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.     

Human immunodeficiency virus 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors may suppress the replication of specific drug-resistant (E138K)RT HIV-1 mutants or select for highly resistant (Y181C-->C181I)RT HIV-1 mutants

Mutant HIV-1 that expresses a Glu138-->Lys substitution in its RT [(E138K)RT] is resistant to the HIV-1-specific RT inhibitor 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)pyrimidine (TSAO). However, cell cultures infected with this mutant were completely protected against virus-mediated destruction by micromolar concentrations of the HIV-1-specific RT inhibitors tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), nevirapine, and bis(heteroaryl)piperazine (BHAP). In contrast, cells infected with a virus mutant that expresses a Tyr181-->Cys substitution in its RT [(Y181C)RT] were not protected by nevirapine and TIBO and were only temporarily protected by BHAP. HIV-1 mutant that emerged under the latter conditions contained a Cys181-->Ile substitution in their RT [(LC181I)RT]. This mutant proved highly resistant to all HIV-1-specific RT inhibitors tested, except for several 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives. When recombinant (C181I)RT was evaluated for susceptibility to the HIV-1-specific RT inhibitors, it was resistant to all inhibitors except the HEPT compounds. Since a (Y181F)RT HIV mutant strain was isolated from cells infected with (Y181C)RT HIV-1 and treated with BHAP, we postulate that the Ile codon was derived from a Cys-->Phe transversion mutation (TGT-->TTT), followed by a Phe-->Ile transversion mutation (TTT-->ATT).     

Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.     

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.     

Synthesis and antibacterial activity of a new series of tetracyclic pyridone carboxylic acids

A novel tetracyclic pyridone carboxylic acid with a thiazolidine ring, 1,2-dihydro-9,1-(epoxymethano)-7-fluoro-8-(4-methyl-1-piperazinyl)-5-oxo -5H- thiazolo[3,2-a]quinoline-4-carboxylic acid (4a), and variants with a nitrogen atom (4b) or carbonyl group (4c) in the place of the 10-position oxygen atom of 4a were prepared and tested for antibacterial activity and inhibitory activity on DNA gyrase from Escherichia coli KL-16. The in vitro antibacterial potency with regard to the 10-position atom was found to be of the following order; O > NCH3 = C = O. The IC50 values for DNA gyrase inhibition activity for the 4a, 4b, and 4c compounds were 0.33, 0.53, and 0.67 g/mL, respectively. The activity of 4a, in which the C-3 methyl group and C-5 of ofloxacin (2a) were connected with a sulfur atom to restrict the conformation of 2a, was more potent than that of 2a against both Gram-positive and -negative bacteria, except for Pseudomonas aeruginosa. Compared to the tetracyclic pyridone carboxylic acid 1a, which has a flat thiazole ring, compound 4a showed comparable or slightly more potent activity against both Gram-positive and -negative bacteria, except for P. aeruginosa.     

The crystallographic structure of the protease from human immunodeficiency virus type 2 with two synthetic peptidic transition state analog inhibitors

The crystal structure of human immunodeficiency virus (HIV) type 2 protease has been determined in complexes with peptidic inhibitors Noa-His-Cha psi [CH(OH)CH(OH)]Val-Ile-Amp (U75875) and Qnc-Asn-Cha psi [CH(OH)CH2]Val-Npt(U92163) (where Noa is naphthyloxyacetyl, Cha is cyclohexylalanine, Amp is 2-aminomethylpyridine, Qnc is quinoline-2-carbonyl, and Npt is neopentylamine), which have dihydroxyethylene and hydroxyethylene moieties, respectively, in place of the normal scissile bond of the natural ligand. The complexes crystallize in space group P2(1)2(1)2(1), with one dimer-inhibitor complex per asymmetric unit and average cell dimensions of a = 33.28 A, b = 45.35 A, c = 135.84 A. Data were collected to approximately 2.5-A resolution. The model structures were refined with resulting R-factors of around 0.19. As expected, the HIV-2 protease structure is approximately C2-symmetric with a gross structure very similar to that of the HIV-1 enzyme. The inhibitors bind in an extended conformation positioned lengthwise in the binding cleft in a manner similar to that found in the HIV-1 protease-inhibitor complexes previously reported. The substitution of the bulkier Ile82 side chain in the HIV-2 protease may help explain the better ability of HIV-2 protease to bind and hydrolyze ligands with small P1 and P1' side groups. It appears that differences in specificity between the proteases of HIV-1 and HIV-2 are not merely a result of simple side chain substitutions, but may be complicated by differences in main chain flexibility as well.     

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.     

Fenfluramine potentiates canine pulmonary vasoreactivity to endothelin-1

Glutamate may act via an N-methyl-D-Aspartate (NMDA)-sensitive receptor site to destroy cholinergic neurons within the nucleus basalis magnocellularis in age-associated neurodegenerative diseases. Multiple interesting properties of the NMDA receptor are relevant to its excitotoxic actions, e.g., glutamate is ineffective unless a glycine (gly) modulatory site is also occupied. Thus, the antagonism of glutamate receptor-related toxicity by blockade of either the NMDA-sensitive recognition site or the gly binding site may therefore have therapeutic applications. The current study investigated the ability of four novel noncompetitive antagonists at these two sites: one NMDA open channel antagonist (MRZ 2/579: 1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride), and three glyB receptor antagonists (MRZ 2/570: 8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline salt; MRZ 2/57: 8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline; MRZ 2/576: 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline) administered acutely, to provide neuroprotection from a NMDA receptor agonist within the nucleus basalis magnocellularis of young rats. Injection of NMDA into the nucleus basalis magnocellularis significantly decreased cortical choline acetyltransferase activity. Acute administration (i.p.) of MRZ 2/579, 2/570, 2/571 and 2/576 provided significant neuroprotection from NMDA.