Inhibition of retinoic acid metabolising enzymes by 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and related compounds

In a search for inhibitors of all-trans retinoic acid (RA)-metabolising enzymes as potential agents for the treatment of skin conditions and cancer we have examined 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (5). Compound (5) is a moderate inhibitor of RA-metabolising enzymes in mammalian cadaverous tissue microsomes and homogenates as well as RA-induced enzymes in cultured human genital fibroblasts and HaCat cells. Overall (5) was more potent than or equipotent with ketoconazole, a standard inhibitor, in the cadaverous systems but less active towards the RA-induced cell culture systems. Examination of the data suggests that RA-induction generates metabolising enzymes not present in the cadaverous systems, which are more susceptible to inhibition by ketoconazole than (5).     

Some 3-(4-Aminophenyl)pyrrolidine-2,5-diones as All- trans -retinoic Acid Metabolising Enzyme Inhibitors (RAMBAs)

A series of (±) -3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3- position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC 50 = 98.8 μM, ketoconazole, 22.15 μM) showed that it was not stereoselective in its inhibition. (±) - (1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC 50 = 20.9 μM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC 50 = 211.6 μM respectively; ketoconazole, 38.8% and 85.95 μM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (±) - (1) was a weak inhibitor (c. 53% at 200 μM) whereas ketoconazole showed high potency (c. 65% at 0.625 μM and 0.25 μM respectively). The nature of the induced target enzyme is discussed.     

Some 3-(4-aminophenyl)pyrrolidine-2,5-diones as all-trans-retinoic acid metabolising enzyme inhibitors (RAMBAs)

A series of (+/-)-3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3-position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC50 = 98.8 microM, ketoconazole, 22.15 microM) showed that it was not stereoselective in its inhibition. (+/-)-(1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC50 = 20.9 microM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC50 = 211.6 microM respectively; ketoconazole, 38.8% and 85.95 microM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (+/-)-(1) was a weak inhibitor (c. 53% at 200 microM) whereas ketoconazole showed high potency (c. 65% at 0.625 microM and 0.25 microM respectively). The nature of the induced target enzyme is discussed.     

Some 1,2-diphenylethane derivatives as inhibitors of retinoic acid--metabolising enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50 = 52.1 microM; ketoconazole, 2.8 microM) and the RA-induced enzyme (100 microM, 65.9% inhibition; ketoconazole, 20 microM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

Some 1,2-Diphenylethane Derivatives as Inhibitors of Retinoic Acid—Metabolising Enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50=52.1?μM; ketoconazole, 2.8?μM) and the RA-induced enzyme (100?μM, 65.9% inhibition; ketoconazole, 20?μM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

Some aryl substituted 2-(4-nitrophenyl)-4-oxo-4-phenylbutanoates and 3-(4-nitrophenyl)-1-phenyl-1,4-butanediols and related compounds as inhibitors of rat liver microsomal retinoic acid metabolising enzymes

Some aryl substituted methyl 2-(4-nitrophenyl)-4-oxo-4-phenylbutanoates generally had poor to moderate inhibitory potency (4-73%) towards rat liver microsomal retinoic acid metabolising enzymes compared with ketoconazole (80%). Conversion to the corresponding 3-(4-nitrophenyl)-1-aryl-1,4-butanediols considerably increased potency (29-78%). The 4-iodophenyl analogue, (30) and the 4-iodo- (45) and 4-methoxyphenyl (46) analogues, were the most potent in both series respectively. The corresponding 5-membered lactones, in the three instances examined, were also potent (52%, 67%, 69%) as were the cis- and trans-isomers of the 5-membered tetrahydrofuran (77%, 65% respectively). Beckmann rearrangement of the oxime methyl 4-(2,4-dichlorophenyl)-4-hydroxyimino-2-(4-nitrophenyl)butanoate (54) gave the expected products (55) and (56), which were potent inhibitors (75%, 74% respectively) of the enzyme whereas the oxime was an activator.     

Some Aryl Substituted 2-(4-Nitrophenyl)-4-oxo-4-phenylbutanoates and 3-(4-Nitrophenyl)-1-phenyl-1,4-butanediols and Related Compounds as Inhibitors of Rat Liver Microsomal Retinoic Acid Metabolising Enzymes

Some aryl substituted methyl 2-(4-nitrophenyl)-4-oxo-4-phenylbutanoates generally had poor to moderate inhibitory potency (4–73%) towards rat liver microsomal retinoic acid metabolising enzymes compared with ketoconazole (80%). Conversion to the corresponding 3-(4-nitrophenyl)-1-aryl-1,4-butanediols considerably increased potency (29–78%). The 4-iodophenyl analogue, (30) and the 4-iodo- (45) and 4-methoxyphenyl (46) analogues, were the most potent in both series respectively. The corresponding 5-membered lactones, in the three instances examined, were also potent (52%, 67%, 69%) as were the cis- and trans-isomers of the 5-membered tetrahydrofuran (77%, 65% respectively). Beckmann rearrangement of the oxime methyl 4-(2,4-dichlorophenyl)-4-hydroxyimino-2-(4-nitrophenyl)butanoate (54) gave the expected products (55) and (56), which were potent inhibitors (75%, 74% respectively) of the enzyme whereas the oxime was an activator.     

Inhibition of platelet thromboxane A2 synthase activity by sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinylmethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 to 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation. Similar effects were obtained by oral administration of 1-5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxygenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cyclooxygenase inhibitors suggesting that its efficacy depended in part on endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of pathophysiological states.     

Retinoic acid metabolism inhibition by 3-azolylmethyl-1H-indoles and 2, 3 or 5-(alpha-azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or alpha-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl) (1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 microM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.     

Antiviral activity of the 3''-amino derivative of (E)-5-(2-bromovinyl)-2''-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.     

In Vitro antifungal activity of 2-(4-substituted phenyl)-3(2H)-isothiazolones

The in vitro antifungal activity of several N2-phenyl-3(2H)-isothiazolones substituted at C4 of the phenyl moiety with heterocyclic nucleus or groups of different physico-chemical properties against four human pathogenic fungi was determined by broth macrodilution method; results were compared with those obtained with itraconazole and ketoconazole. These isothiazolones showed moderate to high activity against some or all tested strains and in comparison with the reference drugs, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g), 5-chloro-2-phenylisothiazol-3-one (1c), 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]-1,4-dihydrotriazol-5-one (1s) and 2-(4-nitrophenyl)isothiazol-3-one (2g) against Aspergillus niger, 5-chloro-2-(4-nitrophenyl)isothiazol-3-one (1g) and 4-[4-(5-chloro-3-oxo-3H-isothiazol-2-yl)phenyl]piperazine-1-carboxamide (1q) against Trichophyton mentagrophytes had comparable activity, compounds 1g and 2g showing higher activity against Microsporum canis. Antifungal activity was favored by the presence of chlorine at C5 of the isothiazolone and/or the presence of nitro group or heterocyclic nucleus at C4 of the phenyl ring and proper hydrophilicity of the molecule.     

Pharmacology of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid), a potent, orally active leukotriene biosynthesis inhibitor.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.     

Effect of 2-(4-aminophenylmethyl)-6-hydroxy-3, 4-dihydronaphthalen-1(2H)-one on all-trans and 13-cis-retinoic acid levels in plasma quantified by high perfomance liquid chromatography coupled to tandem mass spectrometry

The effect of the titled tetralone as a retinoic acid metabolism blocking agent (RAMBA) in vivo in comparison with ketoconazole, a well known cytochrome P450 inhibitor, was studied. Development of a HPLC/MS/MS method for the quantification of retinoic acid levels extracted from rat plasma was used to demonstrate that ketoconazole and the tetralone (100 mg/kg) enhanced the endogenous plasma concentration of retinoic acid. Levels of retinoid were raised from a control value of 0.11 to 0.15 and 0.17 ng/mL after treatment with tetralone and ketoconazole respectively showing that the tetralone and ketoconazole lead to comparable effects, indicating an inhibitory activity of the tetralone on retinoic acid metabolism.     

Ketoconazole as a possible universal inhibitor of cytochrome P-450 dependent enzymes: its mode of inhibition

Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450 dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid 17 alpha-hydroxylase and C17-20 lyase in rat and human testes, 16 alpha-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of 10(-8)M for human testicular enzymes, while the order was 10(-7)-10(-6) M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450 and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450 dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.     

Synthesis and biological evaluation of some 5-arylidene-2-(1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-ones as dual anti-inflammatory/antimicrobial agents

As a part of our ongoing studies in developing new derivatives as dual antimicrobial/anti-inflammatory agents we describe the synthesis of novel 5-arylidene-2-(1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-ones. All newly synthesized compounds were tested for their anti-inflammatory activity using carrageenan mouse paw edema bioassay. Their COX-1/LOX inhibitory activities were also determined. Moreover, all compounds were evaluated for their antimicrobial and antifungal activities against a panel of Gram positive, Gram negative bacteria and moulds. All tested compounds exhibited better antimicrobial activity than commercial drugs, bifonazole, ketoconazole, ampicillin and streptomycin.     

Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.     

Aromatase inhibition by the antifungal ketoconazole

The aromatase inhibitory properties of the antifungal ketoconazole were compared with those of aminoglutethimide. In rat granulosa cells ketoconazole and aminoglutethimide showed IC50 values for aromatase inhibition of 2 X 10(-6) and 6 X 10(-7) M respectively. In the rat, in vivo, ketoconazole was 5 times less potent than aminoglutethimide. In young women, 400 mg of ketoconazole only marginally lowered plasma levels of estradiol-17 beta. It is concluded that ketoconazole is not a compound of choice for clinical use as an aromatase inhibitor.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.