Synthesis of 8-C-glucosylflavones

The syntheses of orientin, parkinsonin A, isoswertiajaponin, and parkinsonin B, which are 8-C-beta-D-glucopyranosyl-3',4',5,7-tetrahydroxyflavone, 5-methyl orientin, 7-methyl orientin, and 5,7-dimethyl orientin, respectively, are reported herein. The C-glucosyl phloroacetophenone derivatives were obtained via a regio- and stereoselective O-->C glycosyl rearrangement. Aldol condensation of the C-glucosyl phloroacetophenone derivatives with 3,4-bisbenzyloxybenzaldehyde afforded the corresponding C-glucosylchalcones. Construction of the flavone system by reaction with I(2)-Me(2)SO, followed by the elimination of the 5-benzyl protecting group in the flavone structure, yielded an orientin derivative and a isoswertiajaponin derivative. Methylation of the orientin derivatives with dimethyl sulfate afforded the parkinsonin A derivative, the isoswertiajaponin derivative, and the parkinsonin B derivative. Finally, hydrogenolysis of these C-glucosylflavone derivatives led to the four 8-C-glucosylflavones. The NMR spectra of these C-glucosylflavones showed a duplication of signals corresponding to a major rotamer, along with a minor one. Based on NOESY experiments in Me(2)SO at ambient temperature, they adopted conformations in which the H-2"and H-4" protons in the glucose moiety were oriented toward the B-ring in the flavone structure.     

Cholest-4-en-3-one-delta 1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Delta(1)-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Delta(1)-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Delta(1)-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Cholest-4-En-3-One-Δ1-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ¹-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ¹-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ¹-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.     

Antibacterial and cytotoxic activity of prenylated bicyclic acylphloroglucinol derivatives from Hypericum amblycalyx

Two new bicyclic acylphloroglucinol derivatives, hypercalyxone A (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 1) and B (1-[5,7-dihydroxy-2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 2), have been isolated from the petroleum ether extract of the aerial parts of Hypericum amblycalyx, together with two further compounds (1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-propan-1-one, 3 and 1-[5,7-dihydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-chroman-8-yl]-2-methyl-butan-1-one, 4), which have been described only as semi-synthetic products. In addition, the known triterpene lup-20(29)-en-3-one was obtained. Structure elucidation was based on 1D and 2D NMR studies, as well as on data derived from mass spectrometry. The four acylphloroglucinol derivatives were evaluated for their cytotoxic and antibacterial activity. All compounds showed moderate cytotoxic activity against KB and Jurkat T cancer cells. Especially compounds 3 and 4 exhibited a strong antibacterial activity against different Gram-positive strains.     

The reactions of palladium(II), thallium(III) and lead(IV) trifluoroacetates with 3beta-acetoxyandrost-5-en-17-one: crystal structure of the first trifluoroacetate bridged 5,6,7-pi-allyl steroid palladium dimer

A number of metal trifluoroacetates were reacted with the olefin 3beta-acetoxyandrost-5-en-17-one (6). Palladium(II) trifluoroacetate afforded bis[micro-trifluoroacetato(alpha-5,7-eta-3beta-acetoxyandrostenyl-17-one)palladium(II)] (20), a new ring B pi-allyl steroid-palladium complex, in quantitative yield. Thallium(III) trifluoroacetate gave 3beta-acetoxy-5alpha-hydroxy-6beta-trifluoroacetoxyandrostan-17-one (16), 3beta-acetoxy-6beta-trifluoroacetoxyandrost-4-en-17-one (9), 3beta-acetoxy-4beta-trifluoroacetoxyandrost-5-en-17-one (10), and 3beta-acetoxy-5alpha,6beta-dihydroxyandrostan-17-one (17). Lead(IV) trifluoroacetate yielded 9, 10 and 16. 3beta-Acetoxy-5alpha,6beta-bis(trifluoroacetoxy)androstan-17-one (15), a new compound, was also formed in this reaction. During the course of the lead(IV) studies the dichlorosteroid 21 and the rearranged allylic oxidation product 24 were formed. Their formation was attributed to the generation of lead(IV) chloride in the reaction. Silver(I) and copper(II) trifluoroacetates proved to be unreactive towards 6.     

Dehydrodinosterol,dinosterone and related sterols of a non-photosynthetic dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinoflagellate Crypthecodinium cohnii contained the 4α-methyl sterols, dinosterol, dehydrodinosterol (4α,23,24-trimethylcholesta-5,22-dien-3β-ol) and the tentatively identified 4α,24-dimethyl-cholestan-3β-ol and 4α,24-dimethylcholest-5-en-3β-ol. The major 4-demethyl sterol was cholesta-5,7-dien-3β-ol which was accompanied by a smaller amount of cholesterol and traces of several other C₂₇,C₂₈ and C₂₉ sterols. In addition, a 3-oxo-steroid fraction was isolated and the major component identified as dinosterone (4α,23,24-trimethylcholest-22-en-3-one). The possible biosynthetic relationships of these compounds are discussed.     

The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.     

Homoisoflavonoids and xanthones from the tubers of wild and in vitro regenerated Ledebouria graminifolia and cytotoxic activities of some of the homoisoflavonoids

Eleven homoisoflavonoids and two xanthones were isolated and characterized from the bulbs of Ledebouria graminifolia. Five of the homoisoflavonoids are new compounds and were identified as: 5-hydroxy-7-methoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-6,7-dimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5,7,8-trimethoxy-3-(4'-hydroxybenzyl)-4-chromanone, 5-hydroxy-3',4',7-trimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one, 5,7-dihydroxy-3',4'-dimethoxyspiro[2H-1-benzopyran-7'-bicyclo[4.2.0]octa-trien]-4-one. Structures were elucidated by extensive 1D, and 2D NMR spectroscopy and HRMS. A method for tissue culture was developed and the bulbs of mature plants were found to contain all the compounds isolated from the wild specimens of L. graminifolia.     

Synthesis of novel 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-ones from thiosemicarbazide precursors

To develop novel biologically active organic compounds possessing a sugar moiety, a series of 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-one were synthesized via reaction of the thiosemicarbazide with ethyl bromoacetate. Their chemical structures were characterized by (1)H and (13)C NMR spectroscopy, elemental analysis and MS. The bioassay results indicated that some of these compound exhibit moderate fungicidal and herbicidal activities. Furthermore, the effect of various solvents at reflux temperature on the reactions of ethyl bromoacetate with the related thiosemicarbazides was investigated.     

Antifungal constituents from the Chinese moss Homalia trichomanoides

Bioautographic assay on TLC plates was adopted to guide the fractionation of the Et2O extract of Homalia trichomanoides (Hedw.) B. S. G., which led to the isolation of the novel p-terphenyl derivative trichomanin (= 4,4'-dihydroxy-1,1':4',1'-terphenyl-2',3',5',6'-tetrayl tetrakis(phenylacetate); 1), together with five known compounds: 3alpha-methoxyserrat-14-en-21beta-ol (2), 3beta-methoxyserrat-14-en-21beta-ol (3), 3beta-methoxyserrat-14-en-21-one (4), atranorin (5), and methyl 2,4-dihydroxy-3,6-dimethylbenzoate (6). Their structures were determined on the basis of spectral data (1D- and 2D-NMR, MS), X-ray crystallographic analysis, and chemical transformation. Compounds 3, 5, and 6 exhibited antifungal activity against Candida albicans, with minimum inhibitory doses (MID) of 2.0, 2.0, and 0.6 microg, respectively.     

Synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxyandrost-5-en-17-one via microbial 7α-hydroxylation

The synthesis of 3β-hydroxy-androsta-5,7-dien-17-one from 3β-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA) via microbial 7α-hydroxylation has been accomplished. At the first stage, 3β,7α-dihydroxy-androst-5-en-17-one was obtained in high yield (71.2%) using a strain of Gibberella zeae VKM F-2600, which was first applied for DHEA conversion. The further route included the substitution of 7α-hydroxyl group with chlorine followed by a dehydrochlorination stage, and required minimal purifications of the intermediate products. The steroids obtained at every step were characterized by TLC_,¹H NMR, MS, UV- and IR-spectrometry.The combination of microbial and chemical steps ensured 54.6% yield of the target 3β-hydroxy-androsta-5,7-dien-17-one from DHEA and can be applied for obtaining novel vitamin D derivatives.     

Three isoflavanones with cannabinoid-like moieties from Desmodium canum

Three further derivatives of 5,7,2',4'-tetrahydroxy-6-methyl isoflavanone have been isolated from the root extract of Desmodium canum and assigned the structures 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(1a,2,3,3a,8b,8c-hexahydro-6-hydroxy-1,1,3a-trimethyl-1H-4-oxabenzo[f]cyclobut[c,d]inden-7-yl)-4H-1-benzopyran-4-one (1) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(6a,7,8,10a-tetrahydro-3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl)-4H-1-benzopyran-4-one (2) 2,3-dihydro-5,7-dihydroxy-6-methyl-3-(3-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-2-yl) 4H-1-benzopyran-4-one (3). The three compounds and the previously isolated chromene 4 all derive from the geranylated precursor 5 by a series of cannabinoid-like oxidative rearrangements.     

An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway

The natural occurrence of 22-hydroxylated steroids in cultured Catharanthus roseus cells and in Arabidopsis seedlings was investigated. Using full-scan gas chromatography-mass spectrometry analysis, (22S)-22-hydroxycampesterol (22-OHCR), (22S,24R)-22-hydroxyergost-4-en-3-one (22-OH-4-en-3-one), (22S,24R)-22-hydroxy-5alpha-ergostan-3-one (22-OH-3-one), 6-deoxocathasterone (6-deoxoCT), 3-epi-6-deoxoCT, 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6-deoxoCT, and 3-epi-28-nor-6-deoxoCT were identified. Metabolic experiments with deuterium-labeled 22-OHCR were performed in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and the metabolites were analyzed by gas chromatography-mass spectrometry. In both C. roseus cells and wild-type Arabidopsis seedlings, [(2)H(6)]22-OH-4-en-3-one, [(2)H(6)]22-OH-3-one, [(2)H(6)]6-deoxoCT, and [(2)H(6)]3-epi-6-deoxoCT were identified as metabolites of [(2)H(6)]22-OHCR, whereas the major metabolite in det2 seedlings was [(2)H(6)]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed that det2 accumulates 22-OH-4-en-3-one. The levels of downstream compounds were remarkably reduced compared with the wild type. Exogenously applied 22-OH-3-one and 6-deoxoCT were found to rescue det2 mutant phenotypes, whereas 22-OHCR and 22-OH-4-en-3-one did not. These results substantiate the existence of a new subpathway (22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-deoxoCT) and reveal that the det2 mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one, which leads to brassinolide biosynthesis.     

Total synthesis of junionone, a natural monoterpenoid from Juniperus communis L., and determination of the absolute configuration of the naturally occurring enantiomer by ROA spectroscopy

Recently, we reported a novel access to 2,2-diethyl-3-[(E/Z)-prop-1-en-1-yl]cyclobutanone by an intramolecular nucleophilic substitution with allylic rearrangement (S(N)i') of (E)-6-chloro-3,3-diethylhept-4-en-2-one. The ring closure reaction was found to proceed with selective syn-displacement of the leaving group. This method was now applied to the total synthesis of junionone, an olfactorily interesting cyclobutane monoterpenoid isolated from Juniperus communis, L. S(N)i' Ring closure of the ketone enolate of (E)-3,3-dimethyl-5-[(2R,3R)-3-methyloxiran-2-yl]pent-4-en-2-one (R,R)-(E)-4' proceeded only after the epoxide moiety had been activated by Lewis acid and led to the junionone precursors (3R)- and (3S)-3-[(1E,3R)-3-hydroxybut-1-en-1-yl]-2,2-dimethylcyclobutanone (S/R,R)-(E)-3. The ratio of syn- and anti-conformers in the transitory molecular arrangement was found to depend on the nature of the Lewis acid. The absolute configuration of both the synthetic as well as the natural junionone, isolated from juniper berry oil, was determined by Raman Optical Activity (ROA) spectroscopy. Our experiments led to a novel synthetic route to both (+)- and (-)-junionone, the first determination of the absolute configuration of natural junionone, and to the development of a practical ROA procedure for measuring milligram quantities of volatile liquids.     

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.     

Homoisoflavanones from Disporopsis aspera

From cytotoxic extracts of the roots of Disporopsis aspera Engl. (Liliaceae) a homoisoflavanone, disporopsin (3-(2',4'-dihydroxy-benzyl)-5,7-dihydroxy-chroman-4-one) (1) and three rare methyl-homoisoflavanones, 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one (2), 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4-one (3) and 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-8-methoxy-chroman-4- one (4) along with five other known compounds, N-trans-feruloyl tyramine (5), adenine (6), 5-(hydroxymethyl)-2-furfural (7), beta-sitosterol (8) and beta-sitosteryl glucopyranoside (9) were isolated. The structures of compounds 1-2 were elucidated by spectral data (1, 2-D NMR and EIMS). The four homoisoflavanones (1-4) were found to be cytotoxic against a series of human cancer cell lines (HCT15, T24S, MCF7, Bowes, A549 and K562) with IC(50) ranging from 15 to 200 microM. Possible biosynthesis routes for homoisoflavonoids (1-4) are discussed.     

Simultaneous determination of 3,3',4',5,7-pentamethylquercetin and its possible metabolite 3,3',4',7-tetramethylquercetin in dog plasma by liquid chromatography-tandem mass spectrometry and its application to preclinical pharmacokinetic study

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)-ESI-MS/MS method was established for the simultaneous determination of 3,3',4',5,7-pentamethylquercetin (PMQ) and its possible metabolite 3,3',4',7-tetramethylquercetin (TMQ) in dog plasma using 4',5,7-trimethylapigenin (TMA) as the internal standard. The plasma sample was pretreated with acetonitrile for protein precipitation and the analytes were separated on an Ultimate XB-CN column (5 μm, 2.1 mm × 150 mm) with the mobile phase consisting of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a positive multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 373.1-312.1 for PMQ, 359.1-344.0 for TMQ and 313.1-298.1 for TMA. The validated concentration ranged from 1.272 to 3060 ng/mL for PMQ and from 10.35 to 1725 ng/mL for TMQ. The lower limit of quantifications for PMQ and TMQ were 1.272 ng/mL and 10.35 ng/mL, respectively. The developed-method was successfully applied for the pharmacokinetic study of PMQ and its metabolite TMQ in dogs following a single oral dose.     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

In vitro metabolism of possible ecdysone precursors by the prothoracic glands of the tobacco hornworm, Manduca sexta

3 beta, 14 alpha-Dihydroxy-5 alpha-7-en-6-one (5 alpha-ketodiol) (1) is metabolized by the prothoracic glands to 2,22-dideoxy-5 alpha-ecdysone (4) and 2-deoxy-5 alpha-ecdysone (3) but not to ecdysone (5) or any other 5 beta-metabolites. Similarly, 3 beta,5 alpha,14 alpha-trihydroxy-cholest-7-en-6-one (5 alpha-ketotriol) (8) is hydroxylated at C-22 and C-25 (9,10) of the side chain. However, 3 beta,14 alhpa-dihydroxy-cholesta-4,7-diene-6-one (ketodienediol) (11) is not metabolized. The absence of 2 beta-hydroxymetabolites for substrates (1) and (8) implies that hydroxylation at C-2 can occur only when the A-B rings are cis fused (5 beta-configuration). By contrast, the enzyme complexes that introduce hydroxyls at C-22 and C-25 do not exhibit a preference for cis over trans fusion and appraently cannot recognize the planar A-B ring configuration.