Synthesis and potent antifolate activity and cytotoxicity of B-ring deaza analogues of the nonpolyglutamatable dihydrofolate reductase inhibitor N-alpha-(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523)

Six new B-ring analogues of the nonpolyglutamatable antifolate N-alpha-(4-a mino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523, 3) were synt hesized with a view to determining the effect of modifications at the 5- an d/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell gr owth inhibition The 5- and 8-deaza analogues were prepared from methyl 2-L- amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N-10-formyl-5-deaza- and -8-deazapteroic acid, respectively. The 5,8-dideaza analogues were prepared from methyl 2-L-[(4-aminobenzoyl)-amino]-5-phthalimidopentanoate and 2,4-d iaminoquinazoline-6-carbonitriles. The K-i for inhibition of human DHFR by the 5-deaza and 5-methyl-5-deaza analogues was about the same as that of 3 (0.35 pM), 11-fold lower than that of aminopterin (AMT, 1), and 15-fold low er than that of methotrexate (MTX, 2). However the K-i of the 8-deaza analo gue was 27-fold lower than that of 1, and that of the 5,8-dideaza, 5-methyl -5,8-dideaza, and 5-chloro-5,8-dideaza analogues was approximately 50-fold lower. This trend was consistent with the published literature on the corre sponding DHFR inhibitors with a glutamate side chain. In colony formation a ssays against the human head and neck squamous carcinoma cell line SCC25 af ter 72 h of treatment, the 5- and 8-deaza analogues were approximately as p otent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Met hyl and 5-chloro substitution was also favorable, with the 5-methyl-5-deaza analogue being 2.5-fold more potent than the 5-deaza analogue. However the effect of 5-methyl substitution was less pronounced in the 5,8-dideaza ana logues than in the 5-deaza analogues. The 5-chloro-5,8-dideaza analogue of 3 was the most active member of the series, with an IC50 = 0.33 nM versus 1 .8 nM for 3 and 15 nM for MTX. The 5-methyl-5-deaza analogue of 3 was also tested at the National Cancer Institute against a panel of 50 human tumor c ell lines in culture and was consistently more potent than 3, with IC50 val ues in the low nanomolar to subnanomolar range against most of the tumors. Leukemia and colorectal carcinoma cell lines were generally most sensitive, though good activity was also observed against CNS tumors and carcinomas o f the breast and prostate. The results of this study demonstrate that B-rin g analogues of 3 inhibit DHFR activity and tumor cell colony formation as w ell as, or better than, the parent compound. In view of the fact that 3 and its B-ring analogues cannot form polyglutamates, their high cytotoxicity r elative to the corresponding B-ring analogues of AMT is noteworthy.