Investigation of the importance of the C-2 oxygen function in the transformation of stemodin analogues by Rhizopus oryzae ATCC 11145

A new stemodinoside, stemodin-alpha-L-arabinofuranoside (5), was isolated from the plant Stemodia maritima. Incubation of stemodin (2) with Rhizopus oryzae ATCC 11145 gave 2 alpha,7 beta,13(S)-trihydroxystemodane (17) and 2 alpha,3 beta,13(S),16 alpha-tetrahydroxystemodane (18) whilst stemodinone (8) afforded 6 alpha,13(S)-dihydroxystemodan-2-one (19). The bioconversion of 2 beta,13(S)-dihydroxystemodane (10) by the fungus yielded 2 beta,7 beta,13(S)-trihydroxystemodane (20) whereas stemod-12-en-2-one (9) provided 7 beta,17-dihydroxystemod-12-en-2-one (21). The results provide useful information about the relationship between the functional groups of the substrates and their potential for bioconversion.     

Stemodane and stemarane diterpenoid hydroxylation by Mucor plumbeus and Whetzelinia sclerotiorum

Incubation of stemodin (1) with Mucor plumbeus ATCC 4740 resulted in the formation of 2alpha,6beta,13-trihydroxystemodane (2), 2alpha,3beta,13-trihydroxystemodane (3), 2alpha,11beta,13-trihydroxystemodane (4) and 2alpha,13,14-trihydroxystemodane (5), while stemodinone (7) afforded 6alpha,13-dihydroxystemodan-2-one (8) and 6alpha,12alpha,13-trihydroxystemodan-2-one (9). Metabolites obtained from the bioconversion of stemarin (11) were 8,13,19-trihydroxystemarane (12) and 2alpha,13,19-trihydroxystemarane (13). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (14) was not transformed by the fungus. Stemodin (1) was incubated with Whetzelinia sclerotiorum ATCC 18687 to produce 2alpha,7beta,13-trihydroxystemodane (6) and 2alpha,11beta,13-trihydroxystemodane (4). Stemodinone (7) was converted to 7beta,13-dihydroxystemodan-2-one (10). Compounds 2, 4, 9, 10, 12 and 13 have not been previously reported.     

Biotransformation of terpenes from Stemodia maritima by Aspergillus niger ATCC 9142.

Incubation of stemodin (1) in cultures of Aspergillus niger ATCC 9142 resulted in the production of 2alpha,3beta,13-trihydroxystemodane (2), 2alpha,7beta,13-trihydroxystemodane (3) and 2alpha,13,16beta-trihydroxystemodane (4), while stemodinone (5) afforded 13,18-dihydroxystemodan-2-one (6) and 13,16beta-dihydroxystemodan-2-one (7). Four novel metabolites were obtained from the bioconversion of stemarin (8) by the fungus, namely 18-hydroxystemaran-19-oic acid (9), 7beta,18-dihydroxystemaran-19-oic acid (10), 7alpha,18,19-trihydroxystemarane (11) and 1beta-hydroxystemaran-19-oic acid (12). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (13) was also transformed to afford 19-N,N-dimethylcarbamoxy-13,17xi,18-trihydroxystemarane (14).     

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.     

Stemodane skeletal rearrangement: chemistry and microbial transformation

Solvolytic rearrangement of the C/D ring system of the tetracyclic diterpenoid stemodinone (2) afforded the compounds 15(13-->12)abeo-13beta-hydroxystemaran-2-one (5) and 15(8-->9)abeo-8beta(H)-12beta-hydroxystachan-2-one (10). Terpene 5 possesses a novel diterpene skeleton. Oxidation of these compounds yielded their respective diketones. Bioconversion of 5 by Rhizopus oryzae yielded 15(13-->12)abeo-7beta,13beta-dihydroxystemaran-2-one (18) while microbial transformation of 10 provided 15(8-->9)abeo-8beta(H)-6alpha,12beta-dihydroxystachan-2-one (19), 15(8-->9)abeo-8beta(H)-7beta,12beta-dihydroxystachan-2-one (20) and 15(8-->9)abeo-8beta(H)-6alpha,12beta,14beta-trihydroxystachan-2-one (21). A rationale for the formation of the rearranged compounds is proposed.     

Biotransformation of diterpenes and diterpene derivatives by Beauveria bassiana ATCC 7159

The biohydroxylation of stemodin and stemodinone by Beauveria bassiana ATCC 7159 gave exclusively 2alpha,13,18-trihydroxystemodane and 13,18-dihydroxystemodan-2-one respectively. Stemarin was converted to the novel 1beta,13,19-trihydroxystemarane and 13-hydroxystemarane-19-carboxylic acid. The synthesis and biotransformation of various derivatives of stemodin have also been studied.     

18-nor-Podocarpanes and podocarpanes from the Bark of Taiwania cryptomerioides

Seven nor- and podocarpane-type diterpenes were isolated from the bark of Taiwania cryptomerioides Hayata, including three 18-nor-podocarpanes: 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatriene (1), 18-nor-1beta,4alpha,13,14-tetrahydroxy-8,11,13-podocarpatrien-7-one (2), 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (3), 1beta,14,19-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (4), 1beta,13,14,18-tetrahydroxy-8,11,13-podocarpatrien-7-one (5), 18-acetoxy-1beta,13,14-trihydroxy-8,11,13-podocarpatrien-7-one (6), and 1beta,14,18-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (7). Their structures were determined by application of 1D and 2D NMR spectroscopy and other techniques. Podocarpane-type diterpenes do not occur extensively in nature, and the presumed oxidative enzyme in this plant will be of interest to identify.     

Diterpenes and sesquiterpenes from the bark of Taxus yunnanensis

Two taxane-type diterpenes, 10beta-acetoxy-2alpha,5alpha,7beta,9alpha-tetrahydroxytaxa-4(20),11-dien-13-one and 2alpha-acetoxy-9alpha-benzoyloxy-5alpha,7beta,10beta,15-tetrahydroxy-11(15-->1)- abeotaxa-4(20),11-dien-13-one, and two new drimane-type sesquiterpenes, 1beta-acetoxy-7-drimen-11alpha-ol-12,11-lactone and 1beta-acetoxy-11,12-epoxy-6-drimen-8alpha,11alpha-diol, were isolated from the bark of Taxus yunnanensis together with 35 known taxane-type diterpenes, a known drimane-type sesquiterpene and a known flavanone.     

Blazeispirane and protoblazeispirane derivatives from the cultured mycelia of the fungus Agaricus blazei

Two blazeispirane derivatives including blazeispirols G and I were isolated from the cultured mycelia of the fungus Agaricus blazei Murill and were established to be (20S, 22S, 23R, 24S)-14 beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-triene-11 alpha,23-diol and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-23,28-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A. Furthermore, four blazeispirol derivatives blazeispirols, U, V, V(1) and Z(1) were isolated form the same source described above. Their structures were determined to be (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxyergosta-4,6,8,11-tetraen-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 alpha,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one, (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-6 beta,7 alpha,23-trihydroxyergosta-4,8,11-trien-3-one and (20S, 22S, 23R, 24S)-14 beta,22:22,25-diepoxy-23-hydroxy-4,5-seco-ergosta-6,8-diene-3,5-dione by extensive 1 D and 2D NMR spectral data.     

Taxanes with C-5-amino-side chains from the needles of Taxus canadensis

Five taxanes with an amino-side chain on C-5 were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 2alpha,7beta,9alpha,10beta,13-pentaacetoxy-11beta-hydroxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),12-diene (1), 2alpha,9alpha-dihydroxy-10beta,13alpha-diacetoxy-5alpha-(3'-methylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (2), 2alpha17-dihydroxy-9alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (3), 2alpha-hydroxy-7beta,9alpha,10beta,13alpha-tetraacetoxy-5alpha-(2'-hydroxy-3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (4), and 9alpha-hydroxy-2alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (5) on the basis of 1D-, 2D-NMR spectroscopic data and high-resolution fast atom bombardment MS analyses. Metabolite (1) was isolated from the needles of the Canadian yew for the first time but had previously been detected in the stems of the Japanese yew, whereas taxanes (2-5) are only now reported. Metabolite (3) is the first reported nitrogen-containing taxane with a 17-hydroxyl substitution.     

Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Biotransformation of ent-3beta,12alpha-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7beta-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-beta face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3beta-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.     

Microbiological transformation of two labdane diterpenes, the main constituents of Madia species, by two fungi

Microbial transformation of 13R,14R,15-trihydroxylabd-7-ene (5) and 13R,14R,15-trihydroxylabd-8(17)-ene (6) by the fungus Debaryomyces hansenii gave 1 (13R,14R,15-trihydroxy-6-oxolabd-8-ene) and 3 (7alpha,13R,14R,15-tetrahydroxy-labd-8(17)-ene), respectively. While, microbial transformation of 5 by Aspergillus niger afforded 2 (3beta,13R,14R,15-tetrahydroxy-labd-7-ene), and 13R,14R,15-trihydroxylabd-8,17-ene (6) gave 3 and 4 (3R,14R,15-3-oxotetrahydroxy-labd-7-ene). The structures of the new compounds, 1 and 2, were assigned by 1D and 2D high-field NMR spectroscopic methods. Antimicrobial activity of these compounds were tested and their MIC were determined.     

Abietane diterpenes from the cones of Cedrus atlantica

Five new abietanes, three of them isolated as the corresponding acetate derivatives, i.e., 9alpha,13alpha-epidioxiabiet-8(14)-en-18-ol, 7alpha,18-diacetoxy, 9beta,13beta-epidioxiabiet-8(14)-ene, 7alpha,18-diacetoxyabiet-8(14)-en-13beta-ol, 7alpha,18-diacetoxy-13beta-methoxyabiet-8(14)-ene, and 13beta-hydroxyabiet-8(14)-en-7-one, were isolated from the neutral part of the hexane extract of the cones of Cedrus atlantica collected in Middle Atlas, Morocco. The structures of these compounds were established by spectroscopic techniques, including 2D NMR spectra, and in the case of 1, by chemical correlation. The cytotoxicity of these abietane diterpenoids was tested against five cell lines.     

Synthesis and bioactivity of C-29 brassinosteroid analogues with different functional groups at C-6

In this paper, we report the synthesis and bioactivity of four synthetic analogues of 28-homobrassinosteroids, in order to evaluate the influence in bioactivity when the C-6 keto group is replaced by different functional groups. The synthetic analogues are 6-deoxo-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,22,23-tetraol], 6alpha-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6alpha,22,23-pentaol], 6beta-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6beta,22,23-pentaol], and [(22R,23R)-6alpha-fluorostigmasta-2alpha,3alpha,22,23-tetraol]. Results indicate that replacement of the 6-keto moiety by an beta or alpha hydroxyl group led to a decrease in activity, whereas the 6-deoxo analogue showed a very low activity, confirming the importance of an electronegative moiety at C-6 to observe hormonal potency. The 6alpha-fluorinated analogue elicited a low activity, similar to that of the 6-deoxo analogue.     

Six new ergosterols from the marine-derived fungus Rhizopus sp

Six new ergosterols, including 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,22-trien-7,15-dione (1), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8,14,22-tetraen-7-one (2), 3beta,15beta-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (3), 3beta,15alpha-dihydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7-one (4), 3beta-hydroxyl-(22E, 24R)-ergosta-5,8(14),22-trien-7,15-dione (5), and 5alpha,8alpha-epidioxy-23,24(R)-dimethylcholesta-6,9(11),22-trien-3beta-ol (6), have been isolated from the marine-derived fungus Rhizopus sp., along with four known ones (7-10). The structures of the new compounds were determined on the basis of extensive spectroscopic data. All compounds were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.     

Labdane diterpenes from Leonurus japonicus leaves

Three labdane diterpenes 15,16-epoxy-6-hydroxylabda-5,8,13(16),14-tretraen-7-one (leojaponin), (9alpha,13S);15,16-diepoxy-7beta-hydroxylabd-14-en-6-one (13-epi-preleoheterin), and (9alpha,13R);15,16-diepoxy-6beta-hydroxylabd-14-en-7-one (iso-preleoheterin) were isolated from the leaves of Leonurus japonicus, in addition to the previously reported preleoheterin. The structure elucidations were made based on analysis of their spectroscopic data.     

Triterpenoids from Gentiana scabra

Five triterpenoids, (20S)-dammara-13(17),24-dien-3-one, (20R)-dammara-13(17),24-dien-3-one, chirat-16-en-3-one, chirat-17(22)-en-3-one and 17beta,21beta-epoxyhopan-3-one, were isolated from the rhizomes and roots of Gentiana scabra together with five known ones, chiratenol, hop-17(21)-en-3-one, hop-17(21)-en-3beta-ol, lupeol and alpha-amyrin. The structures of new compounds were elucidated on the basis of spectroscopic studies.     

Synthesis of (25R)-26-hydroxy-15-ketosterols

(25R)-3beta,26-Dihydroxy-5alpha-cholest-8(14)-en-15-one (1) and (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2) were synthesized from (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-ene (4). Oxidation of 4 with CrO3-3,5-dimethylpyrazole at -20 degrees C gave (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-en-15-one (5) along with (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-16alpha+ ++,17alpha-epoxide (6). Oxidation of 5 with selenium dioxide afforded (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-8(14),16-++ +dien-15-one (7) and (25R)-3beta,26-dibenzoyloxy-5alpha,14beta-choles t-16-en-15-one (8). Selective hydrogenation of 7 followed by hydrolysis in alcoholic potassium hydroxide yielded (25R)-3beta,26-dihydroxy-5alpha-cholest-8(14)-en-15-one (1). Hydrolysis of 5 and 8 in alcoholic potassium hydroxide provided (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2).     

Diterpenoids from Isodon enanderianus

Four ent-kauranoids, 6-epiangustifolin and enanderinanins F-H, as well as 11 known ent-kaurane diterpenoids, macrocalin B, xerophilusin A, trichorabdal A, trichorabdal B, effusin, angustifolin, longikaurin D, longikaurin F, enanderinanin B, xerophilusin G and shikokianin were isolated from the aerial parts of Isodon enanderianus. The new diterpenoids were identified as 6-epiangustifolin (11alpha-hydroxy-6alpha-methoxy-6,19-epoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide), enanderinanin F (19-acetoxy-6,20:6,11beta-diepoxy-6,7-seco-ent-kaur-16-en-15-one-1beta,7-olide), enanderinanin G (1beta,6beta,7beta-trihydroxy-19-acetoxy-16beta-methoxymethyl-7alpha,20-epoxy-ent-kaur-15-one) and enanderinanin H (6beta,7beta,14beta-trihydroxy-1alpha,11beta-acetonide-7alpha,20-epoxy-ent-kaur-16-en-15-one), respectively, on the basis of spectral data, especially by 2D NMR techniques. 6-Epiangustifolin showed significant cytotoxic activity against K562 cell.     

Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740

The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpenoid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1, 14 and ursolic acid (20) were isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2alpha,13-dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl 3beta,7beta,21beta-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3beta,28-dihydroxyurs-12-ene (uvaol) (23) and 3beta,28-bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the micro-organism, however.