2,4-Diaminopyrido[3,2-d]pyrimidine inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii

Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from 2,4-diamino-6-(bromomethyl)-pyrido[3,2-d]pyrimidine hydrobromide (11.HBr) by treatment with the appropriate aniline or N-methylaniline in dimethylformamide at room temperature, with or without NaHCO3 present. Compounds 5-10 were tested as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, and rat liver as a part of a larger effort directed toward the discovery of lipophilic nonclassical antifolates combining high enzyme selectivity and high potency. Of the six analogues tested, the most potent and selective against T. gondii DHFR was 2,4-diamino-6-[(3',4',5'-trimethoxy-N-methylanilono)methyl]pyrido[ 3,2-d d pyrimidine (7), which had an IC50 of 0.0047 microM against this enzyme as compared with 0.026 microM against the rat liver enzyme. The potency of 7 against T. gondii DHFR was similar to that of trimetrexate (TMQ, 1) and piritrexim (PTX, 2) but was > 500-fold greater than that of trimethoprim (TMP, 3). However, while 7 was more selective than either TMQ (19x) or PTX (63x) against this enzyme, its selectivity in comparison with TMP was 8-fold lower. 2,4-Diamino-6-[3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyri midin e (6) was 17-fold less active than 7 and was also less selective. 2,4-Diamino-6-[(3',4'-dichloro-N-methylanilino)methyl]pyrido[3, 2-d]pyrimidine (10) had an IC50 of 0.022 microM against P. carinii DHFR and was comparable in potency to TMQ and PTX. The species selectivity of 10 for P. carinii versus rat liver DHFR was greater than that of either TMQ (21-fold) or PTX (31-fold). On the other hand, even though 10 was slightly more active than TMQ against the P. carinii enzyme, its selectivity was 7-fold lower than that of TMP. Thus, the goal of combining high enzyme binding activity, which is characteristic of the fused-ring compounds TMQ and PTX, with high selectivity for T. gondii and P. carinii DHFR versus rat liver DHFR, which is characteristic of the monocyclic compound TMP, remained unmet in this limited series.     

2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.     

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.     

Nonclassical 2,4-diamino-8-deazafolate analogues as inhibitors of dihydrofolate reductases from rat liver, Pneumocystis carinii, and Toxoplasma gondii

The synthesis and biological activity of 42 6-substituted-2,4-diaminopyrido[3,2-d]pyrimidines (2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in improved yields compared to previous classical analogues using modifications of procedures reported previously by us. Specifically, the S-phenyl-; mono-, di-, and trimethoxyphenyl-; and mono-, di-, and trichlorophenyl-substituted analogues with H or CH3 at the N10 position and methyl and trifluoromethyl phenyl ketone analogues with H, CH3, and CH2C identical to CH at the N10 position were synthesized. The S10 and N10 alpha- and beta-naphthyl analogues along with the N10 CH3 analogues were also synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg); selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference enzyme. Against pcDHFR the IC50 values ranged from 0.038 x 10-6 M for 2,4-diamino-6-[(N-methyl-2'-naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 x 10(-6) M for 2,4-diamino-6[(2',4'-dimethoxyanilino)methyl]pyrido[3,2-d]pyrim idi ne (15). N10 methylation in all instances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was 2,4-diamino-6-[(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine (5) (IC50 0.0084 x 10(-6) M) and the least potent was 2,4-diamino-6[(2'-naphthylamino)methyl]-pyrido[3,2-d]pyrimidine (37) (IC50 0.16 x 10-6 M). N10 methylation afforded an increase in potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2'-chloro-N-methylanilino)-methyl]pyrido[3,2-d] pyrimidine (13), 2,4-diamino-6-[(3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyr pyrimidine (29), and 2,4-diamino-6-[(2',4',6'-trichloroanilino)methyl]pyrido[3,2-d] pyrimidine (32) which combined high potency at 10-8 M along with selectivities of 8.0, 5.0, and 12.4, respectively. The potency of these three analogues are comparable to the clinically used agent trimetrexate while their selectivities for tgDHFR are 17-43-fold better than trimetrexate.     

Selective Pneumocystis carinii dihydrofolate reductase inhibitors: design, synthesis, and biological evaluation of new 2,4-diamino-5-substituted-furo[2,3-d]pyrimidines

Nonclassical antifolates, 2,4-diamino-5-substituted-furo[2, 3-d]pyrimidines 3-12 with bridge region variations of C8-S9, C8-N9, and C8-O9 and 1-naphthyl, 2-naphthyl, 2-phenoxyphenyl, 4-phenoxyphenyl, and 2-biphenyl side chains were synthesized as phenyl ring appended analogues of previously reported 2, 4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines. The phenyl ring appended analogues were designed to specifically interact with Phe69 of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) to afford selective inhibitors of pcDHFR. Additional substituted phenyl side chains which include 2,5-dichloro, 3,4-dichloro, 3,4,5-trichloro, 3-methoxy, and 2,5-dimethoxy analogues 13-17 were also synthesized. The compounds were prepared by nucleophilic displacement of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine(2) with the appropriate thiol, amine, or naphthol. Compound 2 was obtained from 2,4-diamino-6-hydroxypyrimidine and 1, 3-dichloroacetone. The compounds were evaluated as inhibitors against DHFR from P. carinii, Toxoplasma gondii, and rat liver. Two analogues, 2,4-diamino-5-[(2'-naphthylthio)methyl]furo[2, 3-d]pyrimidine (5) and 2,4-diamino-5-[(2'-phenylanilino)methyl]furo[2,3-d]pyrimidine (11) showed significant selectivity and potency for pcDHFR compared to trimethoprim. The X-ray crystal structure of 5 with pcDHFR was also carried out, which corroborated the design rationale and indicated a hydrophobic interaction of the naphthalene ring of 5 and Phe69 of pcDHFR which is responsible, in part, for the more than 18-fold selectivity of 5 for pcDHFR as compared with rat liver DHFR.     

Synthesis and biological activities of tricyclic conformationally restricted tetrahydropyrido annulated furo[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases

The synthesis of seven 2,4-diamino-5,6,7,8-tetrahydro-7-substituted pyrido[4',3':4,5]furo[2,3-d]pyrimidines 1-6 are reported as nonclassical antifolate inhibitors of dihydrofolate reductase (DHFR) and compound 7 as a classical antifolate inhibitor of tumor cells in culture. The compounds were designed as conformationally restricted analogues of trimetrexate. The synthesis was accomplished from the cyclocondensation of 3-bromo-4-piperidone with 2, 4-diamino-6-hydroxypyrimidine to afford regiospecifically 2, 4-diamino-5,6,7,8-tetrahydropyrido[4',3':4,5]furo[2, 3-d]pyrimidine-7-hydrobromide (16). This in turn was alkylated with the appropriate benzyl halide to afford the target compounds 1-6. The classical antifolate 7 utilized 4-(chloromethyl)benzoyl-l-glutamic acid diethyl ester (17) instead of the benzyl halide for alkylation, followed by saponification to afford 7. Compounds 1-6 showed moderate inhibitory potency against DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. The classical analogue 7 was 88-fold more potent against M. avium DHFR than against rat liver DHFR. The classical analogue was also inhibitory against the growth of tumor cells, CCRF-CEM, and FaDu, in culture.     

Isoxazolo-[3,4-d]-pyridazin-7-(6H)-ones and their corresponding 4,5-disubstituted-3-(2H)-pyridazinone analogues as new substrates for alpha1-adrenoceptor selective antagonists: synthesis, modeling, and binding studies

A series of phenylpiperazinylalkyl moieties were attached to monocyclic or bicyclic substituted pyridazinones and the new compounds tested for their affinity towards alpha1-adrenoceptor and its alpha1a, alpha1b and alpha1d subtypes, as well as serotonin 5-HT1A receptor. Several analogues (5, 6, 9, and 10) showed remarkable potency and selectivity towards alpha1a, and alpha1d with respect to alpha1b subtype. None of the test compounds exhibited significant affinity for 5-HT1A receptor. Finally, on the basis of the alpha1-AR subtypes 3D models recently proposed, we have elaborated theoretical interaction models for the new compounds. The theoretical study allowed us to predict the affinity of the new compounds as well as to infer the structural/dynamics determinants of their interaction with the three alpha1-AR subtypes.     

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP-dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP-dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co-eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.     

Thiopyrano[2,3,4-cd]indoles as 5-lipoxygenase inhibitors: synthesis, biological profile, and resolution of 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methoxy]-4,5 -dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]butanoic acid

Leukotriene biosynthesis inhibitors have potential as new therapies for asthma and inflammatory diseases. The recently disclosed thiopyrano[2,3,4-cd]indole class of 5-lipoxygenase (5-LO) inhibitors has been investigated with particular emphasis on the side chain bearing the acidic functionality. The SAR studies have shown that the inclusion of a heteroatom (O or S) in conjunction with an alpha-ethyl substituted acid leads to inhibitors of improved potency. The most potent inhibitor prepared contains a 2-ethoxybutanoic acid side chain. This compound, 14d (2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)methox y]- 4,5-dihydro-1H-thiopyrano[2,3,4-cd]indol-2-yl]ethoxy]-butanoic acid, L-699,333), inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50s of 22 nM, 7 nM and 3.8 microM, respectively). The racemic acid 14d has been shown to be functionally active in a rat pleurisy model (inhibition of LTB4, ED50 = 0.65 mg/kg, 6 h pretreatment) and in the hyperreactive rat model of antigen-induced dyspnea (50% inhibition at 2 and 4 h pretreatment; 0.5 mg/kg po). In addition, 14d shows excellent functional activity against antigen-induced bronchoconstriction in the conscious squirrel monkey [89% inhibition of the increase in RL and 68% inhibition in the decrease in Cdyn (0.1 mg/kg, n = 3)] and in the conscious sheep models of asthma (iv infusion at 2.5 micrograms/kg/min). Acid 14d is highly selective as an inhibitor of 5-LO activity when compared to the inhibition of human 15-LO, porcine 12-LO and ram seminal vesicle cyclooxygenase (IC50 > 5 microM) or competition in a FLAP binding assay (IC50 > 10 microM). Resolution of 14d affords 14g, the most potent diastereomer, which inhibits the 5-HPETE production of human 5-LO and LTB4 biosynthesis of human PMN leukocytes and human whole blood with IC50s of 8 nM, 4 nM, and 1 microM respectively. The in vitro and in vivo profile of 14d is comparable to that of MK-0591, which has showed biochemical efficacy in inhibiting ex vivo LTB4 biosynthesis and urinary LTE4 excretion in clinical trials.     

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.     

Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.