Nonsteroidal progestins and antiprogestins related to flutamide.

From the dual progestin/antiandrogenic properties of certain synthetic steroids (e.g. cyproterone acetate), it was apparent that the progesterone (P) and androgen (A) receptors must have some common ligand binding features. The nonsteroidal antiandrogen (aA) hydroxyflutamide was therefore considered a possible starting point for medicinal chemistry aimed at antiprogestin (aP) activity. Various modifications to the side chain and aryl ring substituents of flutamide yielded both P and aP activity, but always coupled with varying degrees of A or aA activity. Mineralocorticoid activity was present in some structures, but glucocorticoid and antiglucorticoid activities were not detected. Species (rat, rabbit and monkey) and chiral differences presented formidable difficulties in developing simple structure activity patterns, and low ( < 1%) in vitro uterine receptor binding belied in vivo potency of some aPs. One of the most active aPs, ZM172406, the R enantiomer of ZM150271, N-(3-chloro-4-cyanophenyl)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide, had comparable oral potency to mifepristone in rats and monkeys. The racemate ZM150271 was an effective abortifacient during early pregnancy in pigtailed monkeys (3 x 10 mg/kg) but less effective in cynomolgus monkeys. One of the most active progestins (Pn), ZM182345, N-(4-nitro-3-trifluoromethylphenyl)-4-phenyl-2-hydroxy-2-trifluoromet hyl-pentanamide, was at least as potent as P in rats and rabbits but also possessed A activity.

Phase I trial of ZD1694, a new folate-based thymidylate synthase inhibitor, in patients with solid tumors.

PURPOSE: To perform a phase I clinical and pharmacologic study of ZD1694 (Tomudex, Alderley Park, United Kingdom), a new folate-based thymidylate synthase (TS) inhibitor, in patients with advanced malignancy. PATIENTS AND METHODS: From February 1991 to January 1993, 61 patients with a range of solid tumor received 161 courses of ZD1694 given as a single 15-minute intravenous infusion every 3 weeks, at escalating doses from 0.1 to 3.5 mg/m2. Pharmacokinetic (PK) analysis was performed with the first two courses of treatment. There were 33 men and 28 women with a median age of 53 years (range, 21 to 73). Fifty-five patients (90%) had previously received chemotherapy. RESULTS: Reversible liver toxicity and dose-related gastrointestinal (GI) and bone marrow toxicity occurred at > or = 1.6 mg/m2. Liver function usually returned to normal with repeated treatment, but GI and bone marrow toxicities generally became more severe. No renal toxicity was observed. The maximum-tolerated dose (MTD) was 3.5 mg/m2, at which, in addition to antiproliferative toxicities, four of six patients (67%) developed severe malaise that consisted of anorexia, nausea, and asthenia, with rapidly decreasing performance status that limited re-treatment. Abnormal liver function was also seen in four patients (67%). At 3.0 mg/m2, grades III and IV diarrhea were seen in six of 23 patients (26%) and grade IV myelosuppression in two others. Liver toxicity was self-limiting and not associated with severe malaise. Two patients had a partial response to treatment. PK analysis showed that plasma elimination was triexponential, with pronounced variability in the mean terminal half-life (t1/2gamma) for a given dose ranging from 8.2 to 105 hours. There was a linear relationship between dose and both the area under the concentration-time curve (AUC) and maximum concentration (Cmax), but no clear association between these parameters and response or toxicity. CONCLUSION: The dose of ZD1694 recommended for phase II trials is 3.0 mg/m2.

Quinazoline-based thymidylate synthase inhibitors: relationship between structural modifications and polyglutamation.

Quinazoline-based analogues of folic acid are a group of thymidylate synthase (TS) inhibitors that display a wide spectrum of activity for cultured tumour cells, partly due to their differential ability to form polyglutamate metabolites that are (i) more potent TS inhibitors and (ii) not readily effluxed from cells. The rate of cell membrane transport and folylpolyglutamate synthetase substrate activity influence compound polyglutamation. A series of intact-cell assays has been used to determine how specific modifications of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) affect compound polyglutamation. Those containing the 'classical' glutamate structure were usually, but not always, well polyglutamated intracellularly. Replacement of N10 propargyl with smaller aliphatic substituents, particularly when combined with replacement of the benzene ring with thiophene or thiazole heterocycles, was beneficial for antitumour activity through polyglutamate formation. Fluorination of the benzene, particularly if a F was adjacent to the 'bridge region' (3'F or 2',5'diF), also gave compounds with a high dependence on polyglutamation for activity. Those analogues with 2-CH2OH or NH2 substituents were poor substrates for the reduced-folate cell membrane carrier which can account for their reduced polyglutamation rate and hence growth-inhibitory activity. A large decrease or prevention of polyglutamation was achieved by the introduction of CH3, CH2CH3, Br or C1 on C7. The concomitant enhancement in TS inhibition by these modifications gave compounds active under continuous-exposure cell culture conditions. Some ICI 198583 analogues had the glutamate moiety replaced with unnatural amino acids or dipeptides. Only the L-gamma-L-glu analogue (a polyglutamate metabolite of ICI 198583) gave activity entirely attributable to polyglutamate formation.

Quinazoline antifolate thymidylate synthase inhibitors: replacement of glutamic acid in the C2-methyl series.

The synthesis of a series of analogues of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (ICI 198583, 1) is described in which the glutamic acid residue has been replaced by other alpha-amino acids. Most of these analogues were prepared by coupling of tert-butyl-4-(prop-2-ynylamino)benzoate (37) with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline (34) followed by deprotection of the tert-butyl ester to the acid and azide-mediated coupling to the appropriate amino acid or amino acid ester. In cases where the amino acid ester was unreactive with the acid azide, a modification was used in which the quinazolinone moiety was protected as its 3-(pivaloyloxy)methyl derivative. This permitted the generation of the more reactive acid chloride of the p-aminobenzoate unit. In general these modifications result in compounds that have equivalent potency to 1 as inhibitors of isolated TS except where the amino acid lacks a lipophilic alpha-substituent. These compounds appear to require the reduced folate carrier (RFC) for transport into cells, but since they are not converted intracellularly into polyglutamated forms, they have a lower level of cytotoxicity compared to 1. The removal of the alpha-carboxylic acid has given a second set of analogues of 1 which contain simple alkyl amide, benzyl, substituted benzyl, and heterocyclic benzyl amide derivatives. These are considerably less potent than 1 as TS inhibitors but display 1-10 microM cytotoxicities due to the fact that they do not require RFC transport and can presumably readily enter cells by passive diffusion through the cell membrane. Molecular modeling and NMR studies indicated that the incorporation of, respectively, 7-methyl and 2'-fluoro substituents would favor the optimum conformation of these molecules for interaction with the TS enzyme. Accordingly, these substituents were incorporated into selected examples to give the series of analogues 47-55. These all show enhanced (approximately 10-fold) inhibition of TS compared to their unsubstituted counterparts. In the substituted benzylamides (51, 52) and heterocyclic benzyl amides (53-55) the ability to enter cells by passive diffusion results in highly potent (< 1 microM) cytotoxic agents.

The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

Sixteen gamma-linked dipeptide and four L-Glu-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) have been synthesized and evaluated as inhibitors of thymidylate synthase (TS). Z-blocked L-Glu-gamma-L-linked dipeptides and L-Glu-gamma-amides were prepared by condensing alpha-tert-butyl-N-(benzyloxycarbonyl)-L-glutamic acid with the appropriate tert-butyl-protected L-amino acid or amine. The Z group was removed by catalytic hydrogenolysis, and the resulting dipeptides or L-Glu-gamma-amides were condensed with the appropriate pteroic acid analogue trifluoroacetate salt using diethyl cyanophosphoridate as coupling reagent. Deprotection with trifluoroacetic acid in the final step gave the desired quinazoline gamma-linked dipeptides and L-Glu-gamma-amides as their trifluoroacetate salts. Nearly all the dipeptide analogues were potent inhibitors of TS, the best being ICI 198583-gamma-L-2-aminoadipate (IC50 = 2 nM). Several of these dipeptides were found to be susceptible to enzymatic hydrolysis in mice. The quinazoline monocarboxylate L-Glu-gamma-amides, lacking an alpha'-carboxyl group, are less active against TS and L1210 cell growth but are also not susceptible to enzymatic hydrolysis in mice.

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.

Modifications to the bicyclic ring system 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 (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system.

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.

The renal effects of N10-propargyl-5,8-dideazafolic acid (CB3717) and a non-nephrotoxic analogue ICI D1694, in mice.

N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is an analogue of the thymidylate synthase inhibitor, N10-propargyl-5,8-dideazafolic acid (CB3717). CB3717 was found to be active in early clinical studies, but its use was limited by nephrotoxicity. ICI D1694 is a more potent antitumour agent than CB3717 and is also more water soluble. Previous studies have shown ICI D1694 to be non-toxic to the kidney following a single administration but its renal effects after chronic administration are unknown. To assess these effects, and further define the time course and dose relationship of CB3717-induced renal damage, an assay of glomerular filtration rate (GFR) has been developed which can be used in mice and hence in the screening of novel compounds. The 14C-inulin clearance assay developed was used to show a linear relationship between CB3717 dosage and renal damage (r = - 0.989) following a single bolus dose (50-200 mg kg-1), in mice. CB3717-induced renal damage is persistent (greater than 6 weeks) and renal scarring was noted. ICI D1694 has been shown to be non-nephrotoxic following weekly administration of 250 mg kg-1 for 6 weeks. Measurement of GFR has been shown to be a more sensitive indicator of impaired renal function than plasma urea and creatine concentration, and the measurement of plasma creatinine concentration in particular, appears to be without value in the screening of potential nephrotoxins in certain mouse strains.

ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study.

N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is a water-soluble, folate-based thymidylate synthase (TS) inhibitor designed to be a less toxic and more potent analogue of the clinically tested N10-propargyl-5,8-dideazafolic acid. Inhibition of isolated L1210 TS by ICI D1694 is mixed noncompetitive (although tending toward competitive), with a Ki of 62 nM (Kies = 960 nM). The synthetic gamma-polyglutamates are up to 2 orders of magnitude more potent as inhibitors of TS; e.g., the tetraglutamate (glu4) has a Ki of 1.0 nM (Kies = 15 nM). Although inhibitory activity of ICI D1694 toward rat liver dihydrofolate reductase was similar to that of TS (Ki = 92 nM; competitive inhibition) the polyglutamate derivatives did not show enhanced activity. ICI D1694 was also a very potent inhibitor of L1210 cell growth (50% inhibitory activity = 8 nM). L1210 growth inhibition was not observed in the presence of thymidine, consistent with TS being the locus of action. Folinic acid antagonized L1210 growth inhibition in a competitive fashion such that the highest folinic acid concentration used (25 microM) increased the 50% inhibitory activity 6000-fold. When given as a 4-h delayed "rescue", folinic acid was much less effective in antagonizing growth inhibition. These observations are consistent with folinic acid competing with ICI D1694 for uptake into the cell and/or intracellular polyglutamation. The L1210:1565 cell line, which has greatly impaired reduced-folate/methotrexate transport and thus is resistant to methotrexate, was significantly cross-resistant to ICI D1694 (121-fold), suggesting that ICI D1694 is dependent on this uptake mechanism for good cytotoxic potency in L1210 cells. L1210 cells that were incubated for 4 h with 0.1 microM 3H-ICI D1694 accumulated approximately 1.5 microM intracellular 3H, and the high performance liquid chromatography analysis of the cell extracts demonstrated that 96% of the 3H was associated with the ICI D1694 polyglutamate fractions (principally glu4). Upon resuspension in drug-free medium for 24 h, approximately 75% of the cellular 3H was retained, this being the higher polyglutamate pool (glu4-6). In mice, after a single bolus injection of 10 mg/kg of ICI D1694, TS was inhibited greater than 80% for 24 h in ascitic L1210:NCI cells (as measured by the rate of 3H release from [5-3H]deoxyuridine). ICI D1694 cured the L1210:ICR ascitic tumor in mice at 0.4 mg/kg daily for 5 days (maximum tolerated dose, approximately 50 mg/kg).(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Quinazoline antifolate thymidylate synthase inhibitors: bridge modifications and conformationally restricted analogues in the C2-methyl series.

Several C2-methylquinazoline-based antifolates have been prepared in which the C9,N10 bridge has been replaced by the reversed N9,C10 unit. This series was extensively studied by incorporating further substituents at N9 and C10 as well as by modifications to the p-aminobenzoate ring. The C2-methylquinazoline analogues 29, 30, and 31 containing the methyleneoxa, methylenethia, and thia bridge units were also synthesized. In general these isosteric replacements of the bridge unit in the parent C2-methyl-N10-propargylquinazoline antifolate 2 were much less potent as inhibitors of isolated thymidylate synthase (TS) but several were at least as potent as inhibitors of L1210 cell growth in culture. The fusion of the p-aminobenzoate ring into the bicyclic systems 75 and 76 also reduced activity against TS but again gave highly cytotoxic compounds. The cytotoxicities were largely prevented by thymidine, confirming that TS is the major locus.

Quinazoline antifolate thymidylate synthase inhibitors: heterocyclic benzoyl ring modifications.

The synthesis is described of a series of C2-methyl-N10-alkylquinazoline-based antifolates in which the p-aminobenzoate ring is replaced by the heterocycles thiophene, thiazole, thiadiazole, pyridine, and pyrimidine. These were generally elaborated by the reaction of (bromomethyl)quinazoline 18 or its N3-[(pivaloyloxy)methyl]-protected derivative 36 with suitable heterocyclic amines although each heterocyclic system required its own particular synthetic approach. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of L1210 cells in culture. The thiophene system 7 and its related thiazole 8 gave analogues that were considerably more potent than the parent benzene series 2 as inhibitors of L1210 cell growth although in general these heterocycles were somewhat poorer inhibitors of the isolated TS enzyme. The enhanced cytotoxicities of the thiophene and thiazole analogues result, at least in part, from their efficient transport into the cells via the reduced folate carrier mechanism and very good substrate activity for folylpolyglutamate synthetase. The replacement of the C2-methyl group by C2-(fluoromethyl) and C2-(hydroxymethyl) substituents in the thiophene and thiazole series gave derivatives that were only slightly less potent inhibitors of the TS enzyme but which were considerably less cytotoxic.

Non-steroidal antiandrogens. Design of novel compounds based on an infrared study of the dominant conformation and hydrogen-bonding properties of a series of anilide antiandrogens.

Antiandrogenic activity is observed in anilides containing a tertiary hydroxyl group, and these compounds are used to define a pharmacophore in terms of their physicochemical properties. Infrared spectroscopy shows that these anilides exist in a single conformation, which exerts a powerful influence on the hydrogen-bond donor ability of the hydroxyl group in a model system. Arguments are presented which suggest that hydrogen-bonding ability is an important contributor to biological activity. Compounds were synthesized that reproduced these properties in series not containing an amide bond. Such compounds were found to exhibit good antiandrogen activity. We suggest that quantitative information on hydrogen bonding might also be useful in other systems.

The pharmacokinetics of the quinazoline antifolate ICI D 1694 in mice and rats.

N-(5-[N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is an analogue of the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717). CB3717 was found to be an active anticancer agent in early clinical studies, but its use was limited by its relative insolubility at physiological pH. ICI D1694 has been shown to be a more active anticancer agent than CB3717 in model systems, and it is devoid of the acute renal toxicity associated with the administration of the latter drug to mice. In the present study, the pharmacokinetics of ICI D1694 were studied in both mice and rats using reverse-phase HPLC. In rats, ICI D1694 clearance (CL) conformed to a two-compartment open model and was rapid (CL = 10.7 ml min-1 kg-1, t1/2 beta = 30 min). Excretion was mainly biliary (65% of the delivered dose in 4 h vs 12% in urine) in the rat following a 100-mg/kg i.v. bolus. A high degree of protein binding was seen in rat plasma (greater than or equal to 90% over the range of 20-100 microM). In mice, ICI D1694 CL = 27 ml min-1 kg-1 and t1/2 beta = 30 min following 100 mg/kg i.v., which was significantly faster than CB3717 clearance (CL = 6 ml min-1 kg-1, t1/2 beta = 93 min). ICI D1694 was fully bioavailable following i.p. administration (AUC = 3.73 mg ml-1 min i.v. 4.03 mg ml-1 min i.p.), but its bioavailability following oral administration appeared to be low (approximately 10%-20%). Tissue distribution and excretion studies in mice suggested that biliary excretion predominated, confirming the results obtained in rats. Following an i.v. dose of 500 mg/kg ICI D1694 in mice, drug was detectable at 24 h, suggesting the presence of a third phase of plasma clearance. The initial HPLC assay could not detect this third phase following a dose of 100 mg/kg; hence, a more sensitive assay was developed that includes a solid-phase extraction step. The latter assay was used to define the third phase of ICI D1694 clearance in mice, and preliminary studies demonstrated a terminal half-life of 6.5 +/- 2.7 h.

Thymidylate synthase inhibitors: the in vitro activity of a series of heterocyclic benzoyl ring modified 2-desamino-2-methyl-N10-substituted-5,8-dideazafolates.

Heterocyclic para-aminobenzoate modifications of 2-desamino-2-methyl-5,8-dideazafolic acid and a series of its N10-substituted analogs have produced a number of interesting compounds that have enabled a deeper understanding of the biochemical events required for activity in this class of antimetabolite. There is a relationship that has become apparent between compound potency and both uptake via the reduced-folate carrier and FPGS substrate activity. Rapid cellular uptake and metabolism of polyglutamate forms that are approximately 100-fold more potent as inhibitors of TS can translate a modest TS inhibitor such as ICI D1694 into a very potent inhibitor of cell growth (approximately 500- and approximately 10-fold more potent than CB3717 or ICI 198583, respectively). Polyglutamation may therefore act as an almost essential activation step and ICI D1694 may be highly specific for tumors expressing both the reduced-folate carrier and FPGS. Polyglutamation of folate analogs also leads to drug retention which may play a major role in the pharmacodynamics of TS inhibition by ICI D1694 in vivo. Current studies with 3H-ICI D1694 are aimed at demonstrating metabolism to polyglutamates in tumor cells. The serious toxic limitations of CB3717, i.e., liver and kidney toxicities, are not seen with ICI D1694 reflecting the good water solubility of the drug compared with CB3717. The toxicities observed in mice are however to hematological tissues and are due to its TS inhibitory effects. Thus ICI D1694 may elicit toxicities in man more typical of an antimetabolite than of CB3717. The clinical evaluation of ICI D1694 may further our understanding of the role that metabolism to polyglutamates may have in therapeutic activity.

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.

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.

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.

Quinazoline antifolate thymidylate synthase inhibitors: nitrogen, oxygen, sulfur, and chlorine substituents in the C2 position.

The synthesis of 16 new N10-propargylquinazoline antifolates with methylamino, ethylamino, (2-aminoethyl)amino, [2-(dimethylamino)ethyl]amino, (2-hydroxyethyl)amino, (carboxymethyl)amino, dimethylamino, imidazol-1-yl, methoxy, ethoxy, phenoxy, 2-methoxyethoxy, 2-hydroxyethoxy, mercapto, methylthio, and chloro substituents at C2 is described. In general, the synthetic route involved the coupling of diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate (5a) with 6-(bromomethyl)-2-chloro-3,4-dihydro-4-oxoquinazoline in N,N-dimethylformamide with calcium carbonate as the base, displacement of the C2-chloro substituent with nitrogen and sulfur nucleophiles, and deprotection using mild alkali. The C2-ether analogues were most conveniently prepared by coupling 5a with 6-(bromomethyl)-2,4-diakoxy(or diphenoxy)quinazolines. In this series the final deprotection step with aqueous alkali gave simultaneous selective hydrolysis of the C4-alkoxy or C4-phenoxy substituent. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). As a measure of cytotoxicity, they were examined for their inhibition of the growth of L1210 cells in culture. The C2-methoxy analogue 11a was equivalent to the previously described tight binding TS inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717, ICI 155387, 1a) against the TS enzyme and exhibited enhanced potency in culture. The C2-methoxy substituent also gave a 110-fold enhancement in aqueous solubility relative to the C2-amine. These results suggest that 11a will be an interesting compound for further study as a potential antitumor agent in vivo. A further series of 2-methoxyquinazoline antifolates with modified alkyl substituents at N10 is also described. None of these analogues equalled the activity of 11a. Thus the propargyl group appears to be the optimum N10 substituent in both 2-amino- and 2-methoxyquinazoline antifolates.