Formation and retention and biological activity of N10-propargyl-5,8-dideazafolic acid (CB3717) polyglutamates in L1210 cells in vitro

The formation, retention and biological activity of the polyglutamate metabolites of the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) has been investigated in L1210 murine leukaemia cells grown in vitro. CB3717 polyglutamates were measured by HPLC using high specific activity 3H-CB3717. Following the exposure of cells to 50 microM CB3717 for 6, 12 and 24 hr total cellular radioactivity corresponded to 4.5 +/- 1.5, 6.8 +/- 3.6 and 5.9 +/- 3.4 microM drug derived material, respectively. Of this material, greater than 70%, 57 +/- 3% and 51 +/- 5% was in the form of unchanged CB3717 at 6, 12 and 24 hr respectively. The remaining radioactivity was associated with polyglutamate metabolites of CB3717, predominantly the tetra and pentaglutamate forms. Following the removal of extracellular drug after incubation for 24 hr and resuspension in drug free medium, unchanged CB3717 was lost rapidly from the cells such that after 6 hr it accounted for only 5% of total cellular radioactivity. In contrast, levels of CB3717 tetra and pentaglutamates declined solely due to dilution during cell division. Measurement of the whole cell TS activity by 3H-deoxyuridine incorporation into DNA indicated that, despite the loss of unchanged CB3717 from the cell, enzyme activity remained suppressed (less than 10% of control) for at least 24 hr after resuspension in drug free medium. The TS inhibitory activity of the polyglutamated metabolites of CB3717 was investigated using enzyme purified from L1210 cells. As inhibitors, the metabolites were 26-, 87-, 119- and 114-fold more potent than CB3717 as the di-, tri-, tetra- and pentaglutamate forms, respectively. However, as inhibitors of dihydrofolate reductase prepared from rat liver, CB3717 polyglutamates were no more than 5-fold More potent than the parent compound. This study has shown that CB3717 can undergo polyglutamation in tumour cells and that the metabolites are preferentially retained giving rise to prolonged TS inhibition. By virtue of their potent TS inhibitory activity these metabolites are, therefore, most probably the intracellular effectors of CB3717 cytotoxicity.     

Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.     

Potent inhibition of thymidylate synthase by two series of nonclassical quinazolines

The synthesis and biological activity of two series of nonclassical thymidylate synthase (TS) inhibitors are described. The first is a series of 10-propargyl-5,8-dideazafolic acid derivatives (10a-j) and the second is a series of the analogous 2-desamino derivatives (13a-c,k), both bearing a more lipophilic substituent on the phenyl ring than the CO-glutamate of classical antifolates. Compounds 10a-j were prepared in a straightforward manner, generally by treatment of N-[6-(bromomethyl)-3,4-dihydro-4-oxo-2-quinazolinyl]-2,2-dimethylprop anamide (6) with various phenyl-substituted N-propargylanilines (8), followed by deprotection. Compounds 13a-c,k were prepared similarly from [6-(bromomethyl)-4-oxo-3(4H)-quinazolinyl] methyl 2,2-dimethylpropanoate (11). The compounds were tested for inhibition of purified L1210 TS and for inhibition of L1210 cell growth in vitro. Several of these nonclassical analogues approached the TS inhibitory potency of 10-propargyl-5,8-dideazafolic acid (1, CB3717), a glutamate-containing TS inhibitor. 2-Amino target compounds 10a-j were generally potent inhibitors of L1210 TS, with IC50s within the range of 0.51-11.5 microM, compared to 0.05 microM for 1. The order of potency for phenyl substitution at the 4-position in this series was the following: COCF3 greater than or equal to NO2 greater than or equal to CONH2 greater than or equal to COCH3 greater than SO2NMe2 greater than CN much greater than OCF3 greater than or equal to F. The 2-desamino target compounds 13a-c,k also exhibited significant, although diminished, TS inhibition. Both series were growth inhibitory to cells in tissue culture and this inhibition could be reversed by thymidine alone, indicating that the primary target was TS. None of the compounds was a potent inhibitor of dihydrofolate reductase. These studies indicate that the presence of the glutamate moiety in folate analogues is not an absolute requirement for potent inhibition of TS.     

Modulation of anti-metabolite effects. Effects of thymidine on the efficacy of the quinazoline-based thymidylate synthetase inhibitor, CB3717

CB3717 (N-(4-(N-((2-amino-4- hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino ) benzoyl)-L-glutamic acid) is an antitumour agent that inhibits thymidylate synthetase (TS). A dose-dependent fall in plasma thymidine (dThd) (1.43 microM to 0.47 microM) occurred in non-tumour-bearing mice following the administration of CB3717. Further, in mice carrying the L1210/CBRI tumour, the drug's antitumour properties were ablated by co-administration of dThd, an effect consistent with TS being the cytotoxic locus. In vitro studies of protection by dThd against CB3717 cytotoxicity were carried out in an attempt to quantify this reversal. The metabolism of [14C]-dThd was measured in cultures of L1210 cells (10(4)/ml) exposed to a completely cytotoxic dose of CB3717 (50 microM). The cytotoxicity of the drug was only expressed when the dThd concentration (0.5-2 microM) had fallen to less than 0.1 microM in the media. This reduction was due to: (1) dThd incorporation into DNA, (2) catabolism of dThd to thymine. By reducing the initial cell concentration to 10(3)/ml the depletion of dThd was substantially reduced and consequently cells continued to grow for a longer period. The critical concentration of dThd, below which growth in the presence of CB3717 could not be supported was estimated to be between 0.026 and 0.1 microM. Thus even the minimum level of dThd achieved in vivo was still in excess of that required for protection from CB3717 toxicity in vitro. There was a small accumulation of deoxyuridine (dUrd) (approximately 2-fold) in mouse plasma 24 hr after completion of a 5-day course of CB3717 (200 mg/kg) but in vitro studies demonstrated that this was unlikely to modulate CB3717 toxicity in the presence of dThd. We caution against the use of rodent tumour models (or human tumour xenografts) for antitumour or toxicity testing of compounds designed to inhibit the de novo synthesis of thymidylate; they may be misleading because the high dThd levels found in these animals compared with man may mask the cytotoxic effects of these drugs.     

Quinazoline antifolates inhibiting thymidylate synthase: benzoyl ring modifications

Four new analogues of the antifolate N10-propargyl-5,8-dideazafolic acid were prepared that were substituted in the benzoyl ring. The 2'-chloro and 2'-methyl analogues were prepared from the appropriately substituted p-nitrobenzoic acids. The route to the 3'-chloro and 3',5'-dichloro analogues was by chlorination of diethyl N10-propargyl-5,8-dideazafolate and diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate, respectively, using sulfuryl chloride. The compounds were tested for their inhibition of purified L1210 thymidylate synthase (TS), for their inhibition of purified L1210 dihydrofolate reductase (DHFR), and for their inhibition of the growth of L1210 cells in culture. The 2'-chloro substituent reduced the TS inhibition by twofold and the 2'-methyl substituent reduced it by 20-fold; the 3'-chloro and 3',5'-dichloro derivatives were very poor inhibitors. The substituents only slightly affected the DHFR inhibition. None of the compounds improved upon N10-propargyl-5,8-dideazafolic acid in inhibiting the growth of L1210 cells in culture.     

Quinazoline antifolates inhibiting thymidylate synthase: 2-desamino derivatives with enhanced solubility and potency

The poor solubility of the thymidylate synthase (TS) inhibiting antifolate 10-propargyl-5,8-dideazafolic acid has posed problems for its clinical use and is probably responsible for its renal toxicity. The insolubility is caused by the 2-amino-3,4-dihydro-4-oxopyrimidine moiety of the drug which stabilizes the solid state by intermolecular hydrogen bonding. In examining this moiety we have removed the 2-amino group and now report on 2-desamino-10-propargyl-5,8-dideazafolic acid (8e) and four analogues with H, Me, Et, and allyl at N10. 3,4-Dihydro-4-oxo-6-methylquinazoline was solubilized by alkylating the lactam nitrogen with chloromethyl pivalate. Reaction with N-bromosuccinimide gave the corresponding 6-bromomethyl compound, which was coupled with diethyl N-(4-aminobenzoyl)-L-glutamate or the appropriate N-substituted derivative thereof. The quinazoline N3 nitrogen and carboxyl groups in the product were simultaneously deprotected by cold alkali in the final step to give the desired five antifolates. These were tested against L1210 TS and it was found that removal of the 2-amino group caused a slight (3-9-fold) loss of TS inhibition. 8e was only 8-fold a lesser TS inhibitor than the parent drug. Inhibition of rat liver dihydrofolate reductase was reduced by over 1 order of magnitude for three compounds tested. All five analogues were more cytotoxic to L1210 cells in culture than their 2-amino counterparts; 8e was 8.5-fold more active with an ID50 of 0.4 microM. This remarkable result probably owes to increased cellular penetration. 8e was 5-fold more soluble than 1 at pH 5.0 and greater than 340-fold more soluble at pH 7.4.     

Synthesis and antifolate evaluation of the 10-propargyl derivatives of 5-deazafolic acid, 5-deazaaminopterin, and 5-methyl-5-deazaaminopterin.

5-Deaza-10-propargylfolic acid (4), an analogue of the thymidylate synthase (TS) inhibitor 10-propargyl-5,8-dideazafolic acid (PDDF, 1), was prepared via alkylation of diethyl N-[4-(propargylamino)benzoyl]-L-glutamate (7) by 2-amino-6-(bromomethyl)-4(3H)-pyrido[2,3-d]pyrimidinone (15). Bromomethyl intermediate 15 was prepared from the corresponding hydroxymethyl precursor 14 by treatment with 48% HBr. Hydroxymethyl compound 14 was obtained by deamination of reported 2,4-diaminopyrido[2,3-d]pyrimidine-6-methanol (12a) in refluxing 1 N NaOH. Both 12a and its 5-methyl-substituted analogue 12b were converted to versatile 6-bromomethyl intermediates 13a and 13b from which important antifolates may be readily derived. Alkylation of 7 by 13a,b led to 10-propargyl-5-deazaaminopterin (5) and 5-methyl-10-propargyl-5-deazaaminopterin (6). As an inhibitor of TS from H35F/F cells, 4 gave an IC50 value showing it to be approximately 6-fold less inhibitory than PDDF (90 nM for 4 vs 14 nM for PDDF). In in vitro studies, IC50 (microM) values obtained for 4 vs L1210 and S180 of 1.50 and 2.35, respectively, were similar to those obtained for PDDF (2.61 and 1.97). Against HL60 cells, 4 was about 7-fold more cytotoxic than PDDF (IC50 values 0.72 and 5.29 microM). Inclusion of thymidine did not establish TS as the site of cytotoxic action for either 4 or PDDF in the cell lines used. In in vivo tests against L1210 in mice, 4 failed to show therapeutic effect. The 2,4-diamino compounds 5 and 6 were as potent inhibitors of DHFR from L1210 cells as MTX and 7- and 35-fold, respectively, more inhibitory than MTX toward L1210 cell growth. In mediated influx into L1210 cells, 5 and 6 were transported 2.7- and 8.5-fold, respectively, more readily than MTX. Against the EO771 mammary adenocarcinoma in mice, 6 produced greater antitumor effect than MTX. A dose of 36 mg/kg per day for 5 days caused no toxic deaths while the average tumor volume among 10 mice was reduced to 8-9% of that of the control, and 20% of the test animals were rendered tumor free.     

Modulation of dipyridamole action by alpha 1 acid glycoprotein. Reduced potentiation of quinazoline antifolate (CB3717) cytotoxicity by dipyridamole

Dipyridamole potentiates the cytotoxicity of N10-propargyl-5,8-dideazafolic acid (CB3717), an antifolate inhibitor of thymidylate synthase, by inhibiting both thymidine (TdR) salvage and deoxyuridine (UdR) efflux. Dipyridamole binds to the serum component alpha 1acid glycoprotein (alpha 1AGP) and hence the effects of alpha 1AGP on dipyridamole-induced changes in nucleoside transport and CB3717 cytotoxicity have been investigated. Using A549 lung cancer cells in vitro, alpha 1AGP reduced the inhibition of nucleoside transport by dipyridamole in a concentration-dependent manner. Between 10 and 200 times the concentration of dipyridamole was needed to inhibit TdR uptake to the same degree in medium containing 1 mg/ml alpha 1AGP (a physiological concentration) when compared to the uptake in alpha 1AGP-free medium. Although dipyridamole inhibited UdR efflux more than TdR efflux, inhibition of UdR efflux was reduced less than the inhibition of TdR efflux in the presence of 1 mg/ml alpha 1AGP. Thus, clinically achievable levels of dipyridamole (2.5-7.5 microM), even in the presence of physiological alpha 1AGP concentrations, caused significant inhibition of nucleotide uptake and efflux. The cytotoxicity of CB3717 was increased 2-3-fold by 3 and 10 microM dipyridamole in alpha 1AGP-free medium, whereas dipyridamole did not significantly (P greater than or equal to 0.05) potentiate CB3717 cytotoxicity in the presence of 1 mg/ml alpha 1AGP. Measured free dipyridamole levels indicated that the impaired inhibition of nucleoside transport and the lack of potentiation of CB3717 cytotoxicity in the presence of alpha 1AGP was due solely to the binding of dipyridamole to alpha 1AGP. It is concluded that alpha 1AGP levels will be a major determinant of the ability of dipyridamole to modulate the activity of antimetabolites in vivo.     

Quinazoline antifolate thymidylate synthase inhibitors: 2'-fluoro-N10-propargyl-5,8-dideazafolic acid and derivatives with modifications in the C2 position

The synthesis of 2'-fluoro-10-propargyl-5,8-dideazafolic acid and its 2-desamino, 2-desamino-2-hydroxymethyl, and 2-desamino-2-methoxy analogues is described. In general the synthetic route involved the coupling of diethyl N-[2-fluoro-4-(prop-2-ynylamino)benzoyl]-L-glutamate with the appropriate 6-(bromomethyl)quinazoline followed by deprotection with mild alkali. These four compounds together with the 2-desamino-2-methyl analogue were tested for their activity against L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of the L1210 cell line and of two mutant L1210 cell lines, the L1210:R7A that overproduces dihydrofolate reductase (DHFR) and the L1210:1565 that has impaired uptake of reduced folates. Compared with their non-fluorinated parent compounds, the 2'-fluoro analogues were all approximately 2-fold more potent as TS inhibitors. Similarly, they also showed improved inhibition of L1210 cell growth (1.5-5-fold), and this activity was prevented by co-incubation with thymidine. All had retained or improved activity against both the L1210:R7A and L1210:1565 cell lines.     

Methotrexate analogues--XVII. Antitumor activity of 4-amino-4-deoxy-N10-methylpteroyl-D,L-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and D,L-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase (IC50 = 1 nM), inhibited L1210 cell proliferation in culture (IC50 = 0.3 microM), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA (K1 = 190 +/- 70 microM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.     

Quinazoline antifolates inhibiting thymidylate synthase: 4-thio-substituted analogues

We report the synthesis of four new 4-thio-5,8-dideazafolic acid analogues and a 4-(methylthio) analogue structurally related to the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid. Three N10-propargyl-4-thio-5,8-dideazafolic acid analogues had C2 amino, hydrogen, and methyl substituents. A 4-thio and a 4-(methylthio) compound each with hydrogen at C2 and ethyl at N10 were also synthesized. In general, the synthetic route involved thionation of the appropriate 4-oxoquinazoline; the sulfur thus introduced was then protected by methylation. Further protection with a pivaloyl group was required for the quinazoline bearing a 2-amino substituent. The protected quinazolines were treated with N-bromosuccinimide and the resulting 6-(bromomethyl) compounds were then coupled to the appropriate N-monoalkylated diethyl N-(4-aminobenzoyl)-L-glutamate in N,N-dimethylacetamide with calcium carbonate as base. The 4-thio-5,8-dideazafolic acids were obtained by removal of the methylthio group with sodium hydrosulfide, followed by deprotection of the carboxyl groups with cold dilute alkali. For the compound containing a pivaloyl protecting group, hot dilute alkali was used. To obtain the 5,8-dideazafolic acid containing a 4-(methylthio) substituent, the corresponding diester was treated with lithium hydroxide which selectively deprotected the carboxyl groups. The five compounds were tested as inhibitors of L1210 TS. It was found that replacement of the 4-oxygen of the quinazoline moiety by sulfur did not alter the TS inhibition. However, the introduction of a methylthio substituent at position 4 severely impaired TS inhibition. All 4-thio compounds were less cytotoxic to L1210 cells in culture than their 4-oxo counterparts.     

Quinazoline antifolates inhibiting thymidylate synthase: variation of the N10 substituent

The synthesis of 12 new 5,8-dideazafolates with isopropyl, cyclopropylmethyl, 2-fluoroethyl, carbamoylmethyl, phenacyl, 3-fluorobenzyl, 5-uracilylmethyl, carboxymethyl, 2-carboxyethyl, 3-cyanopropyl, 3-hydroxypropyl, and cyanomethyl substituents at N10 is described. In general, the synthetic route involved monoalkylation of diethyl N-(4-amino-benzoyl)-L-glutamate, coupling of the resulting secondary amine with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide in N,N-dimethylacetamide with calcium carbonate as the base, and deprotection using mild alkali. The cyanomethyl derivatives was found to be unexpectedly base labile and was therefore prepared by mild acid deprotection of a di-tert-butyl ester. The compounds were tested as inhibitors of purified L1210 thymidylate synthase (TS). Four members of the series were more potent that the N10-hydrogen compound, but none was superior to the previously described N10-propargyl-5,8-dideazafolic acid. Selected compounds were examined as inhibitors of purified L1210 dihydrofolate reductase (DHFR). As desired, N10 substitution in general reduced DHFR inhibitory activity; these results are discussed. As a measure of cytotoxicity, the compounds were examined for their inhibition of the growth of L1210 cells in culture. None of the new substituents conferred enhanced potency relative to N10-propargyl-5,8-dideazafolic acid (ID50 = 5 microM), which, as the best TS inhibitor and a relatively poor DHFR inhibitor, continues to lead this series.     

Quinazoline antifolates inhibiting thymidylate synthase: computer modelling of the N10 substituent

The synthesis and biological properties of N10-(2,2,2-trifluoroethyl)-5, 8-dideazafolic acid are described. It was fivefold less active as an inhibitor of L1210 thymidylate synthase (TS) than its N10-ethyl congener and sevenfold less active as an inhibitor of the growth of L1210 cells in culture. CNDO calculations were performed on the following N10 substituents in a model fragment of 5, 8-dideazafolic acid: propargyl, ethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, cyanomethyl and methyl. The resulting values of partial charge on the distal terminus of the substituent correlated with the TS inhibition induced by the substituent. In particular, the mildly net positive charge on the acetylenic hydrogen in the propargyl substituent (+0.064) was not matched by any other in the series. N10-propargyl-5, 8-dideazafolic acid continues as the best inhibitor in this series.     

Quinazoline antifolates inhibiting thymidylate synthase: variation of the amino acid

Five new analogues (1c-g) of the antifolate N10-propargyl-5,8-dideazafolic acid (1a) are described in which the benzoyl-L-glutamate moiety was replaced by benzoic acid (desglutamyl-N10-propargyl-5,8-dideazafolic acid), benzoyl-L-aspartate, 4-phenylbutyrate, benzoylglycine, and benzoyl-L-alanine. The esters of the appropriate 4-aminophenyl (benzoyl) starting materials were sequentially alkylated upon nitrogen, first with a propargyl halide and then with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide. Saponification of the antifolate esters so produced gave the desired analogues. The new derivatives (1c-g) and also the known diethyl ester of 1a (1b) were tested for their inhibition of purified L1210 thymidylate synthase (TS) and for their inhibition of the growth of L1210 cells in culture. The TS inhibition of the analogues 1b-g was estimated by calculating the inverse relative potency, defined as the ratio IC50(compound)/IC50(1a). The results obtained were as follows: greater than 62, 84, 9, 333, 21, and 5, respectively. All were thus less inhibitory than 1a. None of the compounds improved upon 1a in inhibiting the growth of L1210 cells in culture.     

Kinetic characteristics of ICI D1694: a quinazoline antifolate which inhibits thymidylate synthase.

The thymidylate synthase (TS) inhibitor ICI D1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-S-glutamic acid) is a structural analogue of the substrate N5,N10-methylenetetrahydrofolate (5,10-CH2FH4) and is currently under clinical evaluation as a treatment for cancer. The compound is shown here to be a mixed non-competitive inhibitor of TS from murine leukemia (L1210) cells when 5,10-CH2FH4 is varied. This result suggests formation of an inactive complex between TS, 5,10-CH2FH4 and the inhibitor. Thus, binding to only one of the two active sites on the TS homodimer may be sufficient to prevent catalysis fully. Treatment of L1210 cells with ICI D1694 is known to cause intracellular accumulation of the tetraglutamate derivative which is shown here to have a 60-fold higher affinity for TS. The IC50 for inhibition of L1210 cell growth is below the Ki value of ICI D1694 for L1210 TS but above that of the tetraglutamate. The formation of polyglutamates and concentration of drug inside cells, therefore, seem to be responsible for biological activity.     

Comparison of the biological effects of selected 5,8-dideazafolate analogues with their 2-desamino counterparts

Three new 5,8-dideaza analogues of folic acid devoid of an amino group at position 2 have been prepared by using synthetic routes patterned after earlier methodologies. They were 2-desamino-5,8-dideazaisofolic acid, 2b, 2-desamino-10-thia-5,8-dideazafolic acid, 2c, and 2-desamino-10-oxa-5,8-dideazafolic acid, 2d. These compounds were found to be 4-6-fold more cytoxic toward L1210 leukemia cells than their 2-NH2 counterparts and to be poor inhibitors of mammalian thymidylate synthase. However, they were only 1.5-3-fold less inhibitory toward dihydrofolate reductase than the analogous compounds containing a 2-NH2 group. The known thymidylate synthase inhibitors 2-desamino-10-propargyl-5,8-dideazafolic acid and 10-propargyl-5,8-dideazafolic acid were included in this study for purposes of comparison.     

Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid

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 N10-propargly-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 folyl-polyglutamate synthetase of the compounds studied having a Vmax/Km nearly 12-fold larger than 1c. Both 1c and 2c were effective inhibitors of the uptake of [3H]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.     

Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.

Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.     

Methotrexate analogues. 19. Replacement of the glutamate side chain in classical antifolates by L-homocysteic acid and L-cysteic acid: effect on enzyme inhibition and antitumor activity

Methotrexate (MTX) and aminopterin (AMT) analogues containing L-homocysteic acid or L-cysteic acid in place of L-glutamic acid were synthesized and tested as inhibitors of dihydrofolate reductase from L1210 cells and folyl polyglutamate synthetase from mouse liver. The ID50 against dihydrofolate reductase was comparable for the MTX and AMT analogues (0.04-0.07 microM), whereas the ID50 against folyl polyglutamate synthetase was 3- to 4-fold lower for the AMT analogues (40-60 microM) than for the MTX analogues (100-200 microM). Thus, N10-substitution has a greater effect on binding to folyl polyglutamate synthetase than dihydrofolate reductase. The cytotoxicity of these compounds was assayed in vitro against L1210 cells, and the AMT analogues again proved more potent (ID50 = 0.03-0.05 microM) than the MTX analogues (ID50 = 0.1-0.4 microM). A similarly increased potency was observed for the AMT analogues against L1210 leukemia in vivo. Though differential cell uptake cannot be ruled out as the basis of increased potency, it is possible that part of the activity of the AMT analogues involves interference with the intracellular polyglutamation of reduced folate cofactors, i.e., that they are "self-potentiating antifolates". Of the four compounds reported, the most active was N-(4-amino-4- deoxypteroyl )-L-homocysteic acid, which produced a 138% increase in life span (ILS) in L1210 leukemic mice when given on a modified bid X 10 schedule at a dose of 2 mg/kg. A comparable ILS was obtained with AMT itself at 0.24 mg/kg. Thus, replacement of gamma-CO2H by gamma-SO3H in the side chain does not decrease therapeutic effect. However, a higher dose is required, presumably to offset pharmacological differences reflecting the inability of the sulfonate group to be polyglutamated .     

Folate analogues. 26. Syntheses and antifolate activity of 10-substituted derivatives of 5,8-dideazafolic acid and of the poly-gamma-glutamyl metabolites of N10-propargyl-5,8-dideazafolic acid (PDDF)

The poly-gamma-glutamyl derivatives of n10-propargyl-5,8-dideazafolic acid (PDDF) with a chain length of up to five glutamate residues were synthesized from N10-propargyl-5,8-dideazapteroic acid by the solid-phase procedure. These compounds were evaluated for their antifolate activity using folate-requiring microorganisms and intact and permeabilized L1210 cells and as inhibitors of dihydrofolate reductase and thymidylate synthase derived from L. casei. The polyglutamylated derivatives of PDDF (1) were more active than the parent compound in inhibiting the growth of L. casei, thymidylate synthesis in permeabilized L1210 cells, and L. casei thymidylate synthase. Two analogues of 5,8-dideazafolic acid (2 and 3), one with a 2-butyne and another with a cyclopropylmethyl substituent at N10, were also synthesized and evaluated for their antifolate activities using the above-mentioned test systems. They were considerably less active than PDDF or its polyglutamylated derivatives. N10-Propargyl-5,8-dideazapteroyl tri-, tetra-, and pentaglutamates were equipotent with 5-fluorodeoxyuridylate as inhibitors of thymidylate synthesis in permeabilized L1210 cells. The polyglutamyl metabolites of PDDF were shown to be the most potent antifolate inhibitors of L. casei and L1210 thymidylate synthases yet described.