Biomedical subjects

S F Queener

Publications and source records attributed to S F Queener.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Animals↗

PubMed1998

2,4-Diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidine analogues of trimethoprim as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Three previously unreported (R,S)-2,4-diamino-5-[(3,4,5-trimethoxyphenyl) alkyl]-6,7-dihydro-5H-cyclopenta[d]pyrimidines 15a-c were synthesized as analogues of trimethoprim (TMP) and were tested as inhibitors of Pneumocystis carinii, Toxoplasma gondii, and rat liver dihydrofolate reductase (DHFR). The length of the alkyl bridge between the cyclopenta[d]pyrimidine and trimethoxyphenyl moiety ranged from one in 15a to three carbons in 15c. The products were tested as competitive inhibitors of the reduction of dihydrofolate by Pneumocystis carinii, Toxoplasma gondii, and rat liver DHFR. Compounds 15a-c had IC50 values of > 32, 1.8 and 1.3 microM, respectively, against P. carinii DHFR, as compared to 12 microM for TMP. Against the T. gondii enzyme, 15a-c had IC50 values of 21, 0.14 and 0.14 microM, respectively, as compared to 2.7 microM for TMP. Inhibitors 15b and 15c with two- and three-carbon bridges were significantly more potent than 15a against all three enzymes. Unlike TMP, 15b and 15c were better inhibitors of the rat liver enzyme than of the microbial enzymes. The potency of 15b and 15c against rat liver DHFR was less than has been reported for the corresponding 6,7-dihydro-5H-cyclopenta[d]pyrimidines with a classical p-aminobenzoyl-L-glutamate side chain as inhibitors of bovine, murine, and human DHFR.

Animals↗

PubMed1997

Comparison of ternary complexes of Pneumocystis carinii and wild-type human dihydrofolate reductase with coenzyme NADPH and a novel classical antitumor furo[2,3-d]pyrimidine antifolate.

The novel furopyrimidine N-(4-{N-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino}benzoyl)-L- glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with wild-type Pneumocystis carinii (pc) and recombinant human wild-type dihydrofolate reductase (hDHFR). These structural data provide the first direct comparison of the binding interactions of the same antifolate inhibitor in the active site for pc and human DHFR. The human ternary DHFR complex crystallizes in the rhombohedral space group R3 and is isomorphous to the ternary complex reported for a gamma-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P2(1) and is isomorphous to that reported for a trimethoprim (TMP) complex. Interpretation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observed for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compared with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a)-C(5)-C(8)-N(9) and C(5)-C(8)-N(9)-C(1'), are -156.5/51.9 degrees and -162.6/51.8 degrees, respectively for h and pc, compared with -146.8/57.4 degrees for MTXT. In each case, the p-aminobenzoylglutamate conformation is similar to that observed for MTXT. In the pcDHFR complex, the active-site region is conserved and the additional 20 residues in the sequence compared with the human enzyme are located in external loop regions. There is a significant change in the nicotinamide ribose conformation of the cofactor which places the nicotinamide O atom close to the 4NH(2) group of MTXO (2.7 A), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen bond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 A contact with the 4NH(2) group of MTXO. Although the novel classical antifolate MTXO is not highly active against pcDHFR, there are correlations between its binding interactions consistent with its lower potency as an inhibitor of h and pcDHFR compared with MTX.

Journal Article↗

PubMed1997

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.

Animals↗

PubMed1997

Nonclassical 2,4-diamino-5-aryl-6-ethylpyrimidine antifolates: activity as inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents.

Twelve novel 2,4-diamino-5-(4'-benzylamino)- and 2,4-diamino-5[4'-(N-methylbenzylamino)-3'-nitrophenyl]-6-ethylp yrimidines bearing 4-substituents on the benzylamino or N-methylbenzylamino aryl ring were synthesized and evaluated as nonclassical inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase (DHFR). Compounds were prepared by reaction of 2,4-diamino-5-(4'-chloro-3'-nitrophenyl)- (8) or 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl)-6-ethylpyrimidine (15) with the appropriate 4-substituted (CO2H, CO2Me, SO2NH2, dioxolan-2-yl, CHO, dimethyloxazolin-2-yl) benzylamine or N-methylbenzylamine derivative. Compounds 25-29 were synthesized from 2,4-diamino-5-{4'-[N-(4"-carboxybenzyl)amino]-3'-nitrophenyl}-6- ethylpyrimidine (10) and the corresponding amine (NH3, MeNH2, Me2NH, piperidine, diethyl L-glutamate) via isobutyl mixed anhydride coupling; hydrolysis of the diethyl L-glutamate 29 afforded the L-glutamate analogue 30. The compounds exhibited potent inhibitory activity against T. gondii (IC50 values 0.0018-0.14 microM) and rat liver (IC50 values 0.0029-0.27 microM) DHFR, with a 4-substituent invariably enhancing binding to both enzymes relative to the unsubstituted benzoprim (5) or methylbenzoprim (6). Modest selectivity for T. gondii enzyme was observed with several analogues, whereas all of the compounds were relatively weak inhibitors of P. carinii DHFR and exhibited no selectivity. Selected analogues were evaluated for in vivo antitumor activity against the methotrexate-resistant M5076 murine reticulosarcoma, with 2,4-diamino-5-{4'-[N-[4"-(N"-methylcarbamoyl)benzyl]-N- methylamino]-3'-nitrophenyl}-6-ethylpyrimidine (14) (Ki for rat liver DHFR = 0.00035 +/- 0.00029 nM) combining significant antitumor activity with minimal toxicity.

Animals↗

PubMed1997

Conformationally restricted analogues of trimethoprim: 2,6-diamino-8-substituted purines as potential dihydrofolate reductase inhibitors from Pneumocystis carinii and Toxoplasma gondii.

Twenty-two 2,6-diamino-8-substituted purines (2-23) were synthesized, in which rotation around the two flexible bonds of trimethoprim (TMP), linking the pyrimidine ring to the side chain phenyl ring, was restricted by incorporation into a purine ring, in an attempt to increase the potency and selectivity of TMP against dihydrofolate reductase (DHFR) from the organisms that often cause fatal opportunistic infections in patients with AIDS, i.e., Pneumocystis carinii (pc) and Toxoplasma gondii (tg). The syntheses of analogues 2-20 were achieved via a one-pot reaction of 2,4,5,6-tetraaminopyrimidine and the appropriately substituted benzaldehyde or phenyl acetaldehyde, in acidic methoxyethanol. Analogues 21-23 were synthesized via nucleophilic displacement of 2,6-diamino-8-(chloromethyl)purine with the appropriate anilines or 2-naphthalenethiol. The compounds were evaluated as inhibitors of pcDHFR and tgDHFR with rat liver (rl) DHFR as the mammalian reference enzyme. Compound 11, the 3',4'-dichlorophenyl analogue, was as potent as TMP and had a selectivity ratio of 13 for pcDHFR, which ranked it as one of the three most selective inhibitors of pcDHFR (compared to rlDHFR) known to date. It also displayed a selectivity ratio of 38 for tgDHFR. None of the other analogues showed any improvement compared to TMP in potency or selectivity. In the preclinical in vitro screening program of the National Cancer Institute, compound 11 showed a GI50 of 10(-6) M for the inhibition of the growth of 17 tumor cell lines.

AIDS-Related Opportunistic Infections↗

PubMed1997

Synthesis and biological activities of conformationally restricted, tricyclic nonclassical antifolates as inhibitors of dihydrofolate reductases.

Seven novel tricyclic pyrimido[4,5-c][2,7]naphthyridones 5-8 and the corresponding naphthyridines 9-11 were synthesized as conformationally restricted inhibitors of dihydrofolate reductase (DHFR) and as antitumor and/or antiinfectious agents. The analogues were designed to orient the side chain trimethoxyphenyl group in different conformationally defined positions in order to explore the effect of the side chain orientation on binding affinity and selectivity for DHFR from various species. The semirigid orientations were achieved by bridging the C5 and N10 of compound 12 with a N-ethyl bridge and by variation of the position of double bonds in rings B and C as well as substitution at the 2',6'-positions of the phenyl ring. The synthesis of compounds 5-11 were accomplished by cyclocondensation of the appropriate keto ester (as the biselectrophile) with 2,4,6-triaminopyrimidine to afford the lactam 5. The dehydrolactams 6 and 7 were prepared by air oxidation and PtO2-catalyzed dehydrogenation of 7, respectively. The dichloro dehydro lactam 8 was obtained by refluxing lactam 5 and/or 6 in POCl3 or a mixture of POCl3/PCl5. Compounds 9-11 were obtained by two methods, direct borane reduction of lactam 5 or 6 or thiation of the dipivoylated lactam 15 followed by reductive dethiation. Compounds 9-11 were interconverted by air oxidation or PtO2-catalyzed reduction/oxidation, respectively. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) with rat liver (rl) serving as the reference mammalian enzyme. In the lactam series 5-8, the most unsaturated analogue 7 showed an IC50 of 86 nM against rlDHFR, almost 100-fold more active than 5 and 3-fold more active than 6. The 2',6'-dichloro dehydro lactam 8 was less active than the corresponding dehydro lactam 6 against rlDHFR. In the naphthyridine series 9-11, the dehydro analogue 10 was more active than 9 against rlDHFR. The fully reduced analogue 11 (as a mixture of cis and trans isomers) was the most active in the naphthyridine series. The analogues were, in general, more inhibitory against rlDHFR than against pcDHFR, or tgDHFR, and thus lacked selectivity. In addition, they were less potent than the bicyclic compounds trimetrexate 3 (TMQ) and piritrixim 4 (PTX).

Animals↗

PubMed1997

Structural studies on bioactive compounds. 28. Selective activity of triazenyl-substituted pyrimethamine derivatives against Pneumocystis carinii dihydrofolate reductase.

Triazenyl-substituted pyrimethamine derivatives 10a-s have been prepared by coupling diazotized 2,4-diamino-5-(3-amino-4-chlorophenyl)-6-ethyl pyrimidine (1c) with a series of secondary amines in aqueous sodium carbonate solution. The triazenes which are stable and poorly soluble as free bases form more soluble, but unstable, salts with alkanesulfonic acids. The lead dimethyltriazene 2,4-diamino-5[4-chloro-3-(3,3-dimethyltriazen-1-yl)phenyl]-6-et hylpyrimidine (4a) forms a crystalline ethanesulfonic acid salt (solvated with 2-propanol), which is protonated at the pyrimidine N-1 position, as determined by X-ray crystallography. The ability of these new triazenes to inhibit Pneumocystis carinii dihydrofolate reductase in vitro has been compared to that of triazene 4a. The most potent and selective compound, 2,4-diamino-5-[3-[3-[2-(acetyloxy)ethyl]-3-benzyltriazen-1-y l]-4- chlorophenyl]-6-ethylpyrimidine (14a), has an IC50 value of 0.17 microM against the microbial enzyme and potentially useful selectivity (rat liver IC50/P. carinii IC50 = 114).

Animals↗

PubMed1997

Effect of N9-methylation and bridge atom variation on the activity of 5-substituted 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.

The effect of N9-methylation and bridge atom variation on inhibitory potency and selectivity of 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases (DHFR) was studied. Specifically three nonclassical 2,4-diamino-5-((N-methylanilino)methyl)pyrrolo[2,3-d]pyrimidines with 2',5'-dimethoxyphenyl (2), 3',4'-dichlorophenyl (3), 1'-naphthyl (4), one classical analogue with a 4'-L-glutamate substituent (10), and four nonclassical 2,4-diamino-5-((phenylthio)methyl)pyrrolo[2,3-d]pyrimidines with 3',4'-dimethoxyphenyl (5), 3',4'-dichlorophenyl (6), 1'-naphthyl (7), and 2'-naphthyl (8) substituents were synthesized. The classical and nonclassical analogues were obtained by displacement of the intermediate 2,4-diamino-5-bromomethylpyrrolo[2,3-d]pyrimidine, 14, with appropriately substituted N-methylaniline, thiophenols, or 4-(N-methylamino)benzoyl-L-glutamate. Compounds 2-8 and 10 were evaluated against Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) DHFRs. The N-methyl and thiomethyl analogues were more inhibitory than their corresponding anilinomethyl analogues (previously reported) against all three DHFRs. The inhibitory potency of these analogues was greater against rlDHFR than against tgDHFR which resulted in a loss of selectivity for tgDHFR compared to the N9-H analogues. The classical N9-methyl analogue 10 was more potent and about 2-fold more selective against tgDHFR than its corresponding desmethyl analogue. All of the analogues, 2-8 and 10, were more selective than trimetrexate (TMQ) against pcDHFR (except 4) and significantly more selective than TMQ against tgDHFR.

Animals↗

PubMed1997

Synthesis and dihydrofolate reductase inhibitory activities of 2,4-diamino-5-deaza and 2,4-diamino-5,10-dideaza lipophilic antifolates.

Two series of nonclassical antifolates (2,4-diamino-5-deaza compounds 2-5 and 5,10-dideaza compounds 6-13) were synthesized as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) organisms that are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity. Syntheses of the target 5-deaza compounds were achieved by initial construction of the pivaloyl-protected 2,4-diamino-6-bromopyrido[2,3-d]-pyrimidine 17 via a cyclocondensation of 2,4,6-triaminopyrimidine with bromomalonaldehyde. Sequential Heck coupling of 17 with styrene followed by ozonolysis afforded the 6-formyl derivative 19. Reductive amination of 19 with 3,4,5-trimethoxyaniline afforded the N10-H analog. The N10-Me and N10-Et analogs were synthesized by nucleophilic displacement of the 6-bromomethyl derivative 22 (obtained from the 6-formyl derivative 19 by reduction and bromination) with the appropriate N-alkylaniline. The trans-5,10-dideaza analogs 6-8 were synthesized via a Heck coupling of the appropriate methoxystyrene with 17, and selective reduction of the resulting 9,10-double bond afforded target compounds 9-11. Further reduction to the tetrahydro derivatives afforded analogs 12 and 13. The 5-deaza N10-Me 3,4,5-trimethoxy analog 3 maintained the best balance of potency and selectivity against both tgDHFR and pcDHFR. Compared to trimethoprim, compound 3 was only slightly less selective but was 300-fold more potent against tgDHFR. The 5,10-dideaza analogs were generally less potent and selective than the 5-deaza compounds.

AIDS-Related Opportunistic Infections↗

PubMed1997

Synthesis and biological evaluation of nonclassical 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines with novel side chain substituents as potential inhibitors of dihydrofolate reductases.

Nine novel 2,4-diamino-5-methyl-6-substituted-pyrido[2,3-d]pyrimidines, 2-10, were synthesized as potential inhibitors of Pneumocystis carinii dihydrofolate reductase (pcDHFR) and Toxoplasma gondii dihydrofolate reductase (tgDHFR). Compounds 2-5 were designed as conformationally restricted analogues of trimetrexate (TMQ), in which rotation around tau 3 was constrained by incorporation of the side chain nitrogen as part of an indoline or an indole ring. Analogue 6, which has an extra atom between the side chain nitrogen and the phenyl ring, has its nitrogen as part of a tetrahydroisoquinoline ring. Analogues 7-9 are epiroprim (Ro 11-8958) analogues and contain a pyrrole ring as part of the side chain substitution on the phenyl ring similar to epiroprim. These analogues were designed to investigate the role of the pyrrole substitution on the phenyl ring of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines. Molecular modeling indicated that a pyrrole substituent in the ortho position of the side chain phenyl ring was most likely to interact with pcDHFR in a manner similar to the pyrrole moiety of epiroprim. Analogue 10, in which a phenyl ring replaced a methoxy group, was synthesized to determine the contribution of a phenyl ring on selectivity, lipophilicity, and cell penetration. The synthesis of analogues 2-4 was achieved via reductive amination of 2,4-diamino-5-methyl 6-carboxaldehyde with the appropriately substituted indolines. The indolines were obtained from the corresponding indoles via NaCNBH3 reductions. Analogues 5-10 were synthesized by nucleophilic displacement of 2,4-diamino-5-methyl-6-(bromomethyl)-pyrido[2,3-d]pyrimidine with the 5-methoxyindolyl anion, 6,7-dimethoxytetrahydroisoquinoline, the appropriately substituted pyrroloaniline or 2-methoxy-5-phenylaniline. The pyrroloanilines were synthesized in two steps by treating the substituted nitroanilines with 2,5-dimethoxy-tetrahydrofuran to afford the nitropyrrole intermediates, followed by reduction of the nitro group with Raney Ni. The analogues were more potent than trimethoprim and epiroprim and more selective than TMQ and piritrexim against pcDHFR and tgDHFR. Compounds 5 and 10 had IC50 values of 1 and 0.64 microM, respectively, for the inhibition of the growth of T. gondii cells in culture, and showed excellent culture IC50/enzyme IC50 ratios, which were correlated with their calculated log P values, indicating a direct relationship between calculated lipophilicity and cell penetration.

AIDS-Related Opportunistic Infections↗

PubMed1997

IMP dehydrogenase from Pneumocystis carinii as a potential drug target.

Mycophenolic acid, a specific inhibitor of IMP dehydrogenase (IMPDH; EC 1.1.1.205), is a potent inhibitor of Pneumocystis carinii growth in culture, suggesting that IMPDH may be a sensitive target for chemotherapy in this organism. The IMPDH gene was cloned as a first step to characterizing the enzyme and developing selective inhibitors. A 1.3-kb fragment containing a portion of the P. carinii IMPDH gene was amplified by PCR with two degenerate oligonucleotides based on conserved sequences in IMPDH from humans and four different microorganisms. Northern hybridization analysis showed the P. carinii IMPDH mRNA to be approximately 1.6 kb. The entire cDNA encoding P. carinii IMPDH was isolated and cloned. The deduced amino acid sequence of P. carinii IMPDH shared homology with bacterial (31 to 38%), protozoal (48 to 59%), mammalian (60 to 62%), and fungal (62%) IMPDH enzymes. The IMPDH cDNA was expressed by using a T7 expression system in an IMPDH-deficient strain of Escherichia coli (strain S phi 1101). E. coli S phi 1101 cells containing the P. carinii IMPDH gene were able to grow on medium lacking guanine, implying that the protein expressed in vivo was functional. Extracts of these E. coli cells contained IMPDH activity that had an apparent Km for IMP of 21.7 +/- 0.3 microM and an apparent Km for NAD of 314 +/- 84 microM (mean +/- standard error of the mean; n = 3), and the activity was inhibited by mycophenolic acid (50% inhibitory concentration, 24 microM; n = 2).

Amino Acid Sequence↗

PubMed1996

2-amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase.

Six novel 2-amino-4-oxo-5-[(substituted phenyl)sulfanyl]pyrrolo[2,3-d]pyrimidines 7-12 were synthesized as potential inhibitors of thymidylate synthase (TS) and as antitumor and/or antibacterial agents. The analogues contain a 5-thio substituent with a phenyl, 4'-chlorophenyl, 3',4'-dichlorophenyl, 4'-nitrophenyl, 3',4'-dimethoxyphenyl, and 2'-naphthyl on the sulfur, and were synthesized from the key intermediate 2-(pivaloylamino)-4-oxo-6-methylpyrrolo[2,3-d]-pyrimidine, 17. Appropriately substituted aryl thiols were appended to the 5-position of 17 via an oxidative addition reaction using iodine, ethanol, and water under conditions which also resulted in the deprotection of the 2-amino group. The compounds were evaluated against human, Lactobacillus casei, Escherichia coli, Streptococcus faecium, and Pneumocystis carinii (pc) TSs and against human, rat liver (rl), pc, and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues with the 3',4'-dichloro and the 4'-nitro substituents in the side chain (9 and 10) were more potent than N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl]-N-prop- 2-ynylamino]benzoyl]-L-glutamic acid (PDDF, 1) and N-[5-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694,2) against human TS. Analogues with the 4'-chloro, 3',4'-dimethoxy, and naphthyl side chains (8, 11 and 12) were more potent than the unsubstituted phenyl analogue (7) but less than 2, 9, and 10 by 1 order of magnitude. They were all poor inhibitors of human, rl, and pc DHFRs (IC50 = 10(-5) M) but moderate inhibitors (IC50 = 10(-6) M) of tg DHFR. The 4-nitro analogue, 10 (EC50 1.5 microM), was comparable to PDDF in its potency as an inhibitor of the growth of the FaDu human squamous cell carcinoma cell line.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

2,4-diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine antifolates as potent and selective nonclassical inhibitors of dihydrofolate reductases.

Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamino)pyrido[2,3-d]pyrimidine antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, (tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they contain a reversal of the C9-N10 bridge present in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate 2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido[2,3-d]pyrimidine (8), followed by reductive amination with the appropriate benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3'4'5'-trimethoxy-substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue of the nonclassical series. Compound 22, the N9 methyl 2'5'-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC 50 = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the mammalian reference, under slightly different assay conditions than those employed for rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent potency (28 nM). Analogue 11, also displayed significant selectivity for sfDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo for the inhibition of the growth of T.gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12 and 21-23 were evaluated as antitumor agents in the National Cancer Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI50s for tumor growth inhibition in the 10 -6 - 10 -7 M range.

Animals↗

PubMed1996

Lipophilic antifolates as agents against opportunistic infections. 1. Agents superior to trimetrexate and piritrexim against Toxoplasma gondii and Pneumocystis carinii in in vitro evaluations.

2,4-Diaminopteridines (21 compounds) and 2,4-diamino-5-methyl-5-deazapteridines (34 compounds) along with three 2,4-diamino-5-unsubstituted-5-deazapteridines and four 2,4-diaminoquinazolines, each with an aryl groups attached to the 6-position of the heterocyclic moiety through a two-atom bridge (either CH2NH, CH2N(CH3),CH2S, or CH2CH2), were synthesized and evaluated as inhibitors of the growth of Toxoplasma gondii in culture and as inhibitors of dihydrofolate reductase enzymes from T. gondii, Pneumocystis carinii, and rat liver. Exceptionally high levels of combined potency and selectivity as growth inhibitors of T. gondii and as inhibitors of the microbial enzymes relative to the mammalian enzyme were found among the 5-methyl-5-deazapteridines but not for the other heterocyclic types. Thirty of the 34 5-methyl-5-deaza compounds gave growth inhibition IC50 values lower than that of pyrimethamine (0.4 microM) with 14 compounds below 0.1 microM, values that compare favorably with those for piritrexim and trimetrexate (both near 0.02 microM). As inhibitors of T gondii DHFR, all but three of the 34 5-methyl-5-deaza compounds gave IC50 values in the order of magnitude with those of piritrexim (0.017 microM) and trimetrexate (0.010 microM), and 17 compounds of this group gave IC50 values versus P. carinii DHFR similarly comparable with those of piritrexim (0.031 microM) and trimetrexate (0.042 microM). Thirteen of these congeners gave both T. gondii growth inhibition and DHFR inhibition IC50 values of 0.10 microM or less, thus indicating facile penetration of the cell membrane. Eleven of these inhibitors of both T. gondii growth and DHFR have selectivity ratios (IC50 rat liver divided by IC50 T. gondii) of 5 or greater for the parasite DHFR. The highest selectivity ratio of nearly 100 belongs to the 5-methyl-5-deaza compound whose 6-substituent is CH2CH2C6H3(OCH3)2-2,5. This compound is over 10(3)-fold more selective for T. gondii DHFR than bridge homologue piritrexim (selectivity ratio 0.088), a compound now in clinical trials. The candidate with CH2NHC6H3(CH3)2-2,5 in the 6-position gave the highest P. carinii DHFR selectivity ratio of 4.0, which is about 60-fold more selective than trimetrexate (0.071) and 80-fold more selective than piritrexim (0.048) toward this enzyme. The 10 best compounds with respect to potency and selectivity includes six compounds bearing 2,5-disubstituted phenyl groups in the side chain (with little, if any, difference in effects of methyl, methoxy, or ethoxy), two side chains bearing 1-naphthyl groups, and two with 5,6,7,8-tetrahydro-1-naphthyl groups. Bridge groups represented in the 10 choice compounds are CH2NH, CH2N(CH3), CH2CH2, and CH2S. The high levels of both potency and selectivity among these agents suggest that in vivo studies now underway may lead to agents that could replace trimetrexate and piritrexim in treatment of toxoplasmosis and P. carinii pneumonia.

Animals↗

PubMed1996

Identification of a class of sulfonamides highly active against dihydropteroate synthase form Toxoplasma gondii, Pneumocystis carinii, and Mycobacterium avium.

Sulfanilanilides with 3',5'-halogen substitutions had Ki values 6- to 57-fold lower than the Ki of sulfamethoxazole when tested against dihydropteroate synthase from Toxoplasma gondii. The compounds acted as competitive inhibitors. These compounds were also active against dihydropteroate synthase from Pneumocystis carinii, Mycobacterium avium, and Escherichia coli but were not significantly more active than sulfamethoxazole. The compounds were significantly more active in culture than were standard agents. Against T. gondii in culture, 50% inhibitory concentrations were 7- to 30-fold lower than that of sulfadiazine; against P. carinii in culture, a concentration of 100 microM caused 33 to 95% inhibition of growth, compared with 9% inhibition with 100 microM sulfamethoxazole.

Animals↗

PubMed1996

6,7-disubstituted 2,4-diaminopteridines: novel inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Four novel, disubstituted diaminopteridines have been identified which antagonize the uptake of a folate precursor (para-aminobenzoic acid) by rat-derived Pneumocystis carinii maintained in short-term axenic culture at concentrations ranging from 4.5 to 26 microM. The compounds were at least 10 to 100 times more active than trimethoprim in this assay. None of these entities exhibited toxicity to mammalian cell lines at < 100 microM. The same structures also caused significant inhibition of Toxoplasma gondii tachyzoite replication within Madin-Darby bovine kidney cells at concentrations ranging from 0.1 to 10 microM. Three of the structures (GR92754, AH10639, and AH2504) were at least an order of magnitude more potent than the standard anti-T. gondii agent, pyrimethamine. All three entities were also significantly more potent and selective than pyrimethamine as inhibitors of T. gondii dihydrofolate reductase (DHFR), with 50% inhibitory concentrations within the range of 0.018 to 0.033 microM. One of these compounds, 6,7-dibutyl-2,4-diaminopteridine (GR92754), was also a potent and selective inhibitor of P. carinii DHFR (50% inhibitory concentration, 0.082 microM). GR92754 is the first DHFR inhibitor described that exhibits greater potency, selectivity, and intracellular activity against both organisms than any of the DHFR agents used clinically, namely, trimethoprim, pyrimethamine, and trimetrexate. This information could provide the starting point for examination of the pharmacokinetic and therapeutic potential of GR92754 and related chemical entities with animal models.

4-Aminobenzoic Acid↗