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The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c , was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b , and 2-amino, 2a , counterparts, as well as N 10 -propargyl-5,8-dideazafolic acid, 1a , and the corresponding 2-desamino, 1b , and 2-desamino-2-methyl, 1c , modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c , respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c , its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folylpolyglutamate synthetase of the compounds studied having a V max / K m nearly 12-fold larger than 1c . Both 1c and 2c were effective inhibitors of the uptake of [ 3 methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.

Six new analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin were synthesized in an effort to obtain enhanced antitumor activity. The modifications included the replacement of the 2-amino group by hydrogen or methyl as well as the inclusion of a methyl substituent at position 9. Based upon activity against L1210 leukemia cells in culture, three of the new analogues together with one compound described previously were evaluated for cytotoxicity in vitro using three human tumor cell lines (Colo 320 DM, Hep G2 and HL-60). The most effective compound was 2-desamino-N9-methyl-5,8-dideazaisoaminopterin (2c) with the HL-60 cells being the most sensitive to its cytotoxic effects. These analogues were evaluated in vitro as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) from human as well as bacterial (Lactobacillus casei) sources. All four of the 4-amino analogues were most effective toward L. casei DHFR compared with human DHFR, with 2-desamino-2-methyl-5,8-dideazaisoaminopterin (2d) and its 9-methyl derivative (2e) having 818- and 430-fold greater selectivity (L. casei/human). Most of the compounds studied were found to be only modest inhibitors of human TS (I50 values = 1.5 to 20 μM) and were therefore at least 40-fold less inhibitory than 10-propargyl-5,8-dideazafolic acid. Nevertheless, reversal of cytotoxicity studies with thymidine, hypoxanthine and folinic acid using the HL-60 cell line suggested that TS is the primary target for these analogues.

The in vitro metabolism of a variety of 5,8-dideazafolate and 5,8-dideazaisofolate analogues by pig liver folylpolyglutamate synthetase and the specificity of the enzyme for some polyglutamate derivatives of these analogues have been investigated. All 4-oxo-quinazoline analogues were metabolized to long chain polyglutamate derivatives, primarily the pentaglutamate, whereas 4-amino-quinazolines were metabolized to a lesser extent, with the accumulation of di- and triglutamate derivatives. This pattern of metabolism was consistent with the large drop in V max K m and V max values for folylpolyglutamate synthetase observed with diglutamate derivatives of 4-aminofolate analogues. The extent of metabolism of the various analogues did not correlate with the relative substrate effectiveness of their parent monoglutamate derivatives. The 5-chloro and 5-methyl substitutions of quinazolines enhanced the addition of glutamate residues to 4-amino derivatives but markedly impaired the metabolism of 4-oxo derivatives.

A series of fourteen 5,8-dideaza analogues of folic and pteroic acids was evaluated for inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR TFase) from chicken liver. Of the 5,8-dideaza folate derivatives studies, 10-oxa-5,8-dideazafolic acid was the most potent inhibitor. The addition of one L-glutamate moiety to the gamma-carboxyl group caused a 6- to 7-fold reduction in Ki in three instances. Two compounds devoid of an L-glutamate were 4- to 6-fold less inhibitory than their parent counterparts possessing one L-glutamate residue.

Data are presented on the systematic analysis of thirty-five quinazoline and substituted triazine compounds as inhibitors of a methotrexate-insensitive form of dihydrofolate reductase purified from methotrexate-resistant L5178Y murine leukemia cells. Several of the compounds were found to be more potent inhibitors of this enzyme activity than was methotrexate. Two of the triazine compounds had IC50 values approaching 10 nM, which is close to that of methotrexate for the normal drug-sensitive dihydrofolate reductase. In addition, some of these compounds, especially the triazines, exhibit a specificity of inhibition for the methotrexate-insensitive enzyme as compared to the normal methotrexatesensitive dihydrofolate reductase derived from the same cell line. These compounds may, therefore, be potentially useful in the treatment of those methotrexate-resistant tumours which express an altered, methotrexate-insensitive dihydrofolate reductase.

A series of eighteen 2,4-diaminoquinazoline analogues of folic, isofolic, pteroic and isopteroic acids having various substituents at position 5 was studied. Each compound was evaluated as an inhibitor of L1210 dihydrofolate reductase, methotrexate influx into L1210 leukemia cells, and growth of methotrexate-sensitive and -resistant L1210 cells in vitro. Bridge reversal at positions 9 and 10 reduced the effectiveness of the classical analogues only with regard to the inhibition of the drug-sensitive cells as compared to methotrexate (MTX). Absence of the glutamate moiety adversely affected the potency of the compounds, particularly when coupled with reversal of the 9,10-bridge. However, the presence of -Cl at position 5 restored significantly the potency of these compounds. The pteroate and isopteroate analogue ethyl esters were generally more effective inhibitors of cell growth than their non-esterified counterparts. Regarding the effects of substituents at position 5, the data suggest that -Cl greater than -CH3 greater than -H for inhibition of methotrexate transport and growth of methotrexate-sensitive L1210 cells. The 5-Cl pteroate analogue and its corresponding ethyl ester were highly effective as growth inhibitors of methotrexate-resistant, transport-defective, L1210 cells in vitro.

Inhibition by a variety of substituted triazines and quinazolines of a methotrexate-insensitive form of dihydrofolate reductase from highly MTX-resistant L5178Y mouse leukemia cells was examined. Some of these compounds were significantly more potent than MTX (up to 100-fold). Two triazenes, terminally substituted with benzenesulfonylfluoride residues, were approximately 30-fold more potent than MTX. Quinazoline analogs of folic acid with alterations in different parts of the molecule varied in their potencies as inhibitors. Although none of the compounds tested was as potent as MTX against MTX-sensitive dihydrofolate reductases, these studies show that some types of folate antagonists have increased specificity against this MTX-insensitive dihydrofolate reductase. This finding increases the likelihood that it may be possible to produce compounds with marked specificity for the insensitive reductase. Such compounds might have utility in antifolate combinations designed to overcome methotrexate resistance.

A selected number of 1,3-diaminobenzo[f]quinazolines and 1,3-diamino-5,6-dihydrobenzo[f]quinazolines, which may be viewed as tricyclic analogues of the lipid-soluble antifolates pyrimethamine (PM), metoprine (DDMP), and etoprine (DDEP), were tested as inhibitors of purified dihydrofolate reductase (DHFR) from WI-L2 lymphoblasts, and as inhibitors of the growth of Streptococcus faecium ATCC 8043 and L1210 murine leukemia cells in culture. In addition, these tricyclic compounds were tested for antimalarial activity against Plasmodium berghei in mice, and for the ability to inhibit the growth of Pneumocystis carinii trophozoites in WI-38 human lung fibroblast cultures in the presence of leucovorin (LV). The most potent analogues were those with chlorine substitution in the ring distal to the 2,4-diaminopyrimidine moiety. Fully aromatic compounds tended to be more active than those in which the 5,6-bond was reduced, suggesting that planarity favors binding to the DHFR active site and may be favorable for cellular uptake. Several of the 2,4-diaminopyrimidine analogues showed greater potency than PM, DDMP or DDEP, and were more nearly comparable to the bicyclic 2,4-diaminopyrimidine antifolates trimetrexate (TMQ) or piritrexim (BW301U), which are known to be selectively toxic to P. carinii in the presence of LV. Two of the tricyclic compounds, 1,3-diamino-8-chlorobenzo[f]quinazoline and 1,3-diamino-9-chlorobenzo[f]quinazoline, proved to have activity similar to TMQ and BW301U in this system.