Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine (PT523) and its B-ring analogues.

The potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) and six of its B-ring (5-deaza, 8-deaza, and 5,8-dideaza) analogues were compared in terms of their ability to: (a) inhibit the growth of CCRF-CEM human leukemic lymphoblasts, and (b) utilize the reduced folate carrier (RFC) in these cells as measured in a competition assay of [(3)H]methotrexate ([(3)H]MTX) influx. The IC(50) values of the hemiphthaloylornithine derivatives against CCRF-CEM cells after 72 hr of drug exposure varied from 0.64 to 1.3 nM as compared with 14 nM for MTX and 4.4 nM for aminopterin (AMT). The K(i) values of these compounds in the [(3)H]MTX influx assay were in the 0.3 to 0.7 microM range as compared with a K(i) of 5.4 microM for AMT and a K(t) of 7.1 microM for MTX. As a group, the affinities of these compounds for the RFC were approximately 10-fold greater than those of their respective glutamate analogues. These results indicate that, in addition to their previously reported tight binding to dihydrofolate reductase, a property contributing to the high potency of PT523 and its B-ring analogs as inhibitors of tumor cell growth is their strong affinity for the RFC.

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(delta)-hemiphthaloyl-L-o rnithine (2, PT523) analogues were synthesized by modifications of the literature synthesis of the corresponding AMT (1) analogues and were tested as inhibitors of tumor cell growth. In growth assays against cultured CCRF-CEM human leukemic cells exposed to drug for 72 h, the IC(50) values of analogues in which N(10) was replaced by CH(2) 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 (IC(50) = 4.4 +/- 1.0 nM) or 2 (IC(50) = 1.5 +/- 0.39 nM). The 10-ethyl-10-deaza analogue of 2 (IC(50) = 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 IC(50) of 3.3 +/- 0.36 nM. In contrast, the analogue of 2 with both an ethyl and a CO(2)Me group at the 10-position had an IC(50) of 54 +/- 4.9 nM, showing this modification to be unfavorable. The 4-amino-1-naphthoic acid analogue of 2 had an IC(50) of 1.2 +/- 0.22 nM, indicating that replacement of the p-aminobenzoic acid (pABA) moiety does not diminish cytotoxicity. The analogues in which the (CH(2))(3) side chain was replaced by slightly longer CH(2)SCH(2) and (CH(2))(2)SCH(2) groups gave IC(50) values of 4.4 +/- 1.1 and 5.0 +/- 0.56 nM and thus were 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 phthaloyl ring had IC(50) values of 7.5 +/- 0.47 and 55 +/- 0.07 nM, confirming the low potency we had previously observed with these compounds against other cell lines. Overall, the results in this study support the conclusion that, while the position of the phthaloyl COOH group and the length of the amino acid side chain in 2 are important determinants of cytotoxic potency, changes in the pABA region and 9, 10-bridge are well-tolerated and can even increase potency.

The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine.

N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.

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

Six new B-ring analogues of the nonpolyglutamatable antifolate Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloy l-L-ornithine (PT523, 3) were synthesized with a view to determining the effect of modifications at the 5- and/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell growth inhibition. The 5- and 8-deaza analogues were prepared from methyl 2-L-amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N10-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-diaminoquinazoline-6-carbonitriles. The Ki 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 lower than that of methotrexate (MTX, 2). However the Ki of the 8-deaza analogue 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 corresponding DHFR inhibitors with a glutamate side chain. In colony formation assays against the human head and neck squamous carcinoma cell line SCC25 after 72 h of treatment, the 5- and 8-deaza analogues were approximately as potent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Methyl 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 analogues 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 cell lines in culture and was consistently more potent than 3, with IC50 values 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 of the breast and prostate. The results of this study demonstrate that B-ring analogues of 3 inhibit DHFR activity and tumor cell colony formation as well 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 relative to the corresponding B-ring analogues of AMT is noteworthy.

Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) modified in the side chain: synthesis and biological evaluation.

Four heretofore undescribed side chain analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523, 4) were synthesized via straightforward methods of antifolate chemistry, and their properties were compared with those of PT523 and two related compounds with the aim of defining the contribution of the hemiphthaloyl-L-ornithine moiety to the exceptional in vitro antitumor activity of this novel non-polyglutamatable aminopterin analogue. The IC50 values of N alpha-(4-amino-4-deoxypteroyl)-N beta-hemiphthaloyl-L-2,3-diaminopropanoic acid (10) and N alpha-(4-amino-4-deoxypteroyl)-N gamma-hemiphthaloyl-L-2,4- diaminobutanoic acid (9) against A549 human non-small-cell lung carcinoma cells in culture were 23 and 22 nM, whereas those of PT523 and N alpha-(4-amino-4-deoxypteroyl)-N epsilon-hemiphthaloyl-L-lysine (8) were 1.3 and 5.2 nM. A decrease in the in vitro activities of 8 and 9 relative to PT523 was also observed against the panel of cell lines used by the National Cancer Institute to screen new drugs. However the potency of 8 and 9 remained several times greater than that of the historical control methotrexate against many of the cell lines in the screening panel. In an in vivo tumor model, SCC-VII murine squamous cell carcinoma, 9 and methotrexate were well tolerated as 5-day continuous infusions at doses of 0.52 and 0.75 mg/kg/day, whereas the highest tolerated dose of PT523 on this schedule was 0.19 mg/kg/day, in agreement with its lower IC50 in culture. To assess the importance of the hemiphthaloyl group in PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-isophthaloyl-L-ornithine (11), N alpha-(4-amino-4-deoxypteroyl)-N delta-terephthaloyl-L-ornithine (12), and N alpha-(4-amino-4-deoxypteroyl)-N delta-(4,5-dichlorohemiphthaloyl)-L-ornithine (13) were also synthesized. The IC50 values of 11 and 12 against A549 cells were 45 and 3300 nM, as compared with 1.3 nM for PT523 and 23 nM for methotrexate. In a clonogenic assay against SCC25 human squamous cell carcinoma cells, the IC50 values of 11 and 12 were 2.9 and 72 nM, as compared with 0.3 nM for PT523 and 27 nM for methotrexate. Thus, activity was decreased by moving the aromatic carboxyl group in PT523 to the meta position and was further diminished by moving it to the para position. The IC50 of the halogenated analogue 13 against SCC25 human head and neck squamous carcinoma cells was 18 nM, suggesting lack of tolerance for this 4,5-disubstitution in the phthaloyl moiety. Our results suggest that the combination of a hemiphthaloyl group and three CH2 groups in the side chain are critical determinants of the potent in vitro activity of PT523.