Microbial hydroxylation of unsaturated, cyclic carboxylic acids protected as benzoxazoles

Microbial hydroxylation of 2-(cyclopent-1-enyl)benzoxazole (1) and 2-(cyclohex-1-enyl)benzoxazole (2) by Cunninghamella blakesleeana DSM 1906 and Bacillus megaterium DSM 32, respectively, gave chiral allylic alcohols 3-(benz-1,3-oxazol-2-yl)cyclopent-2-en-1-ol (3) and 3-(benz-1,3-oxazol-2-yl)cyclohex-2-en-1-ol (4) along with achiral ketones 3-(benz-1,3-oxazol-2-yl)cyclopent-2-en-1-one (5) and 3-(benz-1,3-oxazol-2-yl)cyclohex-2-en-1-one (6). Both allylic alcohols were produced in enantiomeric excesses higher than 99%. The determination of their absolute configurations (S in both cases) is described.     

Optimization of the hydroxylation of 2-cyclopentylbenzoxazole with Cunninghamella blakesleeana DSMZ 1906

Biohydroxylation of 2-cyclopentyl-1,3-benzoxazole with the filamentous fungus Cunninghamella blakesleeana DSMZ 1906 was studied in a 15-l stirred tank reactor. The aim of the work was to avoid substrate limitation through sub-optimal mixing by formation of pellets with a uniform pellet size distribution of 250–500 μm, obtained at an inoculum concentration of 10⁷ spores ml⁻¹ and an agitation rate of 390 rpm. Due to the high toxicity of the educt, 2-cyclopentyl-1,3-benz- oxazole, on the fungus, the medium composition, the time of educt addition, and the educt starting concentration were optimized to reach high educt tolerance and hydroxylation activity. A good maintenance of biotransformation capacity was obtained without excessive loss of activity of the biocatalyst by addition of 30 mg 2-cyclopentyl-1,3-benzoxazole/g biomass (cell dry mass) during the stationary phase in a medium which was optimized in batch fermentations with experimental designs. An increase in product yield and quality (enantiomeric excess) was achieved by developing feeding strategies combining the educt and medium components. The resulting fermentation broth contained 450 mg l⁻¹ of the product (1S,3S)-3-(benz-1,3-oxazol- 2-yl)cyclopentan-1-ol with an enantiomeric excess of 95%, which represents a 48% increase over former reported results. Received: 8 July 1999 / Accepted: 2 October 1999     

Induction and functional role of cytochromes P450 in the filamentous fungi Mortierella alpina ATCC 8979 and Cunninghamella blakesleeana DSM 1906 during hydroxylation of cycloalkylbenzoxazoles

The occurrence and regulation of cytochrome P450 (P450) in Mortierella alpina and Cunninghamella blakesleeana have been studied to elucidate the enzymatic basis by which 2-cyclopentyl-1,3-benzoxazole is hydroxylated to 3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol by these organisms. The occurrence of P450 in M. alpina was first been shown after induction with n-hexane. An assay protocol was developed with n-hexane-induced cells and adapted to the handling of fungal mycelia. This allowed the direct spectral determination of P450 in non-fractionated whole-cell suspensions, and an investigation of its regulation. Small amounts of P450 have been detected in early-stationary-phase cells in the absence of exogenous inducers. Addition of 2-cyclopentyl-1,3-benzoxazole or n-hexane resulted in a significant induction of P450. Induction by n-hexane occurs in all phases of growth but decreases rapidly during the stationary phase. The rate of 2-cyclopentyl-1,3-benzoxazole hydroxylation correlated with the content of substrate-induced P450 but not with the level of n-hexane-induced P450. Hydroxylation rates were significantly diminished in the presence of typical P450 inhibitors, the interaction of which with P450 was shown with isolated microsomes of M. alpina. It is concluded that a P450 enzyme is responsible for the hydroxylation of 2-cyclopentyl-1,3-benzoxazole, but that multiple forms of P450 forms occur. Similarly, a dependence on P450 is shown by spectral as well as by inhibition studies for the hydroxylation of this substrate by C. blakesleeana. Received: 18 August 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Synthesis and immunomodulatory properties of selected oxazolone derivatives

Eleven oxazolone derivatives were synthesized and characterized by (1)H NMR, EI, IR and UV spectroscopic and CHN analysis. Three compounds, 4-[(E)-(4-nitrophenyl)methylidene]-2-phenyl-1,3-oxazol-5(4H)-one (11), 4-[(E)-(4-methoxyphenyl)methylidene]-2-methyl-1,3-oxazol-5-one (12) and 4-[(E)-(4-nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) were screened for phagocyte chemiluminescence, neutrophil chemotaxis, T-cell proliferation, cytokine production from mononuclear cells and cytotoxicity. 4-[(E)-(4-Nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) was found to be the most potent immunomodulator in the series.     

Design, synthesis and biological evaluation of bis(hydroxyphenyl) azoles as potent and selective non-steroidal inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) for the treatment of estrogen-dependent diseases

The 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the reduction of the weakly active estrone (E1) into the most potent estrogen, 17beta-estradiol (E2). E2 stimulates the growth of hormone-dependent diseases via activation of the estrogen receptors (ERs). 17beta-HSD1 is often over-expressed in breast cancer cells. Thus, it is an attractive target for the treatment of mammary tumours. The combination of a ligand- and a structure-based drug design approach led to the identification of bis(hydroxyphenyl) azoles as potential inhibitors of 17beta-HSD1. Different azoles and hydroxy substitution patterns were investigated. The compounds were evaluated for activity and selectivity with regard to 17beta-HSD2, ERalpha and ERbeta. The most potent compound is 3-[5-(4-hydroxyphenyl)-1,3-oxazol-2-yl]phenol (18, IC(50)=0.31 microM), showing very good selectivity, high cell permeability and medium CaCo-2 permeability.     

Molecular docking based screening of neem-derived compounds with the NS1 protein of Influenza virus

AbbreviationsNS1 protein - Non Structural 1 proteinNA - Neuraminidase, HA - Hemagglutinin, M - Matrix, 127-40-2 - 4-[(1E, 3E, 5E,7Z, 9E, 11E, 13E, 15E, 17E)-18-(4-hydroxy-2,6,6-trimethylcyclohex-2-en-1-yl)-3,7,12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17- nonaenyl]-3, 5, 5-trimethylcyclohex-3-en-1-ol, Quercitrin 2 - (3,4-dihydroxyphenyl)-5,7-dihydroxy-3- [(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one, Tiplasinin 2 - [1-benzyl-5-[4-(trifluoromethoxy) phenyl] indol-3-yl]-2-oxoacetic acid, Hyperoside 2 - (3,4-dihydroxyphenyl)-5,7-dihydroxy-3- [(2S,3R,4S,5R,6R)-3, 4, 5-trihydroxy-6- (hydroxymethyl)oxan-2- yl]oxychromen-4-one LGH 4-(2-chloro-4-nitrophenyl)piperazin-1-yl][3-(2-methoxyphenyl)-5-methyl-1,2-oxazol-4-yl]methanone, nRUTIN 2 - (3, 4-dihydroxyphenyl) -5, 7-dihydroxy-3-[(2S, 3R, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-[[(2R, 3R, 4R, 5R, 6S)-3,4,5-trihydroxy- 6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one.     

Discovery of a novel class of 1,3-dioxane-2-carboxylic acid derivatives as subtype-selective peroxisome proliferator-activated receptor alpha (PPARalpha) agonists

A new series of 1,3-dioxane-2-carboxylic acid derivatives was synthesized and evaluated for agonist activity at human peroxisome proliferator-activated receptor (PPAR) subtypes. Structure-activity relationship studies led to the identification of 2-methyl-c-5-[4-(5-methyl-2-phenyl-1,3-oxazol-4-yl)butyl]-1,3-dioxane-r-2-carboxylic acid 4b as a potent PPARalpha agonist with high subtype selectivity at human receptor subtypes. This compound exhibited a substantial hypolipidemic effect in type 2 diabetic KK-A(y) mice.     

Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity

With the appearance of the antifungal resistance, novel antifungal agents need to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of aryltetrazole with 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole or 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole and Michael-type addition of aryltetrazole to phenyl vinyl sulfone. The chemical structures of the synthesized compounds were confirmed by means of ¹H NMR, ¹³C NMR, IR and HRMS spectral data. The compounds were tested against the moulds: Fusarium sambucinum, Fusarium oxysporum, Colletotrichum coccodes, Aspergillus niger, and the yeast Candida albicans. The results showed that among the moulds only C. coccodes was significantly sensitive to all the structures examined. All the tetrazole derivatives acted at the same level against C. albicans and demonstrated a high cell growth inhibition (97–99%) at the concentrations ranging from 16 to 0.0313 μg/mL. The mode of action of 2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c) and 2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d) was established by verifying fungal growth in the presence of osmotic protector-sorbitol. The effect of compound 5c or 5d combined with Fluconazole was determined using the checkerboard method. The calculated fractional inhibitory concentration index (FIC) indicated antagonism (FIC >1). Additionally, survival experiments with lepidopteran Galleria mellonella treated with compounds 5c and 5d were performed and demonstrated the lack of toxicity of these compounds.     

Biotransformation of alpha-isomethylionone to 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one

The main biodegradation product of (+/-)-alpha-isomethylionone (2) with standard activated sludge was characterized as (+/-)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)propan-2-one (1) by its analysis and synthesis. Both enantiomers (1a and 1b) of 1 were synthesized by starting from (R)- and (S)-2,4,4-trimethyl-2-cyclohexen-1-ol (3a and 3b), respectively.     

Oxidation of n-Tetradecane and 1-Tetradecene by Fungi

Cunninghamella blakesleeana (minus strain) and a Penicillium species were grown in a mineral-salts medium containing either n-tetradecane or 1-tetradecene as substrate, and ether extracts of the mycelial mats were analyzed for oxidation products. Extracts from Cunninghamella revealed tetradecanoic acid and 13-tetradecenoic acid from the oxidation of n-tetradecane and 1-tetradecene, respectively, thereby indicating that these hydrocarbons were subject to methyl group oxidation. In contrast to Cunninghamella, the Penicillium oxidized the two substrates by subterminal attacks on methylene rather than methyl groups. This was evidenced by tentative identifications of the following alcohols and ketones from oxidation of the hydrocarbons: tetradecan-2-ol, dodecan-1-ol, tetradecan-2-one, and tetradecan-4-one from n-tetradecane, and tetradecen-4-ol, 13-tetradecen-4-ol, tetradecen-3-ol, 13-tetradecen-4-one, and tetradecen-3-one from 1-tetradecene. A pathway for hydrocarbon oxidation is proposed for subterminal oxidation at the methylene alpha to the methyl group.     

Metabolism of deuterium-labeled jasmonic acid and OPC 8:0 in the potato plant (Solanum tuberosum L.)

The metabolism of deuterium-labeled (+/-)-jasmonicacid and 3-oxo-2-[(Z)pent-2'-enyl]-cyclopentan-1- octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z) pent-2'-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (+/-)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z)-2'-pentenyl]cyclopentan-1-octanoic acid to 5' and 4'-O-glucopyranosyloxyjasmoic acids was strongly suggested.     

Synthesis and antibacterial activity of egonol derivatives

Synthesis of egonol derivatives, 5-(3'-chloropropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 1, 5-(3'-bromopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 2, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propanal 3, 5-(3'-iodopropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 4, 5-[3-(3'-bromopropyloxy) propyl]-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 5, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylmethanoate 6, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propyloleate 7, 5-[3'-hydroxypropyl]-6-bromo-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran 8, 4-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]butanenitrile 9, 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propylbenzoate 10, 5-[3'-hydroxypropyl]-7-methoxy-3-nitro-2-(3',4'-methylenedioxyphenyl)benzofuran 11 and their antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans and Escherichia coli are reported. The starting material egonol 5-[3'-(hydroxy)propyl]-7-methoxy-2-(3', 4'methylenedioxyphenyl)benzofuran was isolated from seeds of Styrax officinalis L. The structural elucidication of these compounds (1-11) was established using 1D ((1)H, (13)C), 2D NMR (HMBC, HMQC, COSY) and LCMS spectroscopic data. While egonol and some synthesised new compounds show similar antibacterial activity and MIC values against S. aureus, B. subtilis, C. albicans and E. coli, other new derivatives show different activity against S. aureus, B. subtilis, C. albicans and E. coli.     

The interaction of per-O-acetylated acyclic 1-(1-butylindol-3-yl)-1-deoxy-ketoses with silylated uracil

The per-O-acetylated open chain derivatives of 1-(1-butylindol-3-yl)-1-deoxy-1-L-sorbose and 1-(1-butylindol-3-yl)-1-deoxy-L-tagatose, which are readily available by alkaline degradation of 1-butylascorbigen followed by acetylation, were used in a nucleoside-type synthesis. The interaction of these ketoses derivatives with bis-(trimethylsilyl)-uracil yielded in each case a mixture of (E)-2,4,5,6-tetra-O-acetyl-1-(1-butylindol-3-yl)-1,3-dideoxy-3-(uracil-1-yl)-L-xylo-hexa-1-enitol and (E)-2,4,5,6-tetra-O-acetyl-1-(1-butylindol-3-yl)-1,3-dideoxy-3-(uracil-1-yl)-L-lyxo-hexa-1-enitol, which were separated by preparative HPLC. The deacetylation of each of these compounds by MeONa in MeOH produced a mixture of 1-(1-butylindol-3-yl)-1,3-dideoxy-4-O-methyl-3-(uracil-1-yl)-alpha-L-sorbopyranose and 1-(1-butylindol-3-yl)-1,3-dideoxy-4-O-methyl-3-(uracil-1-yl)-beta-D-fructopyranose, which were also separated by HPLC, the structures were confirmed by NMR.     

A new 6-C-alkylation from an alkyl mannofuranoside 5,6-cyclic sulfate

Methyl 6-C-alkyl-6-deoxy-alpha-D-mannofuranoside derivatives have been synthesized from methyl 2,3-O-isopropylidene-5,6-O-sulfuryl-alpha-D-mannofuranoside (1). In a Path A, reaction of the 5,6-cyclic sulfate 1 with 2-lithio-1,3-dithiane afforded 2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane (2). Treatment of 2 with n-butyllithium then alkyl iodide gave the corresponding 2-(methyl 5-O-alkyl-6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl )-1,3- dithiane. Reaction of 2 with n-butyllithium and 5,6-cyclic sulfate 1 furnished 2-[methyl 6-deoxy-2,3-O-isopropylidene-5-O-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-manno-furanosid-6-yl)-alpha-D - mannofuranosid-6-yl]-1,3-dithiane. 2-(Methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid- 6-yl)-1,3-dithiane was converted into the lithiated anion, which after treatment with alkyl halide afforded the corresponding 2-alkyl-C-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-1,3- dithiane. In a Path B, 5,6-cyclic sulfate 1 reacted with 2-alkyl-2-lithio-1,3-dithiane derivatives, which led after acidic hydrolysis to 2-alkyl-2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane accompanied by methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranos-5-u loside as the by-product. This methodology was applied to synthesize 2-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-2- (methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane.     

Inhibition of the acyl-CoA desaturases involved in the biosynthesis of Spodoptera littoralis sex pheromone by analogs of 10,11-methylene-10-tetradecenoic acid

The desaturase inhibitory activity of the cyclopropenyl alcohols 9,10-methylene-9-tetradecen-1-ol (9-MTOL), 10,11-methylene-10-tetradecen-1-ol (10-MTOL) and 11,12-methylene-11-tetradecen-1-ol (11-MTOL), which are structural analogs of 10,11-methylene-10-tetradecenoic acid (10-MTA), is reported. At equimolar ratios with respect to the different substrates, the three compounds completely inhibited the three desaturation reactions involved in the biosynthesis of Spodoptera littoralis sex pheromone. The dose-dependence of inhibition was determined for 10-MTA and its alcohol derivative. Both compounds inhibited the transformation of perdeuterated palmitic acid into perdeuterated (Z)-11-hexadecenoic acid and that of (E)-11-tridecenoic acid into (Z,E)-9,11-tridecadienoic acid with similar IC(50) values. The overall results presented in this work support scattered data that neither the free carboxyl groups nor their acyl-CoA esters are a requisite for inhibition of desaturases. Since the synthesis of cyclopropenols is much more convenient than that of cyclopropene fatty acids, this finding is of economical relevance regarding the putative use of cyclopropene derivatives in pest control.     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

A novel antiproliferative agent, phenylpyridineylbutenol, isolated from Streptomyces sp

A novel compound showing antiproliferative effect was isolated from Streptomyces sp. Its structure was determined based on the interpretation of the NMR spectra, and its conformation was elucidated using molecular modeling and 2D NOESY. It was determined to be (E)-4-phenyl-3-(pyridine-2-yl)but-2-en-1-ol.     

Lilac alcohol epoxide: a linalool derivative in Actinidia arguta flowers

Lilac alcohol epoxide (2-(5-methyl-5-(oxiran-2-yl)-tetrahydrofuran-2-yl)propan-1-ol), a previously unreported monoterpene, was identified in the solvent extract of the flowers of seven Actinidia arguta genotypes. The diastereomeric lilac alcohol epoxides co-occurred with the lilac aldehydes and alcohols. Another compound, the lilac diol (2-(5-(1-hydroxyethyl)-5-methyl-tetrahydrofuran-2-yl)propan-1-ol) was synthesised as part of our efforts to identify the lilac alcohol epoxide.     

Synthesis of E- and Z-pyrazolylacrylonitriles and their evaluation as novel antioxidants.

A facile synthesis of (Z)- and (E)-2-(5-arylpyrazol-3-yl)-3-(pyrrol-2-yl)acrylonitriles and (Z)-2-(1,3-diarylpyrazol-5-yl)-3-pyrrol-2-yl)acrylonitriles, and isomerisation of (Z)-2-(5-arylpyrazolyl)acrylonitriles to (E)-2-(5-arylpyrazolyl)acrylonitriles under basic conditions have been reported. (Z)-2-(1,3-Diarylpyrazolyl)acrylonitriles did not undergo isomerisation under the similar conditions. New compounds were identified on the basis of their spectral data (1H-, 13C-, 1H-1H COSY, NOESY, NOE, HMQC NMR, IR, UV and EI mass). The structures of one acrylonitrile and five of their precursor 6-arylpyran-2-ones and cyanomnethylpyrazoles were confirmed by X-ray crystallographic studies. Effects of pyrazolylacrylonitriles and their precursors on rat liver-microsomal lipid peroxidation were evaluated in vitro with a view to establish structure activity relationship and to identify a lead compound.     

Enantiospecific synthesis of carbocyclic (+)-(E)-5-(2-bromovinyl)-2'-deoxyuridine

(+)-1-[(1R, 3S, 4R)-3-hydroxy-4-hydroxymethylcyclopentyl]-5-[(E)-2- bromovinyl]-1H,3H-pyrimidin-2,4-dione 10 was synthesized starting from (+)-endo-5-norbornen-2-yl acetate. This chiral educt was obtained by enzymatic hydrolysis of racemic esters of endo-5-norbornen-2-ol.