4,5-dihydro-1H-pyrazole derivatives with inhibitory nNOS activity in rat brain: synthesis and structure-activity relationships

In an attempt to find new compounds with neuroprotective activity, we have designed, synthesized and characterized 19 new nNOS inhibitors with a 4,5-dihydro-1H-pyrazole structure. Compounds 11r [1-cyclopropanecarbonyl-3-(2-amino-5-chlorophenyl)-4,5-dihydro-1H-pyrazole] and 11e [1-cyclopropanecarbonyl-3-(2-amino-5-methoxyphenyl)- 4,5-dihydro-1H-pyrazole] show the highest activities with inhibition percentages of 70% and 62%, respectively. A structure-activity relationship for the nNOS inhibition can be established from the structural comparison of these new pyrazole derivatives and the described synthetic kynurenines 10.     

Synthesis and molecular modelling of novel substituted-4,5-dihydro-(1H)-pyrazole derivatives as potent and highly selective monoamine oxidase-A inhibitors

This report describes novel pyrazoline derivatives investigated for their ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase. These new synthetic compounds proved to be reversible, potent, and selective inhibitors of monoamine oxidase-A rather than of monoamine oxidase-B, and are promising candidates to further advance drug discovery efforts. The most active compounds show inhibitory activity on monoamine oxidase-A in the 1.0x10(-8)-8.6x10(-9) M range. Moreover, it should be pointed out that for some compounds a high IC50>or=10(-9) M value is associated with a high A-selectivity (Selectivity Index monoamine oxidase-B/monoamine oxidase-A in the 10,000-12,500 range). Further insight to understand enzyme-inhibitor molecular interaction was obtained by docking experiments with the monoamine oxidase-A and monoamine oxidase-B isoforms.     

Synthesis and screening of anti-cancer,antioxidant, and anti-inflammatory activities of novel galloyl pyrazoline derivatives

A novel series of galloyl-2-pyrazoline derivatives were synthesized and screened for their cytotoxic effect against hepatocellular carcinoma (Hep-G2) and colon carcinoma (HCT-116) cells using the MTT assay, proliferative effect on the immune cells RAW macrophage 264.7 using the MTT assay, anti-inflammatory activity through measurement of the accumulation of nitrites using Griess reagent, and for their antioxidant activity through scavenging of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Most of the tested compounds had a concomitant weak cytotoxic effect against both Hep-G2 and HCT-116 cells, except 5-(2-furyl)-4,5-dihydro-1-(3,4,5-trihydroxybenzoyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (IC₅₀ 8.4 μg/mL and 18.6 μg/mL) and 5-(4-cyanophenyl)-4,5-dihydro-1-(3,4,5-trihydroxybenzoyl)-3-(3,4-dimethoxy-phenyl)-1H-pyrazole (IC₅₀ 15.2 μg/mL, 31.5 μg/mL), which exhibited a cytotoxic effect against Hep-G2 and HCT-116 cells, respectively, while only 5-(2,6-dichlorophenyl)-4,5-dihydro-1-(3,4,5-trihydroxybenzoyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole was a significant dose-dependent inducer of macrophage proliferation. On the other hand, all of the tested compounds were significant inhibitors of LPS-stimulated NO generation and potential scavengers of the DPPH radical, except 1-(3,4,5-trihydroxybenzoyl)-4,5-dihydro-5-(4-methoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole.     

Synthesis of 3-(3,4-dimethoxyphenyl)-1 H-1,2,4-triazole-5-thiol and 2-amino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazole derivatives exhibiting anti-inflammatory activity

New 1-acylderivatives of 5-alkylthio-3-(3,4-dimethoxyphenyl)-4H-1,2,4-triazole (5c-f, 6d-f) were synthesized by the acylation of 5-alkylthio-3-(3,4-dimethoxyphenyl)-4H-1,2,4-triazoles (3, 4) with acyl chlorides. The compounds 3, 4 were obtained by the alkylation of 3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole-5-thiol (2) sodium salt with alkyl iodides. Compound 2 and 2-amino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazole (8) were prepared by the treatment of 3,4-dimethoxybenzoylthiosemicarbazide (1) with sodium hydroxide or acetyl chloride (and then sodium hydroxide), respectively. Related 2-acylamino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazoles (7c, e, f) were synthesized by the acylation of compound 8 with acyl chlorides. 3-(3,4-Dimethoxyphenyl)-4-phenyl-4,5-dihydro-1H-1,2,4-triazole-5-thione (9) was N-acylated with acyl chlorides or S-methylated with iodomethane to give 1-acyl-3-(3,4-dimethoxyphenyl)-4-phenyl-4,5-dihydro-1H- 1,2,4-triazole-5-thiones (10a, b) or 3-(3,4-dimethoxyphenyl)-5-methylthio-4-phenyl-4H-1,2,4-triazole (11) respectively. The synthesized compounds 6d, 7a, c, 10a, b, 11 exhibit anti-inflammatory activity.     

Synthesis, molecular modeling studies, and selective inhibitory activity against monoamine oxidase of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)- pyrazole derivatives

A novel series of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazole derivatives have been synthesized and investigated for the ability to inhibit selectively the activity of the A and B isoforms of monoamine oxidase (MAO). All the synthesized compounds show high activity against both the MAO-A and the MAO-B isoforms with Ki values between 27 and 4 nM and between 50 and 1.5 nM, respectively, except for a few derivatives whose inhibitory activity against MAO-B was in the micromolar range. Knowing that stereochemistry may be an important modulator of biological activity, we performed the semipreparative chromatographic enantioseparation of the most potent, selective, and chiral compounds. The separated enantiomers were then submitted to in vitro biological evaluation. The selectivity of the (-)-(S)-1 enantiomer against MAO-B increases twice and a half, while the selectivity of the (-)-(S)-4 enantiomer against MAO-A triples. Both the MAO-A and MAO-B isoforms respectively of the 1O5W and 1GOS models deposited in the Protein Data Bank were considered in the computational study. The docking study was carried out using several computational approaches with the aim of proposing possible binding modes of the MAO enantioselective compounds 1 and 4.     

Effects of monoamine oxidase inhibitors on cocaine discrimination in rats

This study tested the time course of the discriminative stimulus effects of inhibitors of monoamine oxidase alone or in combination with cocaine. Male Sprague-Dawley rats were trained to discriminate cocaine (10 mg/kg, intraperitoneal) from saline using a two-lever choice methodology. The nonselective monoamine oxidase inhibitors tranylcypromine (0.01-5 mg/kg) and phenelzine (1-25 mg/kg), the monoamine oxidase-A selective compound clorgyline (1-25 mg/kg), and the monoamine oxidase-B selective compounds pargyline (0.005-50 mg/kg) and selegiline (1-25 mg/kg) were tested for substitution 15 min or 24 h following administration, and in combination with 10 mg/kg of cocaine 24 and 48 h after administration. At 15 min, selegiline fully substituted for the discriminative stimulus effects of cocaine, whereas all other compounds partially substituted. At 24 h, substitution of cocaine was diminished for all compounds except phenelzine, which produced a greater amount of substitution at 24 h than at 15 min. When cocaine was administered 24 h after clorgyline, selegiline, pargyline, and phenelzine, cocaine-appropriate responding was attenuated at intermediate doses of these drugs, whereas the highest doses did not alter cocaine-lever responding. All compounds except selegiline substantially decreased response rate and produced various adverse effects. At 48 h, the effects of all compounds except phenelzine were markedly reduced. Selectivity for monoamine oxidase-A or monoamine oxidase-B did not predict the ability to substitute for or attenuate the subjective effects of cocaine. These findings suggest that monoamine oxidase inhibitors can modulate the discriminative stimulus effects of cocaine for at least 24 h, and may be useful for treatment of cocaine abuse.     

Determination of the absolute concentrations of monoamine oxidase A and B in human tissues

The concentrations of monoamine oxidase-A and -B were determined in homogenates of human cerebral cortex, caudatus and placenta and in human platelet-rich plasma and platelet membranes by determining the specific binding of tritium-labelled pargyline. The concentrations of the two enzyme forms were similar in both human brain regions examined. Determinations of the minimum quantities of clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition of enzyme activity was found to give overestimates of the amounts of monoamine oxidase-A and -B present due to nonspecific binding of these inhibitors.     

Kynurenamines as neural nitric oxide synthase inhibitors

To find new compounds with potential neuroprotective activity, we have designed, synthesized, and characterized a series of neural nitric oxide synthase (nNOS) inhibitors with a kynurenamine structure. Among them, N-[3-(2-amino-5-methoxyphenyl)-3-oxopropyl]acetamide is the main melatonin metabolite in the brain and shows the highest activity in the series, with an inhibition percentage of 65% at a 1 mM concentration. The structure-activity relationship of the new series partially reflects that of the previously reported 2-acylamido-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acids, endowed with a kynurenine-like structure. Structural comparisons between these new kinurenamine derivatives, kynurenines, and 1-acyl-3-(2-amino-5-methoxyphenyl)-4,5-dihydro-1H-pyrazole derivatives also reported confirm our previous model for the nNOS inhibition.     

Antitumor agents. 217. Curcumin analogues as novel androgen receptor antagonists with potential as anti-prostate cancer agents

A number of curcumin analogues were prepared and evaluated as potential androgen receptor antagonists against two human prostate cancer cell lines, PC-3 and DU-145, in the presence of androgen receptor (AR) and androgen receptor coactivator, ARA70. Compounds 4 [5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one], 20 [5-hydroxy-1,7-bis[3-methoxy-4-(methoxycarbonylmethoxy)phenyl]-1,4,6-heptatrien-3-one], 22 [7-(4-hydroxy-3-methoxyphenyl)-4-[3-(4-hydroxy-3-methoxyphenyl)acryloyl]-5-oxohepta-4,6-dienoic acid ethyl ester], 23 [7-(4-hydroxy-3-methoxyphenyl)-4-[3-(4-hydroxy-3-methoxyphenyl)acryloyl]5-oxohepta-4,6-dienoic acid], and 39 [bis(3,4-dimethoxyphenyl)-1,3-propanedione] showed potent antiandrogenic activities and were superior to hydroxyflutamide, which is the currently available antiandrogen for the treatment of prostate cancer. Structure-activity relationship (SAR) studies indicated that the bis(3,4-dimethoxyphenyl) moieties, the conjugated beta-diketone moiety, and the intramolecular symmetry of the molecules seem to be important factors related to antiandrogenic activity. The data further suggest that the coplanarity of the beta-diketone moiety and the presence of a strong hydrogen bond donor group were also crucial for the antiandrogenic activity, which is consistent with previous SAR results for hydroxyflutamide analogues. When the pharmacophoric elements of dihydrotestosterone (DHT) and compound 4 are superposed, the resulting construct implies that the curcumin analogues may function as a 17alpha-substituted DHT. Compounds 4, 20, 22, 23, and 39 have been identified as a new class of antiandrogen agents, and these compounds or their new synthetic analogues could be developed into clinical trial candidates to control androgen receptor-mediated prostate cancer growth.     

Synthesis of 5-(2-,3- and 4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol derivatives exhibiting anti-inflammatory activity

New S-alkylated 5-(2-,3- and 4-methoxyphenyl)-4H-1,2,4-triazole-3-thiols (5a-c, 6a-c) and 5-(2-,3- and 4-methoxyphenyl)-4-phenyl-4H-1,2,4-triazole-3-thiols (7a-c, 8a-c, 9a-c) were synthesized by the alkylation of 3-(2-,3- and 4-methoxyphenyl)-4,5-dihydro-1H-1,2,4-triazole-5-thiones (3a-c) or 3-(2-,3- and 4-methoxyphenyl)-4-phenyl-4,5-dihydro-1H-1,2,4-triazole-5-thiones (4a-c) with 1-iodobutane or 1-(1,3-benzodioxol-5-yl)-2-bromo-1-ethanone, 2-bromo-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-ethanone and 2-bromo-1-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-1-ethanone. Compounds 3a-c and 4a-c were synthesized by the acylation of thiosemicarbazide or 4-phenyl-3-thiosemicarbazide with 2-, 3- and 4-methoxybenzoyl chlorides and further cyclization of the obtained acylderivatives 1a-c and 2a-c. The synthesized compounds 4a-c, 5a, 6a-c, 7a-c, 8a-c, 9b,c exhibit anti-inflammatory activity.     

Synthesis and antimycobacterial activity of 5-aryl-1-isonicotinoyl-3-(pyridin-2-yl)-4, 5-dihydro-1H-pyrazole derivatives

5-Aryl-1-isonicotinoyl-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazole derivatives were synthesized and tested for their in vitro antimycobacterial activity. The compounds showed an interesting activity against a strain of Mycobacterium tuberculosis and a human strain of M. tuberculosis H4.     

Synthesis and anti-inflammatory activity evaluation of novel 7-alkoxy-1-amino-4,5-dihydro[1,2,4]triazole[4,3-a]quinolines

In this study, a novel series of 7-alkoxy-1-amino-4,5-dihydro[1,2,4]triazole[4,3-a]quinolines were synthesized by using 6-hydroxy-3,4-dihydro-2(1H)-quinolone as the starting material. These compounds were evaluated for anti-inflammatory activity through monitoring their ability to inhibit xylene-induced ear edema in mice. Some of the tested compounds exhibited significant activity, and the compounds 5f (7-(benzyloxy)-4,5-dihydro[1,2,4]triazolo[4,3-a]quinolin-1-amine) and 5i (7-(p-chlorobenzyloxy)-4,5-dihydro[1,2,4]triazolo[4,3-a]quinolin-1-amine) showed the highest anti-inflammatory activity (52% and 58% inhibition, respectively, at 2 h pre-administration) which were comparable to or even slightly more potent than the reference drug ibuprofen (55%). Furthermore, the structure-activity relationship of these 1,2,4-triazole quinolines was demonstrated.     

Antimitotic agents. Chiral isomers of ethyl [5-amino-1,2-dihydro-3-(4-hydroxyphenyl)-2-methylpyrido[3,4-b]pyrazin-7 -yl]carbamate.

Metabolism studies with ethyl [5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7 - yl]carbamate (1) in mice were reported previously to give a hydroxylated metabolite, which was methylated to give a methoxy derivative. The metabolite and its derivatives were considered to be 4-(substituted)phenyl compounds, which have been confirmed by the synthesis of the [1,2-dihydro-3-(4-hydroxyphenyl)- and [1,2-dihydro-3-(4-methoxyphenyl)pyrido[3,4-b]-pyrazin-7-yl]carbama tes (17 and 16). Both the S- and R-isomers of 17 are active in several biological systems, but the S-isomer is more potent then the R-isomer. The difference in activity between the S- and R-isomers of 17 is similar with that observed for S- and R-isomers of 1. As model reactions, several O-substituted derivatives were prepared by alkylation of (RS)-17 with benzyl chloride and condensation of (RS)-17 with butyl isocyanate and (S)-17 with 2-chloroethyl isocyanate.     

2-Arylidene 3,4-dihydro-1 (2H)-naphthalenones with potential platelet anti-aggregating activity: synthesis and structural and pharmacologic study

New 3,4-dihydro-2-arylidene-1-(2H)-naphthalenones derivatives were synthesized and their inhibitory effects in vitro on the arachidonic acid-induced platelet aggregation were evaluated. The 3,4-dihydro-2-[(2,5-dimethoxyphenyl)methylene]-1-(2H)-naphthalenone++ + exhibits the most potent antiaggregating activity; compared with aspirin this compound is three times more active. Its crystal structure has been determined.     

Novel selective PDE4 inhibitors. 1. Synthesis, structure-activity relationships, and molecular modeling of 4-(3,4-dimethoxyphenyl)-2H-phthalazin-1-ones and analogues

A number of 6-(3,4-dimethoxyphenyl)-4,5-dihydro-2H-pyridazin-3-ones and a novel series of 4-(3,4-dimethoxyphenyl)-2H-phthalazin-1-ones were prepared and tested on the cGMP-inhibited phosphodiesterase (PDE3) and cAMP-specific phosphodiesterase (PDE4) enzymes. All tested compounds were found to specifically inhibit PDE4 except for pyridazinone 3b, which showed moderate PDE4 (pIC(50) = 6.5) as well as PDE3 (pIC(50) = 6.6) inhibitory activity. In both the pyridazinone and phthlazinone series it was found that N-substitution is beneficial for PDE4 inhibition, whereas in the pyridazinone series it also accounts for PDE4 selectivity. In the phthalazinone series, the cis-4a,5,6,7,8,8a-hexahydrophthalazinones and their corresponding 4a,5,8,8a-tetrahydro analogues showed potent PDE4 inhibitory potency (10/11c,d: pIC(50) = 7.6-8.4). A molecular modeling study revealed that the cis-fused cyclohexa(e)ne rings occupy a region in space different from that occupied by the other fused (un)saturated hydrocarbon rings applied; we therefore assume that the steric interactions of these rings with the binding site play an important role in enzyme inhibition.     

Enhancement of rat brain cytosolic monoamine oxidase activity by clorgyline. Comparison with (-)-deprenyl and MDL 72145

The presence of unsedimentable forms of monoamine oxidase (EC 1.4.3.4) in liver and brain homogenates has prompted fresh studies on the effects of inhibitors on this cytosolic monoamine oxidase. Clorglycine is a specific monoamine oxidase A inhibitor and (-)-deprenyl and MDL 72145 are specific monoamine oxidase B inhibitors. We investigated the effects of (-)-deprenyl, MDL 72145 and clorgyline on the purified enzyme from mitochondria and cytosolic monoamine oxidase along with high speed cytosol and 1% Triton X-100 treated mitochondrial preparations. Clorgyline enhanced the activity of the purified enzyme several-fold. (-)-Deprenyl and MDL 72145 also enhanced and inhibited the activity of cytosolic monoamine oxidase in a concentration-dependent manner.     

Monoamine oxidase-A selective inhibition in human hypothalamus and liver in-vitro by amiflamine and its metabolites

Amiflamine (FLA 336(+] and its two metabolites, FLA 788(+) and FLA 668(+) were found to be competitive inhibitors of the activity of monoamine oxidase-A in homogenates of human hypothalamus and liver obtained at autopsy. Ki values, determined at pH 7.2, were 1.3, 0.3 and 22 microM (liver) and 0.8, 0.2 and 14 microM (hypothalamus) for amiflamine, FLA 788(+) and FLA 668(+), respectively. Monoamine oxidase-B activity was only weakly inhibited by the compounds.     

The oxidation of tryptamine by the two forms of monoamine oxidase in human tissues

The selective monoamine oxidase inhibitors clorgyline and (-)-deprenyl have been used to determine the activities of monoamine oxidase-A and -B towards tryptamine in several human tissues. The results were compared with those obtained with the A-form-selective substrate 5-hydroxytryptamine, the B-form-selective substrate 2-phenethylamine and the common substrate tyramine. Tryptamine was found to be a substrate for both forms of the enzyme in human liver, kidney cortex and medulla and in seven different brain regions. The Km values of the two forms towards this substrate were similar in all the human tissues examined but the maximum velocities differed. Thus the A-form would contribute approximately 50% of the total monoamine oxidase activity towards this substrate in human cerebral cortex, whereas it would contribute about 60% in kidney cortex and medulla and 75% in liver. These results suggest that both forms of monoamine oxidase would contribute to the metabolism of tryptamine in human tissues and are difficult to reconcile with suggestions that tryptamine excretion may provide a simple index of monoamine oxidase-A inhibition.     

Synthesis and antihypertensive activity of stereoisomers of 4-piperidyl-1,3-dihydro-2-oxo-2H-benzimidazoles. Enhanced potencies of (+) isomers

To elucidate the relationship between the pharmacological activity and stereochemical structure, we resolved 1-[2-(3-,4,5-trimethoxyphenyl)-2-hydroxy-1-methylethyl]-4-(1,3-dihydro-2-oxo-2H -benzimidazol-1-yl)piperidine (1 and 2) and 1-[2-(3,4-dimethoxyphenyl)-2-hydroxy-1-methylethyl]-4-(1,3-dihydro-2-oxo-2H-benzimidazol-yl)piperidine (3), which produced hypotensive effects mainly through their alpha-blocking actions. Threo isomers 1 and 3 were resolved via diastereomeric carbamates. Erythro isomer 2 was obtained by an oxidation and reduction sequence from optically active 1. No significant difference was found between the pharmacological activities of the threo and erythro isomers of the corresponding compounds. However, a clear difference was found between the pharmacological activities of the optical isomers. Difference was most clearly shown in the hypotensive actions of normotensive rats and in alpha-adrenergic blocking activities of isolated rat vas deferens. In these actions, (+) isomers were always more potent than the corresponding (-) isomers.     

Synthesis and selective inhibitory activity of 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives against monoamine oxidase

A novel series of 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives 1-12 have been synthesized and investigated for the ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). The new synthesized compounds 1-12 proved to be more reversible, potent, and selective inhibitors of MAO-A than of MAO-B. Knowing that stereochemistry may be an important modulator of biological activity, we performed the semipreparative chromatographic enantioseparation of the most potent, selective, and chiral compounds, 6 and 11. The separated enantiomers were then submitted to in vitro biological evaluation while increasing their inhibitory activity and A selectivity. The (-)-6 enantiomer shows K(i(MAO-A)) = 2 nM and SI = 165 000, (+)-6 shows K(i(MAO-A)) = 6 nM and SI = 166 666, (-)-11 shows K(i(MAO-A)) = 4 nM and SI = 80 000, and (+)-11 shows K(i(MAO-A)) = 7 nM and SI = 38 571.