V(max)/K(m) of 0.052. As expected from its structure, 15 was a dihydrofolate reductase inhibit or. However its potency was unexceptional (IC50 = 1.2 muM). Compounds 13 and 14 were inactive at concentrations of up to 100 muM, and likewi se showed no activity against thymidylate synthase or glycinamide ribo tide formyltransferase, two other key enzymes of folate-mediated one-c arbon metabolism. Compound 15 was moderately active as an inhibitor of the growth of cultured tumor cells (SCC25 human squamous cell carcino ma), with an IC50 of 0.37 muM (72 hour exposure). By comparison the IC 50 of aminopterin was 0.0069 muM. Thus, even though 15 is a good folyl polyglutamate synthetase substrate, the deep-seated skeletal changes e mbodied in this structure are unfavorable for DHFR binding and may als o be unfavorable for transport into cells.