Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.     

Discovery of N-methyl-4-(4-methoxyanilino)quinazolines as potent apoptosis inducers. Structure–activity relationship of the quinazoline ring

As a continuation of our efforts to discover and develop apoptosis inducing N-methyl-4-(4-methoxyanilino)quinazolines as novel anticancer agents, we explored substitution at the 5-, 6-, 7-positions of the quinazoline and replacement of the quinazoline by other nitrogen-containing heterocycles. A small group at the 5-position was found to be well tolerated. At the 6-position a small group like an amino was preferred. Substitution at the 7-position was tolerated much less than at the 6-position. Replacing the carbon at the 8-position or both the 5- and 8-positions with nitrogen led to about 10-fold reductions in potency. Replacement of the quinazoline ring with a quinoline, a benzo[d][1,2,3]triazine, or an isoquinoline ring showed that the nitrogen at the 1-position is important for activity, while the carbon at the 2-position can be replaced by a nitrogen and the nitrogen at the 3-position can be replaced by a carbon. Through the SAR study, several 5- or 6-substituted analogs, such as 2a and 2c, were found to have potencies approaching that of lead compound N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (1g, EP128495, MPC-6827, Azixa®).     

Occurrence of GTP cyclohydrolase I in Bacillus stearothermophilus

A GTP cyclohydrolase which catalyzes the removal of carbon 8 of GTP as formic acid to yield a single pteridine compound occurs in an obligate thermophile Bacillus stearothermophilus ATCC 8005. The enzyme was purified 5.5-fold. Its molecular weight and Stoke's radius were estimated as 105,000 and 45.3 A, respectively. The Km for GTP was 0.98 microM. The temperature and pH optima for activity were 60-65 degrees C and 8.0-8.4, respectively. No divalent cation was required for the reaction. The pteridine product was 3'-triphosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (dihydroneopterin triphosphate), identified by isolating its immediate derivative, 2',3'-cyclic phosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)pteridine (neopterin cyclic phosphate). The radioactive product from [8-14C]GTP agreed with 14C-formate. Molar ratio of formate release to pteridine formation was 1.0.     

5H-Dibenzo[c,h]1,6-naphthyridin-6-ones: novel topoisomerase I-targeting anticancer agents with potent cytotoxic activity

5H-Dibenzo[c,h]1,6-naphthyridine-6-ones can exhibit potent antitumor activity. The effect of varied substituents at the 5-position of 5H-8,9-dimethoxy-2,3-methylenedioxydibenzo[c,h]1,6-naphthyridine on relative cytotoxicity and topoisomerase I-targeting activity was evaluated. Potent TOP-1-targeting activity is observed when the 5-position is substituted with either a 2-(N,N-dimethylamino)ethyl group, as in 3a, or a 2-(pyrrolidin-1-yl)ethyl substituent, 3c. In contrast, the addition of a beta-methyl group or a beta-hydroxymethyl group to compound 3a, as in 3b and 3j, results in a loss of significant TOP1-targeting activity. While the presence of a 3-(N,N-dimethylamino)propyl substituent at the 5-position or a methyl(2-tetrahydrofuranyl) group allows for retention of TOP1-targeting activity, the 2-(4-methyl-1-piperazinyl)ethyl analogue, 3d, did not exhibit significant activity. Replacement of the N,N-dimethylamino group of 3a with either C(2)H(5) or OH, as in 3f and 3h, respectively, also had a negative impact on both cytotoxicity and TOP1-targeting activity. Treatment of 3a with LAH gave the 5,6-dihydrodibenzo[c,h]naphthyridine, 4a. This dihydro derivative has approximately 2/3 the potency of 3a as a TOP1-targeting agent. Compounds 3a, 3b, 3h, 3i, and 4a were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line, MDA-MB-435, was used in these assays. Compound 3a proved to be effective in regressing tumor growth in vivo when administered either by ip injection or orally 3x week at a dose of 2.0mg/kg. Compound 4a when administered orally 5x weekly at a dose of 40 mg/kg also suppressed tumor growth.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Drug metabolism-based design, synthesis, and bioactivities of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (DDPH) analogs as α₁-adrenoceptors antagonists

1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (DDPH) is a potent α?-adrenoceptor antagonist that is currently under Phase II clinic trials. However, the fast metabolism has restricted its further use. In this paper, 11 DDPH analogs were designed according to the probable metabolism pathways of DDPH, and featured the structures of halogen, methyl, and cyano groups at the 3-, or 4-position of aromatic ring A to block the hydroxylation, and one hydroxyl group at the 3-, or 4-position of aromatic ring B to extend the duration time. These compounds were synthesized in moderate to good yields from the reductive amination of substituted phenoxyacetones with substituted phenylethylamines, and fully characterized with 1H NMR, IR, and HRMS. Biological evaluation indicated that most of the compounds exhibited strong blocking and moderate to good antihypertensive activities. It is clear that the compounds having 4-OH/3-OMe on group B exhibited higher blocking activities and longer duration time than their corresponding analogs having 4-OMe/3-OMe (and also 3-OH/4-OMe). Among them, compound 13 having bromo group at the 4-position of ring A and 4-OH/3-OMe on group B, exhibited the highest blocking activity, whereas compound 17 that had a methyl group at the 4-position of ring A and a hydroxyl group at the 4-position of ring B, was more active than potent DDPH in terms of both blocking and antihypertensive activities. In addition, the possible correlations between the blocking and antihypertensive activities are also briefly discussed.     

Identification and initial structure-activity relationships of a novel non-peptide quinolone GnRH receptor antagonist.

Screening of the Merck sample collection for non-peptide compounds with binding affinity for the rat GnRH receptor led to the identification of the substituted quinolone (1) as a lead compound in the search for a non-peptide GnRH receptor antagonist. Substantial improvements in potency (approximately 300 fold) were achieved by addition of an alkyl amine at the 4-position, a 3,5-dimethylphenyl group at the 3-position and 6-nitro-7-chloro-substitution of the 1 H-quinolone core.     

A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (BH(4)) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V(max)/K(m)) and the coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi-stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 A for the ensemble of docked conformers. Docking of BH(4) analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).     

Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents

The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl)quinazolin-4-amine were examined systematically to explore structure–activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.     

黄芩素的结构修饰以及抗肿瘤活性研究

为了改善黄芩素的抗肿瘤活性。本实验以黄芩素为原料,对其进行结构修饰。首先通过mannich反应,在8位引入胺亚甲基,然后通过酰基化反应在7位(6位)酚羟基上引入不同的疏水性基团。并利用CCK-8法对目标化合物进行抗MCF-7肿瘤细胞的活性评价。结果合成得到了6个目标化合物,通过1HNMR、13CNMR、MS和化学手段相结合的方法确定了其结构,其中化合物2~6为新化合物。实验利用黄酮类邻二酚羟基的特性,通过与氯化锶的络合反应,巧妙而简单的确证了所得目标化合物的酯键是在化合物的7位羟基上。抗MCF-7肿瘤活性实验表明,在黄芩素8位上引入含氮原子的胺亚甲基后活性比先导化合物黄芩素强,在其7位上再引入酯键后3个化合物活性比先导化合物强。     

Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of potency against STS in two in vitro systems is observed for the derivatives: The 4-NO(2) > 2-halogens, 2-cyano > EMATE (unsubstituted)>17-deoxyEMATE > 2-NO(2) > 4-bromo>2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl)= 2,4-di-n-propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or N-unsubstituted sulfamate group (H(2)NSO(2)O-) is a prerequisite for potent and irreversible inhibition of STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-nitroEMATE (2), whose IC(50)s in placental microsomes and MCF-7 cells are respectively 0.8 nM and 0.01 nM.     

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.     

Discovery of 4-aryl-2-oxo-2H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay

As a continuation of our efforts to discover and develop the apoptosis inducing 4-aryl-4H-chromenes as potential anticancer agents, we explored the removal of the chiral center at the 4-position and prepared a series of 4-aryl-2-oxo-2H-chromenes. It was found that, in general, removal of the chiral center and replacement of the 2-amino group with a 2-oxo group were tolerated and 4-aryl-2-oxo-2H-chromenes exhibited SAR similar to 4-aryl-2-amino-4H-chromenes. The 4-aryl-2-oxo-2H-chromenes with a N-methyl pyrrole fused at the 7,8-positions were highly active with compound 2a having an EC(50) value of 13 nM in T47D cells. It was found that an OMe group was preferred at the 7-position. 7-NMe(2), 7-NH(2), 7-Cl and 7,8 fused pyrido analogs all had low potency. These 4-aryl-2-oxo-2H-chromenes are a series of potent apoptosis inducers with potential advantage over the 4-aryl-2-amino-4H-chromenes series via elimination of the chiral center at the 4-position.     

Discovery of N-{N-[(3-cyanobenzene) sulfonyl]-4(R)-(3,3-difluoropiperidin-1-yl)-(l)-prolyl}-4-[(3′,5′-dichloro-isonicotinoyl) amino]-(l)-phenylalanine (MK-0617), a highly potent and orally active VLA-4 antagonist

A series of prolyl-N-isonicotinoyl-(L)-4-aminophenylalanine derivatives substituted at the proline 4-position with cyclic amines was evaluated as VLA-4 antagonists. The ring size and presence or absence of fluorine affected potency and receptor occupancy. The analog with 3,3-difluoropiperidine at the proline 4-position (13) was the most potent compound and had very good duration of receptor occupancy in vitro. The ethyl ester prodrug of 13 demonstrated excellent receptor occupancy after oral dosing in rats.     

Age determination in the adult screwworm (Diptera: Calliphoridae) by pteridine levels

Fluorescence spectrophotometry was used to assess the possible use of pteridines in the compound eyes to estimate the age of adult screwworms, Cochliomyia hominivorax (Coquerel). Factors affecting the quantities of pteridines include temperature and head size. No difference in pteridine levels was found among flies fed protein or carbohydrate. A regression model for estimating the age of female screwworms was constructed. The model uses head capsule size and relative pteridine quantities and assumes a constant body temperature of 30 degrees C. This regression formula has an r2 of 0.74. Our study extends the use of pteridine accumulation for age determination from obligate sanguinivorous Diptera to an autogenous species that feeds facultatively on nectar and wound exudates. The technique appears to provide a valid means to determine age of these flies.     

Synthesis and biological evaluation of 4'-(6,7-disubstituted-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol as potent Chk1 inhibitors

A new series of potent tricyclic pyrazole-based Chk1 inhibitors are described. Analogues disubstituted on the 6- and 7-positions show improved Chk1 inhibition potency compared with analogues with a single substituent on either the 6- or 7-position. Based on the lead compound 4'-(6,7-dimethoxy-2,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-biphenyl-4-ol (2), detailed SAR studies on the 6- and 7-positions were performed. 3'-morpholin-4'-yl-propoxy, pyridin-4'-ylmethoxy, pyridin-3'-ylmethoxy, 2'-(5'-ethyl-pyridin-2'-yl)-ethoxy, pyridin-2'-ylethoxy, (6'-methyl-pyridin-2'-yl)-propoxyethoxy, 2',3'-dihydroxyl-1'-yl-propoxy, and tetrahydro-furan-3'-yloxy have been identified as the best groups on the 6-position when the 7-position is substituted with methoxyl group. Pyridin-2'-ylmethoxy and pyridin-3'-ylmethoxy have been identified as the best substituents at the 7-position while the 6-position bearing methoxyl group. These compounds significantly potentiate the cytotoxicity of DNA-damaging antitumor agents in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and the camptothecin-induced S checkpoints, suggesting that their potent biological activities are mechanism-based through Chk1 inhibition.     

Synthesis of N(1)-substituted analogues of (2R,4R)-4-amino-pyrrolidine-2,4-dicarboxylic acid as agonists, partial agonists, and antagonists of group II metabotropic glutamate receptors.

The chemical synthesis of a series of N(1)-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid [(2R,4R)-APDC] as constrained analogues of gamma-substituted glutamic acids is described. Appropriate substitution of the N(1)-position results in agonist, partial agonist, or antagonist activity at mGluR2, mGluR3, and/or mGluR6.     

Synthesis and SAR study of imidazoquinolines as a novel structural class of microsomal prostaglandin E₂ synthase-1 inhibitors

The imidazoquinoline derivative 1 was found as a novel mPGES-1 inhibitor. Optimization of 1 led to the identification of the 2-chlorophenyl group at the C(2)-position and the quinolone structure at the C(4)-position. Compound 33, the most potent synthesized compound, showed excellent mPGES-1 inhibition (IC(50)=9.1nM) with high selectivity (>1000-fold) over both COX-1 and COX-2.     

Structural requirement of chalcones for the inhibitory activity of interleukin-5

Novel chalcones were found as potent inhibitors of interleukin (IL)-5. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (2b, 78.8% inhibition at 50microM, IC(50)=25.3microM) was initially identified as a potent inhibitor of IL-5. This shows the compatible activity with budesonide or sophoricoside. To identify structural requirements, 26 chalcones were prepared and their inhibitory activities were tested against IL-5. Among them, compound 4-[(E)-3-(2-cyclohexylmethoxy-6-hydroxyphenyl)-3-oxoprop-1-enyl]benzenesulfonamide (2w, 99.5% inhibition at 50microM, IC(50)=1.8microM) shows the most potent activity. The important structural requirements of these chalcone analogs exhibiting the inhibitory activity against IL-5 were recognized as the following. (1) The hydrophobic group such as benzyloxy or cyclohexylmethoxy at 6-position of A ring is necessary. (2) The existence of phenolic hydroxyl at 6-position of A ring is critical. (3) Propenone unit as alpha,beta-unsaturated ketone is essential. (4) Electron withdrawing groups with hydrogen acceptor property at 4-position of B ring enhance the activity and quantitative structure-activity relationship of 2 regarding these substituents was determined.     

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)