Two new sesquiterpene lactones from Ixeris chinensis

Phytochemical investigation of Ixeris chinensis NAKAI (Asteraceae) has resulted in the isolation of a new guaianolide-type sesquiterpene lactone, ixerochinolide (1) as well as the related glucoside, ixerochinoside (2). In addition, the known guaianolides, 8beta-hydroxy-3-oxo-guaia-4(15),10(14),11(13)-trien-1alpha,5alpha,6beta,7alphaH-12,6-olide (8beta-hydroxydehydrozaluzanin), 8beta,15-dihydroxy-2-oxo-guaia-1(10),3,11(13)-trien-5alpha,6beta,7alphaH-12,6-olide (lactucin), 3beta,8alpha,10alpha-trihydroxy-guaia-4(15),11(13)-dien-1alpha,5alpha,6beta,7alphaH-12,6-olide (10alpha-hydroxy-10,14-dihydro-desacylcynaropicrin) and 3beta-D-glucopyranosyloxy-8beta-(p-hydroxyphenylacetyloxy)-guaia-4(15),10(14),11(13)-trien-1alpha,5alpha,6beta,7alphaH-12,6-olide (8-epicrepioside) were identified. The structures were determined on the basis of spectral analyses, especially 1- and 2D NMR. Compound 1 exhibited significant cytotoxicity against human PC-3 tumor cells.     

Trace determination of steroids causing age-related diseases using LC/MS combined with detection-oriented derivatization

With the rapid shift to an aging society in Japan, age-related diseases, such as osteoporosis, dementia and cancer, are sharply increasing. The measurement of steroids related to these diseases in biological fluids and tissues is useful for elucidation of the nature, diagnosis and treatment of such diseases. LC/MS is considered to be the most promising method for this purpose due to its specificity and versatility, but it sometimes does not demonstrate the required sensitivity for trace amounts of steroids, because steroids have a rather low response using either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). To overcome this problem, the author developed detection-oriented derivatization procedures for steroids in LC/MS. For ESI-MS, introduction of a permanently charged moiety is effective. Based on this, 2-hydrazino-1-methylpyridine was developed and used in monitoring prostatic 5alpha-dihydrotestosterone, a good index for the follow-up of patients affected by prostate cancer under androgen deprivation therapy and salivary dehydroepiandrosterone, which is now often designated as an anti-aging hormone. A proton-affinitive Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, was used for the determination of 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], a synthetic prodrug of the active form of vitamin D3, in human plasma, and this new LC/positive-APCI-MS method enabled the pharmacokinetic study of 1alpha(OH)D3 in humans. Electron-capture APCI-MS based on derivatization with 2-nitro-4-trifluoromethylphenylhydrazine was used for the analysis of neurosteroids, which affect brain excitability through action at the neurotransmitter receptors. With this method, the stress-induced rapid biosynthesis of pregnane-type neurosteroids in rat brains was demonstrated.     

A new pyrrolidine derivative and steroids from an algicolous Gibberella zeae strain

A new pyrrolidine derivative, 3-hydroxy-5-(hydroxymethyl)-4-(4'-hydroxyphenoxy)pyrrolidin-2-one (1), and eight known steroids, (22E,24R)-7beta,8beta-epoxy-3beta,5alpha,9alpha-trihydroxyergosta-22-en-6-one (2, a reassigned structure of (22E,24R)-5alpha,6alpha-epoxy-3beta,8beta,14alpha-trihydroxyergosta-22-en-7-one), (22E,24R)-3beta,5alpha,9alpha-trihydroxyergosta-7,22-dien-6-one (3), (22E,24R)-3beta,5alpha-dihydroxyergosta-7,22-dien-6-one (4), (22E,24R)-ergosta-7,22-dien-3beta/,5alpha,6beta-triol (5), (22E,24R)-ergosta-5,22-dien-3beta-ol (6), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (7), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,9(11),22-trien-3beta-ol (8), and (22E,24R)-1(10 --> 6)-abeo-ergosta-5,7,9,22-tetraen-3alpha-ol (9), were isolated from the cultures of Gibberella zeae, an endophytic fungus isolated from the marine green alga Codium fragile. Their structures and relative stereochemistry were elucidated by 1D, 2D NMR and mass spectroscopic techniques. Compound 1 showed cytotoxicity against A-549 and BEL-7402 cell lines.     

Synthesis of deuterium-labeled 16 alpha,19-dihydroxy C19 steroids as internal standards for gas chromatography-mass spectrometry.

[2 beta,7,7,16 beta-2H4]16 alpha,19-Dihydroxyandrost-4-ene-3,17-dione (14) and [7,7,16 beta-2H3]3 beta,16 alpha,19-trihydroxyandrost-5-en-17-one (16), with high isotopic purity, respectively, were synthesized from unlabeled 3 beta-(tert-butyldimethylsiloxy)-androst-5-ene-17 beta-yl acetate (1). The deuterium introduction at C-7 was carried out by reductive deoxygenation of the 7-keto compound 3 with dichloroaluminum deuteride and that at C-2 beta and/or C-16 beta by controlled alkaline hydrolysis of 16-bromo-17-ketone 11 or 12 with NaOD in D2O and pyridine. [7,7-2H2]3 beta-Hydroxyandrost-5-en-17-one (6), obtained from compound 1 by a five-step sequence, was converted to compound 14 or 16 by an eight-step or seven-step sequence, respectively. The labeled steroids 14 and 16 are useful as internal standards for gas chromatography-mass spectrometry analysis of the endogenous levels.     

Tetracyclic triterpenoids from the leaves of Azadirachta indica and their insecticidal activities

A new tetranortriterpenoid, meliatetraolenone [24,25,26,27-tetranor-apotirucalla-(apoeupha)-6alpha-O-methyl, 7alpha-senecioyl(7-deacetyl)-11alpha,12alpha,21,23-tetrahydroxy-21,23-epoxy-2,14,20(22)-trien-1,16-dione] (1) was isolated from the methanolic extract of fresh leaves of Azadirachta indica along with the known compound odoratone (3) which was hitherto unreported from this source. Their structures have been elucidated by spectral studies including 2D NMR. The insecticidal activities of 1 as well as those of odoratone (3) are reported. 1 and odoratone both showed mortality on fourth instar larvae of mosquitoes (Anopheles stephensi) with LC(50) values of 16 and 154 ppm, respectively.     

Pregnane derivatives from Potentilla evestita

A new pregnane derivative, 2,6beta,7beta-trihydroxy-4-methyl-19-norpregna-1,3,5(10)-trien-17-one, has been isolated from the ethyl acetate soluble fraction of Potentilla evestita along with a pregnane derivative, 11alpha,17alpha,21-trihydroxypregna-4,16(22)-diene-3,20-dione, that is reported for the first time as a natural product. Their structures were elucidated with the aid of 1H and 13C NMR spectra and by COSY, HMQC, HMBC and NOESY experiments.     

Biotransformation of phorbol by human intestinal bacteria

Anaerobic incubation of phorbol (1) from Croton tiglium with human intestinal bacteria afforded five metabolites: isophorbol (2), deoxyphorbol (3), 4beta,9alpha,20-trihydroxy-13,15-seco-1,6,15-tigliatriene-3,13-dione (4), 4beta,9alpha,20-trihydroxy-15,16,17-trinor-1,6-tigliadiene-3,13-dione (5) and 4beta,9a,20-trihydroxy-14(13-->12)-abeo-12alphaH-1,6-tigliadiene-3,13-dione (6). All these metabolites (2-6) were identified and characterized by spectroscopic means, including two-dimensional (2D)-NMR. Nine defined strains from the human intestine showed an ability to transform 1 to these metabolites.     

Pregnane- and furostane-type oligoglycosides from the seeds of Allium tuberosum

Two furostane-type steroidal oligoglycosides (1, 2), together with a new pregnane-type oligoglycoside (3), were obtained from the seeds of Allium tuberosum ROTTLER. On the basis of spectroscopic analysis, the structures of three new oligoglycosides (1-3) were elucidated as 26-O-beta-D-glucopyranosyl-(25R)-3beta,22xi,26-trihydroxyl-5alpha-furostane 3-O-beta-chacotrioside, 26-O-beta-D-glucopyranosyl-(25S)-3beta,5beta,6alpha,22xi,26-pentahydroxyl-5beta-furostane 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranoside, and 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl 3beta,5beta,6alpha,16beta-tetrahydroxypregnane 16-(5-O-beta-D-glucopyranoyl-4(S)-methyl-5-hydroxypentanoic acid) ester, respectively.     

Antimutagenic constituents from the thorns of Gleditsia sinensis

Antimutagenic activity-guided fractionation of an extract prepared from the thorns of Gleditsia sinensis LAM. led to the isolation of one triterpenoid and four steroids, which were identified as D:C-friedours-7-en-3-one (1), stigmast-4-ene-3,6-dione (2), stigmastane-3,6-dione (3), stigmasterol (4), and beta-sitosterol (5). Triterpenoid 1 was found for the first time in a natural source and the steroids 2-5 were first isolated from this plant. Stigmasterol was the most active antimutagen, showing 51.2% and 64.2% reduction of the induction factor against the mutagens MNNG and NQO, respectively, in the SOS chromotest. Some NMR data of the steroids 2 and 3 obtained have to be revised.     

Fargosides A-E, triterpenoid saponins from Holboellia fargesii

Five new triterpenoid saponins, fargosides A, B, C, D, and E, were isolated from the roots of Holboellia fargesii. The structures of fargosides A-E were elucidated on the basis of chemical and physicochemical evidence and found to be 3beta,20alpha-dihydroxy-29-norolean-12-en-28-oic acid 3-O-beta-D-xylopyranosyl-(1-->2)-beta-D-glucopyranoside (1), 3beta,20alpha,24-trihydroxy-29-norolean-12-en-28-oic acid 23-O-beta-D-fucopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl-(1-->3)]-beta-D-glucopyranoside (2), 3beta,23-dihydroxy-30-norolean-2,20(29)-dien-28-oic acid 3-O-alpha-L-arabinopyranosyl-(1-->2)-[beta-D-glucopyranosyluronic acid-(1-->3)]-alpha-L-arabinopyranoside (3), 3beta,23-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid 3-O-methyl beta-D-glucopyranosyluronate-(1-->3)-alpha-L-arabinopyranoside (4), and 3beta,23-dihydroxy-olean-12-en-28-oic acid 3-O-methyl beta-D-glucopyranosyluronate-(1-->3)-alpha-L-arabinopyranoside (5), respectively.     

Epoxidation and reduction of DHEA, 1,4,6-androstatrien-3-one and 4,6-androstadien-3beta,17beta-diol

Dehydroepiandrosterone (DHEA) reacted with m-chloroperoxybenzoic acid(m-CPBA) to form 3beta-hydroxy-5alpha,6alpha-epoxyandrostan-17-one (1), but it did not react with 30% H2O2. 1,4,6-Androstatrien-3,17-dione (2) was obtained from DHEA and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. Compound 2 was reacted with 30%H2O2 and 5% NaOH in methanol to give 1alpha,2alpha-epoxy-4,6-androstadien-3,17-dione (3),which was stereoselectively reduced with NaBH4 to form 1alpha,2alpha-epoxy-4,6-androstadien-3beta,17beta-diol (7) and reacted with Li metal in absolute ethanol-tetrahydrofuran mixture to give 2-ethoxy-1,4,6-androstatrien-3,17-dione (8). Compound 2 was also epoxidized with m-CPBA in dichloromethane to afford 6alpha,7alpha-epoxy-1,4-androstadien-3,17-dione (4),which was reacted with NaBH4 to synthesize 6alpha,7alpha-epoxy-4-androsten-3beta,17beta-diol (9).Compound 4 was reduced with Li metal in absolute ethanol-tetrahydrofuran mixture to form 7beta-ethoxy-6alpha-hydroxy-1,4-androstadien-3,17-dione (10). Compound 2 was reduced with NaBH4 in absolute ethanol to form 4,6-androstadien-3beta,17beta-diol (5), which was reacted with 30% H2O2 to give the original compound, but which reacted with m-CPBAto give 4beta,5beta-epoxy-6-androsten-3beta,17beta-diol (6).     

Sesquiterpene lactones from Crepis cameroonica (Asteraceae)

In addition to one known compound, 3beta,8alpha-dihydroxyguaian-4(15),10(14),11(13)-trien-6,12 olide (8-desacylcynaropicrin) (3), two new sesquiterpene lactones have been isolated from the aerial parts of Crepis cameroonica. By means of spectroscopic analysis, the structures and relative configurations of the new compounds were established as 3beta,9beta-dihydroxyguaian-4(15),10(14),11(13)-trien-6,12 olide (1) and 8alpha-hydroxy-4alpha(13),11beta(15)-tetrahydrozaluzanin C (2). The in vitro antimicrobial spectrum of pure compounds and crude extracts are also reported.     

Asporyergosterol, a new steroid from an algicolous isolate of Aspergillus oryzae

Asporyergosterol (1), a new steroid with an E double bond between C-17 and C-20, was identified from the culture extracts of Aspergillus oryzae, an endophytic fungus isolated from the marine red alga Heterosiphonia japonica. Moreover, four known steroids including (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (2), (22E,24R)-3beta-hydroxyergosta-5,8,22-trien-7-one (3), (22E,24R)-ergosta-7,22-dien-3beta,5alpha,6beta-triol (4), and (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (5) were isolated. Structures of these compounds were unambiguously established by spectroscopic techniques and by comparison with literature values. All the isolates exhibited low activity to modulate acetylcholinesterase (AChE).     

Applications of gas chromatography-mass spectrometry in the study of androgen and odorous 16-androstene metabolism by human axillary bacteria

The known involvement of axillary microflora with under-arm odour (UAO) production led us to determine whether the odorous 16-androstene steroids are formed in the axilla by bacterial metabolism of an odourless precursor such as testosterone. Axillary bacteria from 34 men were selectively cultured for aerobic coryneform bacteria (ACB), Micrococcaceae and propionibacteria. Overnight suspensions of bacteria were incubated separately at 37 degrees C for two weeks with radiolabelled testosterone plus unlabelled testosterone (0.5 mg) and 0.5-mg quantities of 4,16-androstadien-3-one (androstadienone) and 5,16-androstadien-3 beta-ol (androstadienol). After extraction and purification by Sep-Pak cartridges and thin-layer chromatography, the eluted steroids were derivatised as the pentafluorobenzyl oximes (PFBO) and tert.-butyl dimethylsilyl (TBDMS) ethers. Saturated analogues were used as internal standards. Selected-ion monitoring electron-impact mass spectrometry was performed at the m/z corresponding to the M+.ion for the PFBO derivatives and the [M - 57]+ ion for the TBDMS ethers. Only ACB produced classical musk-like UAO (UAO + ve) in an in vitro odour-producing system with 29% being UAO -ve. ACB (UAO +ve) metabolised far more (p = 0.001) testosterone than ACB (UAO -ve), the principal metabolites being 5 alpha(beta)-dihydrotestosterone, 5 alpha(beta)-androstane-3,17-dione and 4-androstene-3,17-dione (4-androstenedione). No non-polar 16-androstenes were formed. Micrococcus luteus (ten strains) metabolised testosterone to 4-androstenedione only; propionibacterium spp. did not metabolise testosterone at all. However, incubation of 16-androstenes with ACB gave evidence for 4-ene-5 alpha(beta)-reduction, 3 alpha(beta)-oxido-reduction and epimerisation. In general the direction of transformations favoured formation of the more odorous 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol) from less odorous steroids. Such transformations, in vivo, would not require de novo synthesis of 5 alpha-androstenone or 3 alpha-androstenol and would be consistent with utilisation by ACB of 16-androstenes already present in small quantities in fresh apocrine secretions, which are odourless, to produce a more powerfully smelling mixture on the axillary skin surface.     

Microbial transformation of cortisol and prolyl endopeptidase inhibitory activity of its transformed products

Incubation of cortisol (1) with Gibberella fujikuruoi for 12 days yielded an oxidatively cleaved product, 11beta-hydroxyandrost-4-en-3,17-dione (2), while incubation with Bacillus subtilis and Rhizopus stolonifer yielded the reduced product, 11beta, 17alpha,20,21-tetrahydroxy-(20S)-pregn-4-en-3-one (3). Other reduced products, 11beta, 17alpha, 21-trihydroxy-5alpha-pregnan-3, 20-dione (4) and 3beta, 11beta, 17alpha, 21-tetrahydroxy-5alpha-pregnan-20-one (5) were obtained by incubation of compound 1 with Bacillus cerus. The inhibitory activity of compounds 1-5 against prolyl endopeptidase enzyme (PEP) was also assayed. Compounds 2 (IC50 162.8 microM) and 4 (IC50 157 microM) have shown significant inhibitory activity against PEP.     

Two new triterpenes from the seeds of Trichosanthes cucumeroides

Two new triterpenes named 7-oxodihydrokarounitriol (1) and 7,11-dioxodihydrokarounidiol (2), and one known triterpene, 7-oxodihydrokarounidiol (3), were isolated from the unsaponifiable matter of the seeds of Trichosanthes cucumeroides. The structures of 1 and 2 were elucidated as (3alpha, 11beta, 13alpha, 14beta, 20alpha)-3,11,29-trihydroxy-13-methyl-26-norolean-8-ene-7-one, and (3alpha,13alpha,14beta,20alpha)-3,29-dihydroxy-13-methyl-26-norolean-8-ene-7,11-dione on the basis of extensive NMR (1H, 13C, 1H-1H COSY, DEPT, HMQC, HMBC and NOESY) and MS studies.     

Steroidal sapogenins—LXII 12-methyl hemiketals derived from 12-keto-16-dehydropregnenes

Large-scale degradation of the sidechains of the 12-ketosapogenins, hecogenin and gentrogenin, gave respectively 3β-acetoxy-5,16-pregnene-12,20-dione and 3β-acetoxy-5,16-pregnadiene-12,20-dione. Methanol crystallization of the crude enones gave in small yield the crystalline hemiketals, 3β-acetoxy-12β-hydroxy-12-methoxy-5,16-pregnen-20-one and 3β-acetoxy-12β-hydroxy-12-methoxy-5,16-pregnadien-20-one. The hemiketals exist largely in a hydrogen-bonded state. Their structure proof and infrared and ultraviolet spectra are discussed in detail.     

A cytotoxic and apoptosis-inducing sesquiterpenoid isolated from the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk)

Repeated silica gel and octadecyl silica gel (ODS) column chromatography of the aerial parts of Artemisia princeps PAMPANINI (Sajabalssuk) led to the isolation of a new sesquiterpenoid, 3-((S)-2-methylbutyryloxy)-costu-1(10),4(5)-dien-12,6 alpha-olide (2), along with two previously reported sesquiterpenoids: 8 alpha-angeloyloxy-3beta,4 beta-epoxy-6 beta H,7 alpha H,8 beta H-guaia-1(10),11(13)-dien-12,6 alpha-olide (1, carlaolide B) and 3beta,4 beta-epoxy-8 alpha-isobutyryloxy-6 beta H,7 alpha H,8 beta H-guaia-1(10),11(13)-dien-12,6 alpha-olide (3, carlaolide A). The structure of compound 2 was elucidated by spectroscopic data analysis, including one dimensional (1D) and two dimensional (2D) nuclear magnetic resonance (NMR) experiments. Of the isolates, compound 2 exhibited potent cytotoxicity against human cervix adenocarcinoma cells and induced apoptosis.     

Four new steroid constituents from the waste residue of fibre separation from Agave americana leaves

Three new steroidal saponins, named agamenosides H-J (1-3), and a new cholestane steroid agavegenin D (4) were isolated from the waste residue of fibre separation from Agave americana leaves, together with six known steroids. Structures of the new compounds 1-4 were deduced to be (22S,23S,24R,25S)-24-[(beta-D-glucopyranosyl)oxy]-5alpha-spirostane-3beta,6alpha,23-triol 6-O-beta-D-glucopyranoside (1), (22S,23S,24R,25S)-5alpha-spirostane-3beta,23,24-triol 24-O-beta-D-glucopyranoside (2), (22S,23S,25R,26S)-23,26-epoxy-5alpha-furostane-3beta,22,26-triol 26-O-beta-D-glucopyranoside (3), and (22S,25S)-5alpha-cholestane-3beta,16beta,22,26-tetrol (4), respectively, by means of spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of hydrolytic cleavage.     

Cucurbitacins from Cayaponia racemosa: isolation and total assignment of 1H and 13C NMR spectra

Two new cucurbitane-type triterpenoids, 2beta,3beta,16alpha,20(R),25-pentahydroxy-9-methyl-19-norlanost-5-en-7,22-dione and 2beta,3beta,16alpha,20(R),25-pentahydroxy-9-methyl-19-norlanost-5-en-7,11,22-trione, were isolated from fruits of Cayaponia racemosa. The total (1)H and (13)C chemical shift assignment of these two closely related compounds is described, making use of one- and two-dimensional NMR techniques.