NONCLASSICAL 2,4-DIAMINO-5-ARYL-6-ETHYLPYRIMIDINE ANTIFOLATES - ACTIVITY AS INHIBITORS OF DIHYDROFOLATE-REDUCTASE FROM PNEUMOCYSTIS-CARINIIAND TOXOPLASMA-GONDII AND AS ANTITUMOR AGENTS

Twelve novel 2,4-diamino-5-(4'-benzylamino)- and hylbenzylamino)-3'-ni trophenyl]-6-ethylpyrimidines bearing 4-substituents on the benzylamin o or N-methylbenzylamino aryl ring were synthesized and evaluated as n onclassical inhibitors of Pneumocystis carinii and Toxcoplasma gondii dihydrofolate reductase (DHFR), Compounds were prepared by reaction of 2,4-diamino-5-(4'-chloro-3'-nitrophenyl)- (8) or ino-5-(4'-fluoro-3'- nitrophenyl)-6-ethylpyrimidine (15) with the appropriate 4-substituted (CO2H, CO2Me, SO2NH2, dioxolan-2-yl, CHO, dimethyloxazolin-2-yl) benz ylamine or N-methylbenzylamine derivative. Compounds 25-29 were synthe sized from 2,4-diamino-5-{4'-[N-(4 oxybenzyl)amino]-3'-nitrophenyl}-6- ethylpyrimidine (10) and the corresponding amine (NH3, MeNH2, Me2NH, p iperidine, diethyl L-glutamate) via isobutyl mixed anhydride coupling; hydrolysis of the diethyl L-glutamate 29 afforded the L-glutamate ana logue 30. The compounds exhibited potent inhibitory activity against T . gondii (IC50 values 0.0018-0.14 mu M) and rat liver (IC50 values 0.0 029-0.27 mu M) DHFR, with a 4-substituent invariably enhancing binding to both enzymes relative to the unsubstituted benzoprim (5) or methyl benzoprim (6). Modest selectivity for T. gondii enzyme was observed wi th several analogues, whereas all of the compounds were relatively wea k inhibitors of P. carinii DHFR and exhibited no selectivity. Selected analogues were evaluated for in vivo antitumor activity against the m ethotrexate-resistant; M5076 murine reticulosarcoma, with 2,4-diamino- 5-{4'-[N-[4 ]-N-methylamino]-3'-nitrophenyl}-6-ethylpyrimidine (14) (K -i for rat liver DHFR = 0.000 35 +/- 0.000 29 nM) combining significan t antitumor activity with minimal toxicity.