Detection of active oxygen species in biological systems

1. Cypridina luciferin analogues, 2-methyl-6-(p-methoxyphenyl)-3,7- dihydroimidazo[1,2-a]pyrazin-3-one (MCLD) and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one(CLA ), react with O2- or 1O2 to emit light in visible region. Such chemiluminescences were used for the detection of O2- or 1O2 in activated leukocyte systems and myeloperoxidase (granulocyte-extract) + Br- + H2O2 systems in vitro. 2. The mechanisms of MCLA (CLA)-dependent luminescence is described in detail. Superoxide generated from sinusoidal cells in acute ethanol intoxication of rats was detected by MCLA-dependent luminescence from the surface of perfused rat liver (organ luminescence). 3. Furthermore, with alive animals, O2- generated in the lung of rats with necrotized pancreatitis and that in the stomach of rats after ischemia/reperfusion were detected by their organ luminescences.     

Synthesis and pharmacological activity of triazolo[1,5-a]triazine derivatives inhibiting eosinophilia

In continuation of our previous work on eosinophilia inhibitors, we synthesized an additional series of inhibitors, which consisted of 5-amino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole derivatives and a newly developed series of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives. We evaluated their inhibitory activity on the airway eosinophilia model, which was induced by the intravenous (iv) injection of Sephadex particles. In the 1,2,4-triazole series with various substituents at the 3 position of the triazole ring such as 2-furyl, pyridyl, and phenoxy, none of derivatives had comparable activity to the previously reported compound GCC-AP0341, 5-amino-3-(4-chlorophenyl)-1-[(methylamino)thiocarbonyl]-1H-1,2, 4-triazole. In the triazolo[1,5-a]triazine series, 2-(4-chlorophenyl)-6-methyl-1,2,4-triazolo[1,5-a]-1,3, 5-triazine-7(6H)-thione (3h) was highly potent, and when given orally it had an ID50 value of 0.3 mg/kg, which is comparable to that of GCC-AP0341. The fact that the structure-activity relationship of these two series was quite similar suggests that a common substructure, such as the 1,2,4-triazole ring with a substituted phenyl ring at the 3 position and a thiocarbonyl moiety at the 1 position, could contribute to the activity. Our selected compound 3h was less active than GCC-AP0341 in the antigen-induced hyper-responsiveness model in guinea pigs; however, we plan to carry out further studies on eosinophil functions, especially on their activation, using our two compounds, 3h and GCC-AP0341.     

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.     

Determination of horseradish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one (MCLA) was observed at 462 nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100 nmol/L under the optimum condition, H2O2 (10 mmol/L) and MCLA (10 mumol/L) at pH 5.8.     

Preparation and steric structure elucidation of saturated or partially saturated isoindolo[2,1-a][3,1]benzoxazinones, -[1,2-b][2,4]benzoxazepinones, cyclopentane- and -cyclohexane[b]pyrrolo[2,1-a][3,1]-benzoxazines

The reactions of 2-carboxybenzaldehyde (1) with 1,3- or 1,4-aminoalcohols (2a-i, 3a,b) were used to prepare partially or fully saturated tetra- and pentacyclic compounds containing a condensed 1,3-oxazino- or oxazepinoisoindolone moiety and one terminal saturated carbocycle. Isoindolo[2,1-a][3,1]benzoxazinones (4a-d, 6, 7), stereoisomeric isoindolo[1,2-b][2,4]benzoxazepinones (5a-c) hexahydrocyclopentane[b]pyrrolo[1,2-a][3,1]-benzoxazinone (10a,b), octahydroindolo[1,2-b]- and decahydroindolo[1,2-a]benzoxazinone (11a,b and 12a,b) and related pentacyclic derivatives (4e-g) were prepared. The diastereomers 5a-c differ in the ring annelation or in the position of the NCHO hydrogens and annelational hydrogens. The stereostructures of these compounds were elucidated by means of 1H and 13C NMR spectroscopy, including DNOE, DEPT, 2D-HSC measurements and X-ray analysis.     

A potent 5-hydroxytryptamine receptor (5-HT2A) antagonist, DV-7028, delays arterial thrombosis development in rats

In our study, we demonstrated that DV-7028: (3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6, 7,8,9-tetrahydro-2H-pyrido [1,2,-a]-1,3,5-triazine-2, 4(3H)-dione maleate)--a selective 5-HT2A receptor antagonist, inhibited thrombus formation in the arterial thrombosis model and was completely ineffective in the prevention of venous thrombosis in the rat. In washed platelets prelabelled with 3H-serotonin, DV-7028 inhibited, in a dose-dependent manner, the collagen-induced secretion of serotonin. However, the uptake of serotonin into platelets was not affected by this substance. Administration of DV-7028 also inhibited platelet aggregation in the whole blood and platelet-rich plasma (PRP) induced by collagen, and diminished serotonin-induced aggregation of rat platelets in the presence of a sensitizing but nonaggregating amount of ADP, whereas it did not modify aggregation in PRP when induced by ADP. DV-7028 caused a concentration-dependent, almost parallel shift to the right of the concentration-response to serotonin for its pressor effect in the rat perfused tail artery. The present data demonstrate that DV-7028 exhibits 5-HT2A receptor antagonistic properties in the rat cardiovascular system, exhibits antithrombotic effect in the model of arterial but not venous thrombosis in rats. These results constitute further evidence of the possible importance of serotonin as a mediator of platelet thrombosis in arteries. Moreover, they can provide a useful tool for the prevention of various thrombotic diseases.     

Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1

A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[a]pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene, 7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[c]phenanthrene, 2-amino-6-methyldipyridol[1,2-a:3',2'-d]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.     

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 3. Discovering bioisosteres of the imidazo[1,2-a] pyridine moiety

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.     

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 4. Discovery of novel frameworks mimicking the active conformation

In recent articles we reported the identification of a series of 8-[[2, 6-dichloro-3-[N-methyl-N-[(E)-(substituted)acryloylglycyl]amino]++ +benzy l]oxy]-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists. Optimization of the terminal glycine part and the imidazo[1,2-a]pyridine moiety led to the discovery of a clinical candidate (5, FR173657). With the aim of completion of the structure-activity relationship (SAR) study, we next investigated the roles of the substituents on the central phenyl ring. The results suggested that the 2,6-dichloro or 2, 6-dimethyl groups may play important roles in regulating the conformations of the 1- and 3-substituents and also may interact with hydrophobic pockets of the B2 receptors. Furthermore, according to the results of a molecular modeling study reported in part 1 of this series, we designed and synthesized a series of sterically constrained analogues by replacing the N-methylamide group with cis-amide-like rigid moieties. We discovered several bioisosteres and chemically proved that the N-methylamide moiety adopts the cis-amide form in the active conformation. Extensive chemical modification led to the identification of a novel class of highly potent and orally active non-peptide B2 antagonists represented by a pyrrole derivative (52a, FR193517). Compound 52a inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50s of 0.37 and 0.56 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration.     

Synthesis and immunotropic activity of some 2-aminobenzimidazoles, Part 4

2-Aminobenzimidazole reacted with selected esters of alpha, beta-unsaturated acids and alpha, beta-unsaturated ketones to form derivatives of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-2-ones, 1,4-dihydropyrimido[1,2-a]benzimidazoles, and 2-acetylaminobenzimidazole. 2-Cinnamoylaminobenzimidazole, 4-phenyl-1,2,3,4-tetrahydropyrimido[1,2,-a]benzimidazol-2-on e, 4-(benzimidazol-2-ylamino)-4-phenylbutan-2-one, and 4-methyl-2-phenyl-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole were tested for their potential activity in immunological assays in the mouse model. Compound 1, at a dose of 100 micrograms/mouse, significantly inhibited the humoral immune response and, at the same time, stimulated the cellular type of that response. The proliferative response of mouse splenocytes to concanavalin A was inhibited only at a higher dose (2 micrograms/ml). The immunotropic activity of 4, on the other hand, was uniformly strongly inhibitory in all applied tests. The suppressive activity of 4 was lower in the cellular immune response and proliferation tests than that of cyclosporin A.     

Cycle length-dependent action potential duration and contractile force at various [Ca2+]o in guinea pig papillary muscle

The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses were examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole[ and its metabolite, SK&F 105561 [2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2 -a] imidazole] are dual 5-lipoxygenase (5-LO) and cycloxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.     

Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives

A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK.     

Research on heterocyclic compounds, Part 40. 2-Phenylimidazo[1,2-a]pyridine-3-carboxylic acid derivatives: synthesis and antiinflammatory activity

A series of 2-phenylimidazo[1,2-a]pyridine-3-carboxylic esters, acids, and amides were synthesized and pharmacologically tested in order to evaluate their antiinflammatory and analgesic activity and their ulcerogenic action on the gastro-intestinal tract. The most active member of this series of compounds was found to be 6-methyl-2-phenylimidazo[1,2-a]pyridine-3-carboxylic acid (5c).     

Biliary cystadenoma of the liver

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O2.-, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2'-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe2+-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

Dermatomyositis and nasopharyngeal carcinoma

Four novel series of 4(3 H)-quinazolinone derivatives have been synthesized by cyclization of the intermediate 3-aryl-2-(6-aryl-2-cyclohexen-1-on-5-yl)-4(3 H)-quinazolinones 3a-f with hydrazine, phenylhydrazine, hydroxylamine and thiourea. The products are 3-aryl-2-(6-aryl-3-methyl-1 H-4,5-dihydrobenzo[d]pyrazol-4-yl)-4(3 H)-quinazolinones 4a-f; 3-aryl-2-(6-aryl-3-methyl-1-phenyl-1 H-4,5-dihydrobenzo[d]pyrazol-4-yl)-4(3 H)-quinazolinones 4g-1; 3-aryl-2-(6-aryl-3-methyl-4,5-dihydrobenzo[d]-1,2-oxazol-4-yl)-4(3 H)-quinazolinones 5a-f, and 3-aryl-2-(7-aryl-4-methyl-5,6-dihydro-2(1 H)thioxoquinazolin-5-yl)-4(3 H)-quinazolinones 6a-f. Some of these compounds showed anti-inflammatory activity comparable to or higher than that of the reference compound proquazone.     

Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells

A novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism involves the oxidation of non-K-region trans-dihydrodiols by dihydrodiol dehydrogenase (DD) to yield PAH o-quinones whose cytotoxicity and genotoxicity are unknown. The cytotoxicity of several PAH o-quinones derived from this reaction [naphthalene-1,2-dione (NPQ), benzo[a]pyrene-7,8-dione (BPQ), and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBAQ)] was examined in rat (H-4IIe) and human (Hep-G2) hepatoma cells which are known to express DD. 2-Methylnaphthalene-1,4-dione (menadione), a known cytotoxic p-quinone, was used as a positive control. Hepatoma cells (1 x 10(6) cells/mL) were exposed to PAH o-quinones (1-100 microM) for 0-4 h, and cell viability and survival were measured and related to O2.- production and changes in redox potential [GSSG/GSH and NAD(P)+/NAD(P)H]. Three different modes of cytotoxicity were observed: (1) NPQ (no bay region) and DMBAQ (methylated bay region) were as cytotoxic as menadione in reducing cell survival but had less effect on cell viability. These o-quinones adversely affected GSH levels and the redox state of the cell and caused an increase in the production of O2.- in cell suspensions. This cytotoxicity was not enhanced by dicoumarol (10 microM), a DT-diaphorase inhibitor, implying that this enzyme is unable to prevent these PAH o-quinones from entering one-electron redox-cycles. (2) BPQ (bay region only) was the least cytotoxic of the PAH o-quinones studied. BPQ decreased cell viability (< 40% at 20 microM) but did not adversely affect cell survival or the redox state of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

32P-postlabeling analysis of the DNA adducts of 6-fluorobenzo[a]pyrene and 6-methylbenzo[a]pyrene formed in vitro

Studies of benzo[a]pyrene (BP) and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by polycyclic aromatic hydrocarbons. Substitution of BP at C-6 with fluorine to form 6-fluorobenzo[a]pyrene (6-FBP) or a methyl group to form 6-methylbenzo[a]pyrene (6-CH3BP) decreases tumorigenicity compared to BP. BP, 6-FBP, and 6-CH3BP formed adducts with DNA when (1) they were activated by 3-methylcholanthrene-induced rat liver microsomes, (2) they were activated by horseradish peroxidase (HRP), (3) their 7,8-dihydrodiols were activated by microsomes, or (4) the radical cation of BP, 6-FBP, or 6-CH3-BP was directly reacted with DNA. With microsomes, 6.5 mumol of [3H]6-FBP/mol of DNA-P and 10 mumol of [14C]6-CH3BP/mol of DNA-P were bound vs 15 mumol of [3H]BP. With microsomes, two major 6-FBP adducts and some minor adducts were obtained. One major adduct coincided with that from 6-FBP-7,8-dihydrodiol. With microsomes, the minor 6-FBP adducts coincided with the adducts obtained from 6-FBP radical cation plus DNA and the major adduct of HRP-activated 6-FBP. With microsomes, 6-CH3BP showed adducts similar to some formed with HRP and one from 6-CH3BP radical cation. 6-CH3BP-7,8-dihydrodiol produced a small amount of one adduct that did not coincide with any from 6-CH3BP. The adducts of 6-FBP appear to be formed mostly through the diolepoxide pathway, whereas those of 6-CH3BP appear to arise mostly via one-electron oxidation.     

Regulation of E2F-1 gene expression in avian cells

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.     

Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides

Covalent adducts between serum albumin and low molecular weight organic electrophiles are formed with a high degree of regioselectivity mostly for nucleophilic amino acid residues located in subdomains IIA and IIIA. Previous studies have indicated that diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) may target residues in a different subdomain. The regioselectivity of PAH epoxide and diol epoxide binding was examined in this study by reaction of human serum albumin in vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-epoxide (2), 1,2-dihydrochrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (7), 3,4-dihydrobenzo[c]phenanthrene-3,4-diol 1,2-epoxide (8), 11,12-dihydrobenzo[g]chrysene-11,12-diol 13,14-epoxide (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (10) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and benzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were chromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociation (CID) tandem mass spectrometry. Product ion spectra revealed that adducts fragmented predominantly by cleavage of the peptide-PAH bond with retention of charge by the peptide as well as by the hydrocarbon. Peptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond. Longer peptide sequences established site selectivity by virtue of their uniqueness, while shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His146; epoxide 3 and diol epoxides 7-9 reacted predominantly with Lys137. Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possibilities was Lys159, which is also located in subdomain IB. The results, taken together with previous findings, demonstrate that the reaction of polycyclic aromatic hydrocarbon epoxides with human serum albumin is highly selective for a small number of residues in subdomain IB.