Mass spectrometric study of the enzymatic conversion of cholesterol to (22R)-22-hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol, and pregnenolone, and of (22R)-22-hydroxycholesterol to the lgycol and pregnenolone in bovine adrenocortical preparations. Mode of oxygen incorporation.

Incubation of cholesterol with a bovine adrenocortical mitochondrial acetone-dried powder preparation yielded (22R)-22-hydroxycholesterol (I), (20R,22R)-20,22-dihydroxycholesterol(II), and pregnenolone (III) which were conclusively identified by combined gas chromatography-mass spectrometry. Incubations with [4-14C]cholesterol yielded I, II, and III with specific activities (determined from partial mass-spectral scans) not significantly different from those of the used substrate or the cholesterol reisolated after the incubation, demonstrating that the isolated compounds arose mostly, if not entirely, from the substrate cholesterol. Incubations in an 18O-enriched atmosphere yielded I, II, and III with 18O at position C-22, C-20 and C-22, and C-20, respectively, providing evidence that the hydroxyl groups of the side chain of I and II and the C-20 oxygen atom of III originated from molecular oxygen. The distribution of the oxygen atoms in II after incubation with 18O2 and 16O2 (devoid of 16O18O) proved that the hydroxyl groups of the side chain of II were introduced from two different molecules of oxygen, consistent with a sequential hydroxylation of cholesterol. No (20S)-20-hydroxycholesterol was found. Incubation of I in an 18O-enriched atmosphere afforded II and III with 18O at C-20.     

Cytotoxic derivatives of (22R,23R)-22,23-dihydroxystigmastane

(22R,23R)-22,23-dihydroxystigmast-4-en-3-one, (22R,23R)-22,23-dihydroxystigmast-4-en-3,6-dione, (22R,23R)-3beta,5alpha,6beta,22,23-pentahydroxystigmastane, (22R,23R)-5alpha,6alpha-oxido-3beta,22,23-trihydroxystigmastane, (22R,23R)-5beta,6beta-oxido-3beta,22,23-trihydroxystigmastane, and (22R,23R)-3beta,6beta,22,23-tetrahydroxystigmast-4-ene were synthesized. Their cytotoxicities were comparatively studied using the MCF-7 line of carcinoma cells of human mammary gland and cells of human hepatoma of the Hep G2 line.     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

Facile Synthesis of (22R, 23R)-Homobrassinolide

A synthesis of (22R,23R)-homobrassinolide is described. The LC and the chemical correlation studies for the oxidation product of a stigmasterol-like side chain with osmium tetroxide are mentioned. A stereochemical view for the mechanism of osmium tetroxide oxidation of the side chain is proposed.     

Cytotoxic derivatives of (22R,23R)-dihydroxystigmastane

(22R,23R)-22,23-dihydroxystigmast-4-en-3-one, (22R,23R)-22,23-dihydroxystigmast-4-en-3,6-dione, (22R,23R)-3β,5α,6β,22,23-pentahydroxystigmastane, (22R,23R)-5α,6α-oxido-3β,22,23-trihydroxystigmastane, (22R,23R)-5β,6β-oxido-3β,22,23-trihydroxystigmastane, and (22R,23R)-3β,6β,22,23-tetrahydroxystigmast-4-ene were synthesized. Their cytotoxicities were comparatively studied using the MCF-7 line of carcinoma cells of human mammary gland and cells of human hepatoma of the Hep G2 line.     

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.     

Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.     

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies.

Heating (20R)-3beta,20,26-trihydroxy-27-norcholest-5-en-22-one (1) with hydrazine and KOH at 160 degrees C completely converted the steroid to a diastereoisomeric mixture of the new (20R,22RS)-27-norcholest-5-ene-3beta,20,22-triols (2). Exclusive formation of 2 suggests that the expected Wolff-Kishner reduction to a methylene group at the C-22 ketone in 1 was diverted to the C-26 position by a 1,5-hydride shift. All attempts under acid conditions failed to produce a C-22 phenyl hydrazone from 1. However, reaction of 1 was reacted with phenylhydrazine in hot KOH, gave the C-26 phenylhydrazone 4 as the sole product. Evidently, under alkaline conditions, first a hydride ion undergoes an intramolecular transfer from the C-26 CH(2)OH group to the C-22 ketone in 1, and then the phenylhydrazine traps the newly formed aldehyde. To examine this hypothesis, we constructed computer-simulated transition state models from quantum chemical calculations and then compared data from these models with NMR measurements of the reaction mixtures containing 2. The NMR data showed that the C-22 diastereoisomers of 2 are formed in a nearly 1:1 ratio exactly as predicted from the energy-optimized transition states, which were calculated for intramolecular 1,5-hydride shifts that produced each of the two C-22 diastereoisomers. Accordingly, these results support the hypothesis that an intramolecular 1,5-hydride shift mechanism promotes complete conversion of 1 to 2 under classical Wolff-Kishner reduction conditions.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.     

Inhibition of adrenocortical cytochrome P-450scc by (20R)-20-phenyl-5-pregnene-3 beta,20-diol

The effects of the cholesterol analogue, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), on the catalytic and spectral properties of purified bovine adrenocortical cytochrome P-450scc were investigated. In contrast to results with cholesterol and (20R)-20-hydroxycholesterol, no conversion of 20-PPD to pregnenolone could be detected; instead, 20-PPD was found to be a potent inhibitor of cytochrome P-450scc. Kinetic analyses showed that the inhibition is reversible and competitive with respect to cholesterol with an apparent Ki = 30nM. Spectral binding studies with ferricytochrome P-450scc showed that 20-PPD formed a 1:1 complex with the enzyme, having an absorption spectrum similar to that produced by (20R)-20-hydroxycholesterol. These results indicate that 20-PPD binds with very high affinity to the substrate site on cytochrome P-450scc. The finding that the phenyl side chain is readily accommodated suggests the presence in this site of an open pocket which may be normally occupied by C-22 to C-27 of the cholesterol side chain.     

Competitive inhibition of cytochrome P-450scc by (22R)- and (22S)-22-aminocholesterol. Side-chain stereochemical requirements for C-22 amine coordination to the active-site heme

Two diastereomeric aminocholesterols, (22R)-22-aminocholesterol and (22S)-22-aminocholesterol, are both found to be potent inhibitors of the biosynthesis of pregnenolone from cholesterol by purified bovine mitochondrial P-450scc. Both steroids are competitive versus cholesterol, but the stereochemically correct analog (22R)-22-aminocholesterol is bound approximately 1000 times more tightly than (22S)-22-aminocholesterol. The dissociation constants are 25 nM and 13 microM, respectively. Direct comparisons between spectroscopic and enzymatic properties of the two enzymesterol complexes and the 22-amino-23,24-bisnor-5-cholen-3 beta-ol complex are made, underlining the importance of the stereochemistry at the C-22 position.     

Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R

We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2-4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2-4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.