D-homoandrostanes. 4. The incubation of some D-homo-5 alpha-androstanes with Rhizopus nigricans

Nine dioxygenated D-homoandrostanes were incubated with Rhizopus nigricans to investigate the effect of D-ring modification on microbiological hydroxylation. Structure determination of the products by NMR spectroscopy, and in certain cases independent synthesis of their oxidised products, showed that in contrast to 5 alpha-androstanes the majority of the compounds were hydroxylated in the "reverse" mode, and only D-homo-5 alpha-androstane-3,17-dione was hydroxylated in the "normal" mode to any extent. Stereospecific ring D-hydroxylation at C(17 alpha) was observed for both D-homo-5 alpha-androstane-3,6- and 3,7-diones.     

Reaction of (13S)-13-iodo-6beta-methoxy-3alpha,5-cyclo-13,14-seco-5alpha-androstane-14,17-dione with hydroxylamine and its application to the synthesis of new 13,14-seco steroids

The synthesis of 13,14-seco steroids starting from easily available (13S)-13-iodo-6beta-methoxy-3alpha,5-cyclo-13,14-seco-5alpha-androsta-14,17-dione is described. The C-17 ketone was converted regioselectively into its oxime with simultaneous stereoselective deiodination at C-13. The remaining C-14 carbonyl group was then reduced stereoselectively with Ca(BH4)2. The configurations at the relevant stereocenters of the thus obtained hydroxy oxime were determined by X-ray analysis. Successful regeneration of the C-17 carbonyl group was achieved by treatment of the corresponding oxime acetate with TiCl3.     

Synthesis of 6-oxy functionalized campest-4-en-3-ones: efficient hydroperoxidation at C-6 of campest-5-en-3-one with molecular oxygen and silica gel

As a reference compound library for the investigation of biosynthesis of brassinosteroids, focused on a pathway from campesterol (1) to campestanol (2), 6-oxy functionalized campest-4-en-3-ones as well as campest-5-en-3-one (7) and campestane-3,6-dione were prepared from 1. Oxidation of 1 with pyridinium chlorochromate buffered by calcium carbonate gave 5-en-3-one (7) in 76% yield. Treatment of 7 with silica gel under an oxygen atmosphere in ethyl ether at room temperature produced efficient hydroperoxidation at the C-6 position to give 6alpha-hydroperoxycampest-4-en-3-one and 6beta-hydroperoxycampest-4-en-3-one in 34% and 49% yields, respectively. These compounds were converted to 6alpha-hydroxycampest-4-en-3-one and 6beta-hydroxycampest-4-en-3-one by reduction with triethyl phosphite. This provided the first example of the practical use of hydroperoxidation at C-6 of a Delta(5(6))-unsaturated 3-oxo-steroid with molecular oxygen and silica gel. On the other hand, oxidation of 1 with pyridinium chlorochromate in the absence of calcium carbonate gave campest-4-ene-3,6-dione in 64% yield. This compound was then converted in a highly stereoselective manner to campestane-3,6-dione with A/B trans ring junction by reduction with titanium (III) chloride in 85% yield.     

The absolute configuration of prunioside A from Spiraea prunifolia and biological activities of related compounds

The stereochemistry of prunioside A isolated from Spiraea prunifolia was determined by chemical transformations and NMR spectral data analysis. The configurations at C-5 and C-6 were determined to be 5S and 6R by application of the modified Mosher's method, CD analysis, and 13C NMR spectroscopic data analysis of an acetonide derivative. Other compounds related to prunioside A have inhibitory effects on the synthesis of nitric oxide in LPS-stimulated macrophage-like RAW 264.7 cells.     

Short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione a novel cytotoxic marine oxysterol

The first and short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione 1 a metabolite from marine algae, has been achieved in six steps from readily available diosgenin 5. Selective deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et(3)SiH and catalytic amount of B(C(6)F(5))(3) to produce compound 9 in high yield. Oxidation of 11 with PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydrogenation and deprotection furnished the 3,6-diketo steroid 1.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Synthesis of C-5-thioglycopyranosides and their sulfonium derivatives from 1-C-(2'-oxoalkyl)-5-S-acetylglycofuranosides

1-C-(2'-oxoalkyl)-5-S-acetylglycofuranosides of L-arabinose, D-ribose, and D-xylose were converted to 1-C-(2'-oxoalkyl)-5-thioglycopyranosides by base treatment. The transformation was achieved through beta-elimination to an acyclic alpha,beta-conjugated aldehyde (ketone or ester), followed by an intramolecular hetero-Michael addition by the 5-thiol group. The cycloaddition was highly stereoselective in favor of an equatorial 1-C-substitution. The resultant C-5-thioglycopyranosides were further converted to the sulfonium salts by treatment with cyclic sulfate and methyl iodide. Two sulfonium isomers were obtained due to the presence of both S-axial and S-equatorial substitutions. We observed that the chemical shifts of both C-1 and C-5 in the S-axial substituted sulfonium sugars are always shifted up-field (5-10 ppm) in comparison to those in the S-equatorial substitutions (deltaC 49-53 ppm vs 42-45 ppm at C-1 and 37-42 ppm vs 32-35 ppm at C-5), which provides an easy way for determination of the stereochemistry.     

Crystal structures of 10alpha-gon-5-enes from the synthetic pathway to desogestrel

X-ray crystallographic studies performed on the product of the ketalization reaction of 13beta-ethyl-11alpha-hydroxy-gon-5-ene-3,17-dione have lead to the unequivocal assignment of the 10alpha stereochemistry to C10, showing that an inversion of configuration occurred during formation of the 3,17-diketal. From the Swern oxidation of this compound, 11alpha-(methylthio)methoxy-10alpha-gonene was obtained as the major product instead of the desired 11-ketone. Modeling studies showed that the configurational instability at C10 is determined by the presence of the 11alpha-hydroxyl group.     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

Synthesis of 21-amino-5-pregnene-3,20-dione bysethylene ketal

21-Amino-5-pregnene-3,20-dione bisethylene ketal was obtained in good yield by lithium aluminum hydride reduction of 21-azido-5-pregnene-3,20-dione bisethylene ketal. The azido bisethylene ketal was synthesized by the sequence: deoxycorticosterone → deoxycorticosterone 21-p-toluene-sulfonate → 21-azidoprogesterone → 21-azido-5-pregnene-3, 20-dione bisethylene ketal. The structure of the title compound was confirmed by its conversion to the known 21-acetylaminoprogesterone. 21-Amino-5-pregnene-3,20-dione bisethylene ketal is a stable aminosteroid which is a useful intermediate for the synthesis of C-21 nitrogen derivatives of progesterone.     

Antimalarials based on the dioxane scaffold of plakortin. A concise synthesis and SAR studies

In our search for new antimalarial agents inspired by natural products, we describe herein the synthesis, the evaluation of in vitro antiplasmodial activity, and the SAR studies for a series of endoperoxide antimalarials based on the plakortin scaffold. These simplified analogues are characterized by: (i) a 3,6-dihydro-1,2-dioxin ring or a 1,2-dioxane ring disubstituted at C-4 and C-5; (ii) a pentyl substituent at C-6 (‘western’ alkyl side chain) and they have been prepared from commercially available material using simple reactions.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and -2. A critical role for serine 530 and valine 349.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.     

Conversion of 15α-hydroxy-11-oxo-progesterone to 3β,11α,15β-trihydroxy-5-pregnen-20-one

3β,11α,15β-Trihydroxy-5-pregnen-20-one, an intermediate required for the synthesis of the oogoniols, has been prepared from 15α-hydroxy-11-oxo-progesterone in an overall yield of 16%. The three isomers (at C-11, C-15) of the pregnene were also prepared.     

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.     

The 5-O-beta-D-galactofuranosyl-containing peptidophosphogalactomannan of Penicillium charlesii. Characterization of the mannan by 13C NMR spectroscopy

Penicillium charlesii secretes a galactofuranosyl and phosphodiester-containing peptidophosphogalactomannan (pPGM). A linear mannan was prepared from pPGM by treatment with 48% aqueous HF which selectively cleaves galactofuranosyl and phosphodiesters; treatment with alkaline borohydride releases the mannan from the polypeptide. Mannan from P. charlesii cultured in D-[1,2-13C2]glucose contained mannopyranosyl residues which were enriched in 13C at both C-1 and C-2 and, to a lesser extent, at C-5 and C-6. The mannan was examined with a combination of 13C NMR INADEQUATE pulse sequence and selective 13C saturation to assign the resonance frequency of anomeric carbons directly coupled to specific C-2 signals. Three species of mannosyl residues, each substituted with a glycosidic linkage at C-2, and a fourth species substituted at C-6 and not substituted at C-2 were observed. Mannan obtained from P. charlesii cultured in D-[6-13C]glucose contained mannopyranosyl residues which were enriched in 13C primarily in C-6. The mannan was examined by DEPT 13C NMR to determine the number of species which were substituted at C-6. Mannan, treated as described above, contained a 1----6-linked mannopyranosyl species. pPGM contains minor quantities of at least four other substances attached to hydroxymethyl groups of the hexosyl residues.     

Ring-B functionalized androst-4-en-3-ones and ring-C substituted pregn-4-en-3-ones undergo differential transformation in Aspergillus tamarii KITA: ring-A transformation with all C-6 substituted steroids and ring-D transformation with C-11 substituents

The fungus Aspergillus tamarii transforms progesterone 1 into testololactone 5 in high yield through a four-step enzymatic pathway which is flexible to a range of steroidal substrates. To date, no studies have investigated the fate of C-6 (ring-B) and C-11 (ring-C) functionalized steroidal substrates on metabolism. Remarkably all of the C-6 functionalized substrates underwent reductive metabolism on ring-A in contrast to C-11 functionalized steroids where only ring-D oxidative or reductive transformation occurred. In order to discern the precise role of the functional groups in directing metabolism 6-ketoprogesterone 10 with functionality at C-6 and the ring-D methyl ketone underwent reductive and oxidative transformation on both terminal A and D rings showing that this functionality was directing metabolism. Androst-4-en-3,6-dione 12 devoid of ring-D functionality underwent reductive metabolism on ring-A proving that the C-6 functionality was directing metabolism to this ring with the ring-D methyl ketone responsible for generating transformation at this position. Functionality at C-11 exclusively controlled entry into and degree of metabolism on the lactonization pathway. These novel findings may have important bearing in the future understanding of structure activity relationships in revealing new metabolic pathways and further affords a unique opportunity for generation of novel bioactive steroidal compounds.     

Metabolism of androst-4-en-3,17-dione by the filamentous fungus Neurospora crassa

Microbial transformation of androst-4-en-3,17-dione (AD; I) using Neurospora crassa afforded six metabolites; 6beta,14alpha-dihydroxyandrost-4-en-3,17-dione (II), 6beta,9alpha-dihydroxyandrost-4-en-3,17-dione (III), 7alpha-hydroxyandrost-4-en-3,17-dione (IV), 9alpha-hydroxyandrost-4-en-3,17-dione (V), 14alpha-hydroxyandrost-4-en-3,17-dione (VI), and androst-4,6-dien-3,17-dione (VII). The steroid products were assigned by interpretation of their spectral data such as (1)H NMR, (13)C NMR, FTIR, and mass spectroscopy. The characteristic transformations observed were C-6beta, C-7alpha, C-9alpha, C-14alpha hydroxylations, and C6-C7 dehydrogenation. The best fermentation condition was found to be 6-day incubation at 25 degrees C and pH value of 5.0-6.5 according to TLC profiles. Time course study showed the accumulation of V and VI from the third day and IV from the fourth day of the fermentation. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 3.5mM in one batch. Biotransformation was completely inhibited in a concentration above 7.0mM.     

Relevance of the C-5 position to schweinfurthin induced cytotoxicity

The schweinfurthins are an intriguing group of anti-proliferative agents that display low nanomolar activities against several cell types, including the human-derived glioblastoma cell line SF-295, but have little impact on other cell lines even at micromolar concentrations. This activity has inspired the synthesis of seven of the natural schweinfurthins, all with the correct absolute stereochemistry, and a variety of analogues designed to probe different facets of the pharmacophore. Reported herein is the synthesis of several new schweinfurthin analogues varied at the C-5 position along with data on their biological activity in the NCI 60 cell-line assay.     

Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.