Analogues of the potent nonpolyglutamatable antifolate N-alpha-(4-amino-4-deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, p-aminobenzoyl moiety, or 9,10-bridge: Synthesis and in vitro antitumor activity

Seven N-alpha-(4-amino-4-deoxypteroyl)-N-sigma-hemiphtha (2, PT523) analogu es were synthesized by modifications of the literature synthesis of the cor responding AMT (1) analogues and were tested as inhibitors of tumor cell gr owth. in growth assays against cultured CCRF-CEM human leukemic cells expos ed to drug for 72 h, the IC50 values of analogues in which N-10 was replace d by CH2 and CHMe were found to be 0.55 +/- 0.07 and 0.63 +/- 0.08 nM, and thus these analogues are more potent than 1 (IC50 = 4.4 +/- 1.0 nM) or 2 (I C50 = 1.5 +/-: 0.39 nM). The 10-ethyl-10-deaza analogue of 2 (IC50 = 1.2 +/ - 0.25 nM) was not statistically different from 2 but was more potent than edatrexate, the 10-ethyl-10-deaza analogue of 1, which had an IC50 of 3.3 /- 0.36 nM. In contrast, the analogue of 2 with both an ethyl and a CO2Me g roup at the 10-position had an IC50 of 54 +/- 4.9 nM, showing this modifica tion to be unfavorable. The 4-amino-1-naphthoic acid analogue of 2 had an I C50 Of 1.2 +/- 0.22 nM, indicating that replacement of the p-aminobenzoic a cid (pABA) moiety does not diminish cytotoxicity. The analogues in which th e (CH2)(3) Side chain was replaced by slightly longer CH2SCH2 and (CH2)(2)- SCH2 groups gave IC50 values of 4.4 +/- 1.1 and 5.0 +/- 0.56 nM and thus we re somewhat less potent than the parent molecule. However the analogues in which the aromatic COOH group was at the meta and para positions of the pht haloyl ring had IC50 values of 7.5 +/- 0.47 and 55 +/- 0.07 nM, confirming the low potency we had previously observed with these compounds against oth er cell lines. Overall, the results in this study support the conclusion th at, while the position of the phthaloyl COOH group and the length of the am ino acid side chain in 2 are important determinants of cytotoxic potency, c hanges in the pABA region and 9,10-bridge are well-tolerated and can even i ncrease potency.