New modified trichothecenes accumulated in solid culture by mutant strains of Fusarium sporotrichioides.

Mutant strains of Fusarium sporotrichioides NRRL 3299 deficient in the ability to synthesize T-2 toxin were examined on solid rice medium. Five novel alicyclic trichothecenes were isolated: 11 alpha-hydroxytrichodiene; tricho-9-ene-2 alpha,3 alpha,11 alpha-triol; tricho-9-ene-2 alpha,3 alpha,8 alpha,11 alpha-tetraol; tricho-9-ene-2 alpha,3 alpha,8 beta,11 alpha-tetraol; and tricho-9-ene-2 alpha,3 alpha,11 alpha,16-tetraol.     

Bioconversion of possible T-2 toxin precursors by a mutant strain of Fusarium sporotrichioides NRRL 3299.

Liquid cultures of a mutant strain of Fusarium sporotrichioides NRRL 3299 that accumulates trichodiene rather than T-2 toxin converted tricho-9-ene-2 alpha,3 alpha,11 alpha-triol, trichotriol (tricho-10-ene-2 alpha,3 alpha,9 alpha-triol), tricho-10-ene-2 alpha,3 alpha,9 beta-triol, 3 alpha-hydroxytrichothecene, and 3 alpha-acetoxytrichothecene to T-2 toxin. Other possible oxygenated precursors of T-2 toxin, including trichodiol (tricho-10-ene-2 alpha,9 alpha-diol), trichothecene, 4 alpha-hydroxytrichothecene, and 15-hydroxytrichothecene, were not metabolized. The results indicate that in the biosynthesis of T-2 toxin by F. sporotrichioides, (i) oxygenation at C-3 occurs prior to the second cyclization, (ii) this second cyclization involves two steps that may be nonenzymatic, and (iii) oxidation at C-3 precedes that at C-4 or C-15.     

Bioconversion of possible T-2 toxin precursors by a mutant strain of Fusarium sporotrichioides NRRL 3299

Liquid cultures of a mutant strain of Fusarium sporotrichioides NRRL 3299 that accumulates trichodiene rather than T-2 toxin converted tricho-9-ene-2 alpha,3 alpha,11 alpha-triol, trichotriol (tricho-10-ene-2 alpha,3 alpha,9 alpha-triol), tricho-10-ene-2 alpha,3 alpha,9 beta-triol, 3 alpha-hydroxytrichothecene, and 3 alpha-acetoxytrichothecene to T-2 toxin. Other possible oxygenated precursors of T-2 toxin, including trichodiol (tricho-10-ene-2 alpha,9 alpha-diol), trichothecene, 4 alpha-hydroxytrichothecene, and 15-hydroxytrichothecene, were not metabolized. The results indicate that in the biosynthesis of T-2 toxin by F. sporotrichioides, (i) oxygenation at C-3 occurs prior to the second cyclization, (ii) this second cyclization involves two steps that may be nonenzymatic, and (iii) oxidation at C-3 precedes that at C-4 or C-15.     

Sterols from the fungus Lactarium volemus

Seven ergostane-type sterols and two mono-glucosides were isolated from the ethyl acetate soluble fraction of Lactarium rolemus. Three are previously unknown, i.e. 3-O-beta-D-glucopyranosyl-22E,24R-5alpha,8alpha-epidioxyergosta-6,22-diene, 3-O-beta-D-glucopyranosyl-22E,24R-5beta,8beta-epidioxyergosta-6,22-diene and 22E,24R-ergosta-7,22-diene-3beta,5alpha,6beta,9alpha-tetraol. The structural elucidation of these compounds was mainly achieved by spectroscopic methods.     

Isolation and characterization of two new trichothecenes from Fusarium sporotrichioides strain M-1-1.

Two new trichothecenes were isolated along with T-2 toxin, neosolaniol, and HT-2 toxin from the culture filtrate of Fusarium sporotrichioides strain M-1-1. The structures of the new toxins were characterized to be 4 beta, 8 alpha-diacetoxy-12,13-epoxytrichothec-9-ene-3 alpha, 15-diol (designated NT-1) and 4 beta-acetoxy-12,13-epoxy-trichothec-9-ene-3 alpha,8 alpha,15-triol (designated NT-2).     

Studies of the oxysterol inhibition of tumor cell growth

The oxysterols 3 beta-hydroxy-5 alpha-cholest-8-en-11-one, 3 beta-hydroxy-5 alpha-cholest-8-en-7-one, 3 beta-hydroxy-5 alpha-cholest-8(14)-en-7-one, 3 beta-hydroxy-4,4'-dimethylcholest-5-ene-7 one, 4,4'-dimethylcholest-5-ene-3 beta, 7 alpha-diol, 4,4'-dimethylcholest-5-ene-3 beta, 7 beta-diol, lanost-8-ene-3 beta, 25-diol, 25-hydroxylanost-8-en-3-one, 9 alpha, 11 alpha-epoxy-5 alpha-cholest-7-en-3 beta-ol, 3 beta-hydroxycholest-5 alpha-en-22-one, and 3 beta-hydroxycholest-5-en-22-one oxime were evaluated with respect to their ability to inhibit cell growth. All of the sterols were found to possess cytotoxicity when incubated with hepatoma (HTC) and lymphoma (RDM-4) cells in culture at 10-30 microM concentrations.     

Stereospecific 1,4-addition to an alpha,beta-unsaturated steroidal epoxide: syntheses of new 15-oxygenated sterols

3 beta-Benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene (1) is a key intermediate in the synthesis of C-7 and C-15 oxygenated sterols. Treatment of 1 with benzoyl chloride resulted in the formation of 3 beta,15 alpha-bis-benzoyloxy-7 alpha-chloro-5 alpha-cholest-8(14)-ene (2). Reaction of 2 with LiAlH4 or LiAlD4 resulted in the formation of 5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3a) or [14 alpha-2H]5 alpha-cholest-7-ene-3 beta,15 alpha-diol (3b). Diol 3b was selectively oxidized by Ag2CO3/celite to [14 alpha-2H]5 alpha-cholest-7-en-15 alpha-ol-3-one (4). Treatment of 1 with MeMgI/CuI gave 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 alpha-diol (5). Selective oxidation of 5 with pyridinium chlorochromate (PCC)/pyridine or oxidation with PCC resulted in the formation of 7 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one (6) and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3,15-dione, respectively. Reduction of 6 with LiAlH4 yielded 5 and 7 alpha-methyl-5 alpha-cholest-8(14)-ene-3 beta,15 beta-diol (6). Reaction of 1 with benzoic acid/pyridine gave 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-en-15 alpha-ol (9). Treatment of 9 with LiAlH4 or ethanolic KOH resulted in the formation of 5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol (10). Dibenzoate 9, upon brief treatment with mineral acid, gave 3 beta-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (11). Oxidation of 9 with PCC yielded 3 beta,7 alpha-bis-benzoyloxy-5 alpha-cholest-8(14)-ene-15-one (12). Ketone 12 was also prepared by the selective hydride reduction of 5 alpha-cholest-8(14)-en-7 alpha-ol-3,15-dione (13) to give 5 alpha-cholest-8(14)-ene-3 beta,7 alpha-diol-15-one (14), which was then treated with benzoyl chloride to produce 12.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Synthesis of the marine epoxy sterol 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol

The synthesis of 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol (1), a highly oxygenated marine sterol containing a 9,11-epoxide moiety in the nucleus, is described. Epoxy sterol 1 was synthesized from cholesta-5,7-dien-3 beta-ol. Oxidation of this sterol with m-chloroperbenzoic acid followed by hydrolysis and acetylation furnished 5 alpha-cholest-7-ene-3 beta,5,6 alpha-triol 3,6-diacetate (2). Mercuric acetate dehydrogenation of diacetate 2, followed by oxidation with manganese dioxide and epoxidation with m-chloroper-benzoic acid, afforded 9 alpha,11 alpha-epoxy-3 beta,5-dihydroxy-5 alpha-cholest-7-en-6-one (5). Reduction of 5 with lithium aluminum hydride gave the desired compound 1. The structures of all synthetic intermediates were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A reassignment of resonances for carbons 1, 8, and 15 in the 13C NMR spectrum of 1, based on 2D-NMR correlation spectroscopy, has been accomplished.     

Triterpenoidal glycosides from Justicia betonica

dFrom the aerial portion of Justicia betonica L., four triterpenoidal glycosides (justiciosides A-D) were isolated. Their structures were established through chemical and NMR spectroscopic analyses as olean-12-ene-1beta,3beta,11alpha,28-tetraol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, olean-12-ene-1beta,3beta,11alpha,28-tetraol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, 11alpha-methoxy-olean-12-ene-1beta,3beta,28-triol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, 11alpha-methoxy-olean-12-ene-1beta,3beta,28-triol 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside, respectively.     

Cytotoxic sesquiterpenes from Ligularia platyglossa

Four sesquiterpene lactones including an eremophilenolide dimer, named as biligulaplenolide, 1, 8beta-hydroxy-1-oxo-(14alpha,15alpha eremophil-7(11),9(10)-dien-12,8alpha-olide, 2, 1-hydroxy-2-oxo-(14alpha,15alpha eremophil-1(10),7(11),8(9)-trien-12,8-olide, 3, 4alpha,8beta,9alpha-trihydroxy- 5alphaEta-7(11)-eudesmen-12,8alpha-olide, 4, along with two known ones, 10alpha-hydroxy-1-oxo-eremophil-7(11),8(9)-dien-12,8-olide, 5, and furanoeremophil-1(10)-ene-2,9-dione, 6, were isolated from the underground organs of Ligularia platyglossa (Franch.) Hand.-Mazz. Their structures were elucidated by spectroscopic methods including single-crystal X-ray diffraction analysis (2 and 3). Their in vitro cytotoxicities against seven cancer cell lines (BGC-823, A549, HL-60, B16, SMMC-7721, BEL7402, Hela) were evaluated. Compounds 2, 3, 5 showed cytotoxic activities on HL-60 cancer cells with IC50 in the range of 24.0 to 51.1 microM, whereas compound 3 exhibited only weak cytotoxic activity against the B16, BEL7402 and Hela cancer cells. Flow cytometric analysis indicated that compound 3 induces Hela cells to apoptotic death after 48 h treatment with 0.38 mM of this compound.     

Metabolism of prostaglandins D2 and F2 alpha in primary cultures of rat hepatocytes

3H-Labeled prostaglandins D2 and F2 alpha rapidly degraded to more-polar metabolites in primary cultured rat hepatocytes. The metabolites of prostaglandins D2 and F2 alpha accumulated in the culture medium. The metabolites extracted by ethyl acetate at pH 3 were purified by silicic acid column and thin-layer chromatography of silica gel, and were analysed by gas chromatography-mass spectrometry. The major metabolites from prostaglandin D2 were identified as dinor-prostaglandin D1 (7 alpha,13-dihydroxy-9-ketodinorprost-11-enoic acid) and tetranor-prostaglandin D1 (5 alpha,11- dihydroxy-7-ketotetranorprost-9-enoic acid). Those from prostaglandin F2 alpha were identified as dinor-prostaglandin F1 alpha (7 alpha,9 alpha,13-trihydroxydinorprost-11-enoic acid), tetranor-prostaglandin F1 alpha (5 alpha,7 alpha,11-trihydroxytetranorprost-9-enoic acid) and 9 alpha,11 alpha,15-trihydroxyprost-13-ene-1,20-dioic acid. These data indicate that prostaglandins D2 and F2 alpha mainly degraded by beta-oxidation, which is the same process as reported earlier for prostaglandins E1 and E2, and that prostaglandin F2 alpha was also subjected to omega-oxidation.     

Cholestane rhamnosides from the bulbs of Ornithogalum saundersiae.

Phytochemical examination of the bulbs of Ornithogalum saundersiae yielded six cholestane rhamnosides, two of which had previously been isolated from the same plant material. However, detailed spectroscopic analysis of the aglycone led us to revise the configuration of the C-11 hydroxyl group of the latter two and reassign their structures as (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-alpha-L-rhamnopyranoside and (22S)-cholesta-5,24-diene-3 beta,11 alpha,16 beta,22-tetrol 16-O-alpha-L-rhamnopyranoside, respectively. The other four are new naturally occurring constituents and their structures were determined to be (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-(2,3-di-O-acetyl-alpha-L-rhamnopyranoside), (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-{2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-alpha-L-rhamnopyran oside}, (22S)-cholest-5-ene-3 beta,11 alpha,16 beta,22-tetrol 16-O-{2-O-acetyl-3-O-(p-methoxybenzoyl)-alpha-L-rhamnopyranoside} and (22S)-cholesta-5,24-diene-3 beta,11 alpha,16 beta,22-tetrol 16-O-(2,3-di-O-acetyl-alpha-L-rhamnopyranoside), respectively. The isolated compounds were evaluated for their cytostatic activity against leukemia HL-60 cells.     

Reduction of aromatic steroidal A rings by lithium in ethyl amine.

The reduction of 3-methoxy-estra-1,3,5(10)-trien-17beta-ol (6) and 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol (2) by lithium in ethyl amine in the absence of a proton source is described. Both reductions, contrary to the reports of previous investigators, which indicated the 4-ene to be the main reaction product, gave a complex mixture of products. In the case of the reduction of 2, which is an intermediate in the synthesis of the progestagen desogestrel (1), we obtained the expected known 13-ethyl-gona-4-ene-11alpha,17beta-diol (4) in small amounts and three new steroidal monoenes, 13-ethyl-gona-5(10)-ene-11alpha,17beta-diol (11), 13-ethyl-gona-5(6)-ene-11alpha,17beta-diol (12), and 13-ethyl-gona-1(10)-ene-11alpha,17beta-diol (13). These compounds were characterized as the 11,17-diacetates with the 5(10)-ene 11 being the major compound.     

Isolation and structure of a cytotoxic epoxy sterol from the marine mollusc Planaxis sulcatus

A novel epoxy sterol isolated from the marine mollusc Planaxis sulcatus has been identified as 9 alpha,11 alpha-epoxycholest-7-ene-3 beta,5 alpha,6 beta-triol by spectrometric methods.     

The chemistry of 9 alpha-hydroxysteroids. 3. Methods for selective formation and dehydrations of 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one

Two methods to produce the 17-cyanohydrin, using potassium cyanide in acetic acid/methanol or acetone cyanohydrin with aqueous sodium hydroxide, were followed with 9 alpha-hydroxyandrost-4-ene-3,17-dione, both providing 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one. The selectivity of one of these methods, that which uses acetone cyanohydrin, is not in agreement with a comparable reaction with the 9 alpha-unsubstituted androst-4-ene-13,17-dione to give the 17 alpha-cyano-17 beta-hydroxy product, as reported in the literature and confirmed by us. The 9 alpha-hydroxy and 17 alpha-hydroxy groups were used for the regioselective introduction of 9(11)- and 16(17)-double bonds by dehydrating 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one under different conditions.     

Two new taxanes from the needles and branches bark of Taxus cuspidata

Two new taxanes were isolated from the MeOH extract of the needles and branches bark of the Japanese yew, Taxus cuspidata. The structures were established as (2alpha,5alpha,7beta,9alpha,10beta,13alpha)-5,10,13,20-tetraacetoxytax-11-ene-2,7,9-triol (1) and (2alpha,5alpha,9alpha,10beta)-2,9,10-triacetoxy-5-[(beta-D-glucopyranosyl)oxy]-3,11-cyclotax-11-en-13-one (2) on the basis of in-depth 1D- and 2D-NMR analyses. Compound 2 is the first example of a transannular taxane glycoside isolated from a natural source.     

21beta-Hydroxy-oleanane-type triterpenes from Hippocratea excelsa

Stem bark of Hippocratea excelsa afforded six pentacyclic triterpenes, five oleanane and one ursane types. They were identified as 11beta,21beta-dihydroxy-olean-12-ene-3-one (2), 3alpha,11alpha,21beta-trihydroxy-olean-12-ene (3), 3alpha,21beta-dihydroxy-11alpha-methoxy-olean-12-ene (4), 3alpha,21beta-dihydroxy-olean-9(11),12-diene (5), 3alpha,21beta-dihydroxy-olean-12-ene (6) and 3alpha,21beta-dihydroxy-11alpha-methoxy-urs-12-ene, isolated as its diacetate derivative (7), as well as 3alpha,21beta-dihydroxy-olean-12-ene (1) previously isolated from the root bark. The known alpha- and beta-amyrin, oleanoic and ursolic acids, trans-polyisoprene, and the ubiquitous beta-sitosterol were also isolated. Structures were elucidated on the basis of spectroscopic analyses, including homo- and heteronuclear correlation NMR experiments (COSY, ROESY, HSQC and HMBC) and comparison with literature data. The antigiardial activity of compounds 2-5 was not significant.     

Two novel C29-5beta-sterols from the stems of Opuntia dillenii

Two novel C29-5beta-sterols, opuntisterol [(24R)-24-ethyl-5beta-cholest-9-ene-6beta,12alpha-diol] (1) and opuntisteroside [(24R)-24-ethyl-6beta-[(beta-d-glucopyranosyl)oxy]-5beta-cholest-9-ene-12alpha-ol] (2), together with nine known compounds, beta-sitosterol (3), taraxerol (4), friedelin (5), methyl linoleate (6), 7-oxositosterol (7), 6beta-hydroxystigmast-4-ene-3-one (8), daucosterol (9), methyl eucomate (10) and eucomic acid (11), were isolated from the stems of Opuntia dillenii collected in Guizhou Province, China. Their structures were elucidated mainly by spectroscopic analysis. The absolute configuration of 1 were deduced from comparative 1H NMR data of the (S)- and (R)-methoxyphenyl acetate derivatives. Compounds 6-8, 10 and 11 were isolated from O. dillenii for the first time.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.