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.     

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.     

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.     

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.     

The biochemical pharmacology of the thymidylate synthase inhibitor, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a more water-soluble analogue of the quinazoline-based thymidylate synthase (TS) inhibitor, N10-propargyl-5,8-dideazafolic acid (CB3717). A 3-fold loss in TS inhibitory activity (murine and human TS, Ki = 10 nM) was accompanied by a 40-fold increase in growth inhibitory potency against L1210 and W1L2 cells in vitro (IC50 = 0.085 and 0.05 microM, respectively) when compared with CB3717. In L1210 cells a concentrative uptake mechanism was demonstrated for [3H]ICI 198583 (Kt = 2.9 microM). The L1210:1565 cell line, with an impaired ability to transport reduced folates or methotrexate (MTX), was resistant (100-fold relative to the wild-type L1210 line) to ICI 198583 (but not CB3717) and did not take up [3H]ICI 198583 significantly. The measurement of folylpolyglutamate synthetase (FPGS) substrate activity demonstrated a Km of 40 microM for ICI 198583 and a Vmax/Km (relative to folic acid) of 3.5. The formation of intracellular polyglutamate derivatives was demonstrated in both L1210 (mouse) and WIL2 (human) cells grown in vitro after exposure to 1 microM [3H]ICI 198583. In L1210 cells, by 4 hr, approximately 50% of the intracellular 3H(approximately 1 microM) was found as polyglutamate forms of ICI 198583, principally as tri- and tetraglutamates. After 24 hr the ICI 198583 polyglutamate pool had expanded, the tetraglutamate metabolite predominated and there was significant formation of the pentaglutamate. Upon resuspension of L1210 cells in drug free medium, ICI 198583 was largely lost from the cells but the polyglutamates were preferentially retained, after 24 hr approximately 70% remained. Synthetic ICI 198583 polyglutamates were shown to be up to 100-fold more potent as inhibitors of isolated TS than the parent compound. Following in vivo administration (500 mg/kg i.v.) ICI 198583 was cleared rapidly from the plasma of mice (T1/2 beta = 16 min, clearance = 42 mL/min/kg). Despite this clearance there was prolonged, dose-dependent inhibition of TS in L1210:NCI cells in vivo. Thus, following 500 mg/kg i.v. the flux through TS was inhibited by greater than 80% for at least 24 hr. Administration of five doses at 5 mg/kg daily of ICI 198583 to L1210:ICR tumour-bearing mice resulted in greater than 60% of the mice being cured, a 10-fold improvement in potency over CB3717. The maximum tolerated dose (MTD) for ICI 198583 using this schedule was greater than 500 mg/kg/day compared with 200 mg/kg/day of CB3717.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Quinazoline antifolate thymidylate synthase inhibitors: alkyl, substituted alkyl, and aryl substituents in the C2 position

Modification of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]-L-glutamic acid (1a) has led to the synthesis of quinazoline antifolates bearing alkyl, substituted alkyl, and aryl substituents at C2. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(alkylamino)benzoyl]-L-glutamate with a C2-substituted 6-(bromo-methyl)-3,4-dihydro-4-oxoquinazoline followed by deprotection using mild alkali. Good enzyme inhibition and cytotoxicity were found with compounds containing small nonpolar groups in the C2 position with the 2-desamino-2-methyl analogue 3a being the most potent. Larger C2 substituents were tolerated by the enzyme, but cytotoxicity was reduced. Highly potent series were followed up by the synthesis of a number of analogues in which the N10 substituent was varied. In this manner a number of interesting TS inhibitors have been prepared. Although none of these was more potent than 1a against the isolated enzyme, over half of the compounds prepared were more potent as cytotoxic agents against L1210 cells in culture. The potential of such compounds as useful antitumor agents was further enhanced by the finding that the improved aqueous solubilities of compounds such as 3a over 1a were reflected in vivo in that 3a was at least 5 times less toxic to mice than 1a.     

Chemistry and antitumor evaluation of selected classical 2,4-diaminoquinazoline analogues of folic acid

A series of six 2,4-diaminoquinazoline analogues of folic acid which bear close structural resemblance to methotrexate, 1a, were synthesized by unequivocal routes. Three of these have not been described previously, while complete structural characterization of the remaining compounds is presented for the first time. Each of the compounds was a potent inhibitor of dihydrofolate reductase (DHFR) from rat liver or L1210 leukemia cells having I50 values in a range similar to that of 1a. However, a wide divergence in inhibitory activity toward the growth of human gastrointestinal adenocarcinoma or L1210 leukemia cells in vitro was observed. Compounds having a normal folate configuration at positions 9 and 10 were more inhibitory than their isomeric reversed-bridge counterparts. The N-formyl modifications were the least active of the compounds studied. Unsubstituted or N-methyl modifications competed effectively with tritiated 1a for uptake into L1210 leukemia cells, while N-formyl modifications did not. Against an L1210 cell line resistant to 1a by virtue of altered transport and overproduction of DHFR, partial but not complete cross-resistance was observed for certain analogues. Of the three compounds selected for in vivo evaluation against L1210 leukemia in mice, two had a similar level of antitumor activity to that of 1a. The compound 5,8-dideazamethopterin, 2b, however, was slightly more active than 1a but at substantially reduced dose levels.     

Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid and related compounds

The chemical synthesis of three close analogues (2-4) of N10-propargyl-5,8-dideazafolate (PDDF) is described. The quinazoline ring of 2 and 4 was constructed from the pivotal intermediate 9 in a novel and unambiguous manner during the final step of the synthesis under very mild conditions. 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate (DMPDDF) (2) was a strong inhibitor of human and Lactobacillus casei thymidylate synthases, whereas 2-desamino-2-(trifluoromethyl)-N10-propargyl-5,8-didezafolate (3) and 2-desamino-2,3-dimethyl-N10-propargyl-5,8-dideazafolate (4) were only weak inhibitors of this enzyme. DMPDDF exhibited excellent growth inhibition of Manca human lymphoid leukemia and H35 hepatoma cells in culture. The inhibitor activities of 2 were 43- and 65-fold greater than that of PDDF, respectively, in these cell lines. H35R cells that are resistant to methotrexate by virtue of a transport defect were cross resistant to DMPDDF but not to PDDF. H35FF cells which have 70-fold greater amounts of thymidylate synthase compared to H35N cells were 130-fold resistant to DMPDDF. Furthermore, the toxicity of DMPDDF to H35 hepatoma cells could be completely reversed by thymidine, establishing its locus of action as thymidylate synthase. Transport studies in vitro established that DMPDDF effectively inhibits MTX influx into H35 hepatoma cells, whereas PDDF has no effect on MTX transport in this cell line. These data suggest that the greater activity of DMPDDF relative to PDDF is partly due to the ability of the former compound to enter cells via the MTX/reduced folate transport system. Enzyme inhibition data of 4 suggest that the presence of N3H in DMPDDF is essential for binding to thymidylate synthase.     

Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.     

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.     

Potent dual thymidylate synthase and dihydrofolate reductase inhibitors: classical and nonclassical 2-amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine antifolates

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.     

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.     

N-[4-[[(3,4-dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid, a novel A-ring analogue of 2-desamino-5,8-dideazafolic acid.

N-[4-[[(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid ("2-aza-2-desamino-5,8- dideazafolic acid", ADDF) was synthesized from 2-amino-5-methylbenzamide via a four-step sequence consisting of diazotization, benzylic bromination, condensation with dimethyl N-(4-aminobenzoyl)-L-glutamate, and ester hydrolysis. ADDF was an inhibitor of recombinant mouse thymidylate synthase; inhibition was competitive with 5,10-methylenetetrahydrofolate as variable substrate (Ki = 2.3 microM). It was a substrate for murine folylpolyglutamate synthetase with kinetic characteristics (Km = 28 microM) comparable to those of aminopterin, and it inhibited the growth of L1210 cells in culture (IC50 = 0.52 microM). The structural modification of the A-ring embodied in ADDF appears to offer a novel, heretofore unexplored approach to the design of TS inhibitors.     

Quinazoline antifolate thymidylate synthase inhibitors: benzoyl ring modifications in the C2-methyl series

The synthesis of nine new 2-methyl-10-propargylquinazoline antifolates with substituents in the p-aminobenzoyl ring is described. In general the synthetic route involved the coupling of the appropriate ring-substituted diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth L1210 cells in culture. Compared to the parent compound 1a the 2'-fluoro analogue 2a exhibited enhanced potency in both systems whereas the 3'-fluoro analogue 3a showed enhanced growth inhibitory properties against L1210 cells despite being a poorer inhibitor of the isolated enzyme. Chloro, hydroxy, methoxy, and nitro substituents in the 2'-position were also well tolerated by the enzyme but failed to give enhanced growth inhibition. The series was extended to cover analogues of the 2'-fluoro, 3'-fluoro, 2'-chloro, 2'-methyl, 2'-amino, 2'-methoxy, and 2'-nitro derivatives with modified alkyl substituents at N10.     

Synthesis and biological activities of 5-trifluoromethyl-5'-azido-2',5'-dideoxyuridine and 5-trifluoromethyl-5'-amino-2',5'-dideoxyuridine.

5-Trifluoromethyl-2'-deoxyuridine (1) was tosylated with p-toluenesulfonyl chloride in dry pyridine at 3 degrees to give 5-trifluoromethyl-5'-O-(p-tolylsulfonyl)-2'-deoxyuridine (2), which was converted to 5-trifluoromethyl-5'-azido-2',5'-dideoxyuridine (3) by reacting with lithium azide in N,N-dimethylformamide at 85-90 degrees for 2 h. Compound 3 was then hydrogenated in ethanol-water (1:1, v/v) at room temperature and 35 psi of hydrogen pressure, using 10% palladium on charcoal as cstalyst, to yield 5-trifluoromethyl-5'-amino-2',5'-dideoxyuridine (4). Compound 4 is about fourfold less potent than compound 1 as an antiviral agent but is about 40-fold less toxic to the host Vero cells. Thus the therapeutic index of compound 1 has been improved by a factor of 10 by replacement of the 5'-hydroxyl with an amino group. Compound 1, however, is more than 100-fold more inhibitory to Sarcoma 180 cells in culture relative to compound 4. Compound 3 is markedly less potent than compound 1 or 4 as either an antiviral or an antineoplastic compound.