Cyanopyrazoles as analogs of purine precursors--II. Effects on macromolecular synthesis and cell cycle progression

The cytotoxic and cytokinetic effects, and in vitro inhibition of macromolecular synthesis by cyanopyrazoles were studied using Friend leukemia and Ehrlich ascites tumor cells. At concentrations in the range of 2.5 mM to 50 microM analog 3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (I) was highly cytotoxic and completely inhibited thymidine, uridine and leucine incorporation into macromolecular material. 24 hr incubation of FL cells with cytostatic concentrations of compound I (in the range of 2 to 0.5 microM) resulted in an accumulation of cells in the G2 + M phase. Analogs N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole (II) and 3(5)-amino-4-cyanopyrazole (III) were not cytotoxic at concentrations up to 5 mM and did not substantially inhibit precursor incorporation into macromolecules but exhibited a cytostatic activity. These compounds caused a decrease of FL cells in the G2 + M phase and an accumulation in the S phase. Analogs I and II displayed a similar in vivo inhibitory effect on thymidine incorporation into DNA in EAT cells. The results indicate that the cytotoxicity of cyanopyrazoles correlates with their ability to inhibit precursor incorporation into macromolecular material. On the other hand, the cytostatic action of compound I is not coupled to a block of nucleic acid synthesis.     

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues

The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.     

Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors

The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedl?nder reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.     

Structurally diverse 5-substituted pyrimidine nucleosides as inhibitors of Leishmania donovani promastigotes in vitro

The following structurally diverse 5-substituted-2'-deoxyuridine nucleosides displayed potent in vitro antileishmanial activity: 5-formyl, 5-(2,2,-dicyanovinyl)-, 5-(2-cyano-2-ethoxycarbonylvinyl), 5-(2-cyano-2-methoxycarbonylvinyl)-, 5-(2-amino-3-cyano-5-oxo-5,6,7,8-tetrahydro-4H-chromen-4-yl)- and related congeners, and the 5-(3-methyl-5-oxo-1-phenyl-4,5-dihydro-4H-pyrazol-4-ylidene) group.     

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts.

Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

Synthesis and mass spectrometric fragmentation characteristics of imidazole ribosides-analogs of intermediates of purine de novo synthetic pathway

Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides--dephosphorylated substrates of the enzymes--in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH3, CO2, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.     

Friedländer reaction on 2-amino-3-cyano-4H-pyrans: Synthesis of derivatives of 4H-pyran [2,3-b] quinoline, new tacrine analogues

The unprecedented Friedländer reaction of densely functionalized 2-amino-3-cyano-4H-pyrans (1) with cyclohexanone has afforded in one step and good yield 5-amino-4-aryl-3-ethoxycarbonyl-2-methyl-6,7,8,9-tetrahydro-4H-pyran[2,3-b]quinolines (2), novel amino-substituted fused pyran derivatives. These compounds are new tacrine analogues.     

Inhibition of hepatic lipogenesis by α-cyano-4-hydroxycinnamate

In agreement with its well-known inhibition of mitochondrial carrier-mediated pyruvate transport, α-cyano-4-hydroxycinnamate elevates pyruvate and lactate levels in suspensions of isolated rat hepatocytes, whereas it lowers citrate levels and causes strongly depressed rates of fatty acid synthesis with glucose as carbon precursor. It stimulates the oxidation of exogenous fatty acids and inhibits their esterification.α-Cyano-4-hydroxycinnamate also impairs fatty acid synthesis from substrates (acetate, octanoate) that bypass mitochondrial pyruvate transport. Cholesterol synthesis from acetate, a process utilizing the same cytosolic acetyl-CoA pool as does fatty acid synthesis, is hardly affected by α-cyano-4-hydroxy-cinnamate. These observations suggest an inhibitory site of action of α-cyano-4-hydroxycinnamate located in the fatty-acid biosynthetic pathway itself. This suggestion has been confirmed by demonstrating the inhibition of purified rat-liver acetyl-CoA carboxylase by α-cyano-4-hydroxycinnamate at concentrations prevailing in the intact cell upon incubation with this compound.Maximal inhibition of purified acetyl-CoA carboxylase requires about 20 min of preincubation of the enzyme with α-cyano-4-hydroxycinnamate. Fatty acid synthesis from acetate in the intact cells is further diminished after an incubation time of 20 min.The inhibition by α-cyano-4-hydroxycinnamate of fatty acid synthesis from acetate can be partially overcome by insulin. Possible interactions of the inhibitor and the hormone at the level of acetyl-CoA carboxylase are discussed.It is concluded that α-cyano-4-hydroxycinnamate does not provide a simple and unequivocal tool to distinguish between actions of effectors on hepatic fatty acid synthesis per se and on the glycolytic pathway.     

Acyclic pyrazolo[3,4-d]pyrimidine nucleoside as potential leishmaniostatic agent

A new synthesis of 6-amino-1-hydroxyethoxymethyl-4 (5H)-oxopyrazolo[3, 4-d]pyrimidine (4) has been mentioned. Compound 4 exhibited inhibition of amastigotes of Leishmania donovani to the extent of 89 % at 30 microg/mL, whereas iso-guanine analogue 5 had the inhibition only to the extent of 52.8% at 100 microg/mL in vitro. In hamster model the maximum inhibitory response for compound 4 against amastigotes multiplication was observed to be 94% at 50 mg/kg single dose for 5 consecutive days.     

Metabolism in vivo of furazolidone: evidence for formation of an open-chain carboxylic acid and alpha-ketoglutaric acid from the nitrofuran in rats

The in vivo metabolism of an antibacterial nitrofuran, furazolidone [N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone] was investigated. When the nitrofuran was administered orally to rats, two new-type nitrofuran metabolites, N-(4-carboxy-2-oxobutylideneamino)-2-oxazolidone and alpha-ketoglutaric acid, were isolated from the urine, together with 3-(4-cyano-2-oxobutylideneamino)-2-oxazolidone and N-(5-acetamido-2-furfurylidene)-3-amino-2-oxazolidone. In addition, the present study showed that the corresponding aminofuran was an intermediate in the conversion of furazolidone to these metabolites.     

Synthesis of nucleosides as potential inhibitors of purine biosynthesis.

The synthesis of analogues of 4-carboxy-5-amino-1-beta-D-ribofuranosylimidazole-5'-phosphate (CAIR) in which the carbonyl group is replaced by a grouping of tetrahedral geometry is described. These are planned as transition state inhibitors of SAICAR synthetase, the enzyme which converts CAIR into its amide with L-aspartic acid.     

Synthesis, crystal structure determination, and biological properties of the DNA-dependent protein kinase (DNA-PK) inhibitor 3-cyano-6-hydrazonomethyl-5-(4-pyridyl)pyrid-[1H]-2-one (OK-1035)

The first reported synthesis of the DNA-PK inhibitor 3-cyano-6-hydrazonomethyl-5-(4-pyridyl)pyrid-[1H]-2-one (OK-1035) is described. The structure of OK-1035 was validated by X-ray crystallography. An IC(50) value of 100 microM was determined for inhibition of DNA-PK, and this is approximately 12-fold higher than that reported previously.     

GABA receptor antagonists and insecticides: common structural features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltrioxabicyclooctanes

Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole trifluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy, bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with supplemental enhancement by the 3-cyano and 4-ethynyl substituents.     

Study on derivatives of 5-amino-4-acylamino-1H-pyrazole as inhibitors of furin

A series of 5-amino-1H-pyrazoles was synthesized and studied as inhibitors of furin. The most potent compound, 5-amino-4-acetylamino-3-(4-methylphenylamino)1H-pyrazole, was found to retard the activity of furin by mixed-type inhibition with K = 288 microM. These findings permit to plan new ways for chemical modifications of the 5-amino-1H-pyrazole structure and design more potent furin inhibitors of non-peptide nature.     

Chemo- and stereoselective synthesis of benzocycloheptene and 1-benzoxepin derivatives as alpha-sympathomimetic and anorexigenic agents

The synthesis and pharmacological evaluation of cis- and trans-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ols 4a-c and 5a-c and cis- and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ols 4d-f and 5d-f were carried out. Chemo- and stereoselective synthesis of 5a-f was achieved by reduction of corresponding alpha-amino ketones 3a-f with LiAl(t-BuO)3H. cis-4-Amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 4d and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 5d exhibited marked anorexigenic activity in mice at a dose of LD50 800 and 500 mg/kg and ED50 75 and 55 mg/kg, respectively, while the analog cis-2,3-dihydroxy-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol 8 showed typical alpha-sympathomimetic activity.     

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5-(t-butyl)-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2 -one], LS 82-556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] and M&B 39279 [5-amino-4-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.     

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes)

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.     

Novel ring transformation of 5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-isoxazole-4-carbaldehyde with 1,2-diaminobenzenes to 3-cyano-1,5-benzodiazepine C-nucleosides

Syntheses of 3-cyano-7- and 8-substituted-4-(beta-D-ribofuranosyl)-1H-1,5-benzodiazepines were reported. Treatment of isoxazole carbaldehyde with 1,2-diamino-4-nitrobenzene in chloroform gave a Schiffs base, 4-(2-amino-5-nitrophenyl)iminomethyl-5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)isoxazole in 82% yield with no trace of the other regioisomer. The cyclocondensation of the resulting Schiffs base in benzene containing trifluoroacetic acid (TFA) gave 3-cyano-8-nitro-4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1H-1,5-benzodiazepine in 49% yield. The same reaction of isoxazole carbaldeyde with 1,2-diamino-4-methoxy- and 4-chlorobenzenes afforded the corresponding Schiffs bases. Extending the reaction time for Schiffs base gave the corresponding cyanobenzodiazepines in good yields. Debenzoylation of the compounds with sodium methoxide produced deprotected C-nucleosides.     

A novel class of AMPA receptor allosteric modulators. Part 1: design, synthesis, and SAR of 3-aryl-4-cyano-5-substituted-heteroaryl-2-carboxylic acid derivatives

The synthesis and initial SAR studies of novel, highly potent positive allosteric modulators of AMPA receptors based on 3-(4-tert-butylphenyl)-4-cyano-5-methylsulfanyl-thiophene-2-carboxylic acid (6a) are described. SAR studies at the thioether moiety indicated that substitution at this position was mandatory and better potency was achieved with small groups.