Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra

The complex sterol mixture isolated from $\text{A, nigra}$ was found to contain a low level of Δ⁴-3-keto steroids, 5β-stanols and 4α-methyl sterols in addition to regular (4-demethyl) sterols. The following new marine sterols were isolated and identified using MS and 360 MHz NMR: 5β-cholest-22E-en-3β-ol, 24S-methyl-5β-cholest-22E-en-3β-ol, 24-methylene-5β-cholestan-3β-ol, both epimers at C-24 of 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol, 4α, 22ξ, 23ξ-(or 24ξ-)trimethyl-5α-cholest-8(14)-en-3β-ol and (22S, 23S, 24S)-4α-24-dimethyl-22, 23-methylene-5α-cholestan-3β-ol. The latter sterol and 23-demethylgorqosterol have opposite configurations at C-22, C-23, and C-24; the Δ⁸⁽¹⁴⁾ sterol has an unprecedented side chain.     

Brassinosteroids and sterols from a green alga,Hydrodictyon reticulatum: Configuration at C-24

Two brassinosteroids, (24S)-24-ethylbrassinone [(22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-ethyl-5α-cholestan-6-one] and 24-epicastasterone [(22R,23R,24R)-2α,3α,22,23-tetrahydroxy-24-methyl-5α-cholestan-6-one] have been identified from Hydrodictyon reticulatum. Examination of the sterols of this alga has established that 24-ethylcholesterol is predominantly the 24α-epimer, but 24-methylcholesterol is a mixture of the 24α- and 24β-epimers. Thus, similarity with respect to the C-24 configuration was observed between the brassinosteroids and 4-demethylsterols.     

Three new 5,10-epoxygermacranolides from Liatris chapmanii and Liatris gracilis

Examination of Liatris chapmanii (T + G) Kuntze led to the isolation of two new germacranolides, chapliatrin (1a) and isochapliatrin (1b). Liatris gracilis Pursh gave chapliatrin and acetylchapliatrin 1c). The stereochemistry assigned to C-3, C-4 and C-10 is tentative. All three compounds possess the hitherto-unreported 5,10-oxygen linkage. L. gracilis also gave the benzofuran euparin (2) and the flavones hispidulin (dinatin, 3a and 3′,6-dimethoxy-4′,5,7-trihydroxyflavone (3b). L. chapimanii also gave 5-hydroxy-3′,4′,6,7-tetramethoxyflavone (3c).     

Triterpenes from Douglas fir sapwood

The saponified ether-soluble extractives of Douglas fir sapwood contained (24R)- 4α,14α,24-trimethyl-9β,19-cyclo-5α-cholestan-3β-ol(24R-cyclocucalanol),a new natural product; 4α,14α-dimethyl-9β,19-cyclo-24-methylene-5α-cholestan-3β- ol (cycloeucalenol); and (24R)-4α,24-dimethyl-5α-cholest-7-en-3β-ol (24R- methyllophenol); this is the first time they have been reported from Douglas fir.     

Metabolism of 4-cholesten-3-one to 5α-cholestan-3-one by leaf homogenates

Young leaf homogenates of Digitalis purpurea, Cheiranthus cheiri and Strophanthus kombe' were incubated at 30° for 2 hr with 4-cholesten-3-one-[4-¹⁴C] in a buffered medium containing an NADPH generating system. A single identical metabolite was observed with each tissue. The metabolite was identified by co-chromatography and co-crystallization to constant specific activity as 5α-cholestan-3-one. Leaf homogenates of D. purpurea exhibited the greatest metabolic activity. Addition of non-radioactive progesterone effectively competed with the radioactive substrate, markedly reducing the yield of 5α-cholestan-3-one.     

A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoromethyl)dioxirane

Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5α-cholestan-3β-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO₃, pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.     

Reaction of acylated methyl arabinosides with hydrogen bromide

Treatment of methyl tri-O-acetyl-β-D-arabinopyranoside (1a) with hydrogen bromide in benzene or in acetic acid gave, in addition to the pyranosyl bromide (2a), a considerable proportion of tri-O-acetyl-D-arabinofuranosyl bromide (5). Similar treatment of methyl tri-O-benzoyl-β-D-arabinopyranoside (1b) gave a good yield of the pyranosyl bromide (2b); no furanoid derivative was formed. Ring contraction also took place when methyl 4-O-acetyl-2,3-di-O-benzoyl-β-D-arabinopyranoside (7) was treated with hydrogen bromide, whereas the isomeric 3-O-acetyl-2,4-di-O-benzoyl compound (12) gave the pyranosyl bromide 13 in high yield. Thus, methyl pyranosides with an O-acetyl group at C-4 undergo ring contraction on treatment with hydrogen bromide. The corresponding compounds with O-benzoyl groups at C-4 gave pyranosyl bromides only.     

Synthesis and reactions of 1′,2:4,6-di-o-isopropylidene-sucrose

The reaction of sucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A) gave, after acetylation followed by chromatography, 1′,2:4,6-di-O-isopropylidenesucrose tetra-acetate (1) in 15% yield. The structure of 1 was determined on the basis of p.m.r. and mass spectrometry, and by chemical transformations. Treatment of 1 with aqueous acetic acid afforded sucrose 3,3′,4′,6′-tetra-acetate 2. Reacetalation of 2 using reagent A gave 1 in 80% yield. The p.m.r. spectrum of 2 confirmed the presence of hydroxyl groups at C-2 and C-4. The following sequence of reactions showed that the remaining two hydroxyl groups were located at C-6 and C-1′. Selective tritylation of 2 gave 1′,6-di-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (3) as the minor, and 6-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (4) as the major, product. When tritylation was carried out under forcing conditions, 2 gave 3 as the major product. Acetylation of 4 afforded 6-O-tritylsucrose hepta-acetate. Mesylation of 2 gave the tetramethanesulphonate 5, which afforded the 6-dcoxy-6-iodo derivative 6 on treatment with a refluxing solution of sodium iodide in butanone. Treatment of 3 with methanesulphonyl chloride in pyridine gave the disulphonate 7, which on detritylation followed by acetylation gave 2,4-di-O-methanesulphonylsucrose hexa-acetate (9). Treatment of 9 with sodium benzoate in hexamethylphosphoric triamide displaced the 4-sulphonate, with inversion of configuration, to give the galacto derivative 10.     

Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates

The (25R)- and (25S)-epimers of C₂₇ 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C₂₄allo-cholic acid performate with NaBH₄/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I₂/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C₁₈ column and their structural characteristics, particularly the chiral center at C-25, delineated using ¹H and ¹³C NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of allo-bile acids from cholesterol.     

Synthesis of 5-beta-D-ribofuranosylcytosine (pseudo-cytidine) and its alpha isomer

The dilithio derivative of 2,4-di-O,N-trimethylsilylcytosine was condensed with 2,4:3,5-di-O-benzylidene-D-ribose to give a mixture of the protected, epimer at C-1′ pentitols 5 and 6; in addition, a compound substituted at N-3 or N-4, whose structure was not elucidated, was also obtained. The epimers were treated with acid to give 4-amino-2-hydroxy-5-(β-and α-D-ribofuranosyl)pyrimidine (10 and 12). The n.m.r. spectrum of 10 corresponds predominantly to the C-2′ endo structure. On the other hand, the n.m.r. spectrum of 12 presents couplings identical with those of the “α pseudo-uridine”. On nitric deamination, each isomer gave in a highly preponderant yield the corresponding pseudo-uridine at C-1′.     

Dérivés d'acides 3-(β-d-érythrofuranosyl)-pyrazolecarboxyliques

Treatment of 2,5-anhydro-1-bromo-1-deoxy-2,3-O-isopropylidene-1-p-nitrophenylhydrazono-d-ribose with methyl acetylenecarboxylate gave methyl 3-(2,3-O-isopropylidene-β-d-erythrofuranosyl)-1-p-nitrophenylpyrazole- (8) and 5-carboxylate (9). Amidification at C-5 of 8 was easier than at C-4 of 9. Similarly, dimethyl 3-(2,3-O-isopropylidene-β-d-erythrofuranosyl)- 1 - p-nitrophenylpyrazole-4,5-dicarboxylate gave specifically a 5-carbamoyl derivative, the structure of which was established by comparison of the ¹³C-n.m.r.spectrum with those of a series of glycosylpyrazoles. The correlation between the experimental values of the chemical shifts of the carbon atoms of the pyrazole ring and the values calculated by addition of the contributions of the various groups linked to the ring was better (R 0.98) than the correlations obtained by calculation by the CNDO/2 method of the total electron population (R 0.92) or of the π-electron population of each carbon atom (R 0.85).     

A-nor steroids. 1. The stereochemistry of A 2-carbomethoxy-A-nor-cholestan-3-one obtained via the Dieckmann condensation

The Dieckmann condensation of dimethyl 3, 4-$\text{seco}$-5α-cholestan-3, 4-dioate ($\text{1}$), using sodium methoxide in benzene under reflux for two hours, is shown to give 2α-carbomethoxy-A-nor-5α-cholestan-3-one ($\text{2}$). Confirmation of the stereochemistry of the β-keto ester $\text{2}$ was obtained through its sodium borohydride reduction product, 2α-carbomethoxy-A-nor-5α-cholestan-3β-ol ($\text{4}$).     

Monoclonal antibody recognition of abscisic Acid analogs

Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1′ and C-2′. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1′, but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2′ methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross-reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1′ as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies.     

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihdroxyeicosatetraenoic acids generated by ω /ω − 1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B₄ in double-bond geometry alone. The other two differed from leukotriene B₄ in olefin geometry and C-5 configuration. Epoxidation at the 14,15-olefin resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydroxylation gave two diastereomeric dihydroxyeicosatetraenoic acids.     

Microbial transformations of flavanone by Aspergillus niger and Penicillium chermesinum cultures

As a result of biotransformation of flavanone (1) by the strain Aspergillus niger MB (being the UV mutant) and by the wild strain Penicillium chermesinum 113 the products of hydroxylation at C-6 (2) and C-4′ (5) were obtained. Additionally, three dihydrochalcones with hydroxyl groups at C-2′ (4), C-2′ and C-5′ (3) and C-2′ and C-4 (6) were formed.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

Synthesis of branched-chain,pyrrolidino-sugar derivatives related to apiose

3-C-(Acetamidomethyl)-1,2-O-isopropylidene-β-l-threofuranose (4) and the 3-acetate (5) have been prepared in high yields from mono-O-isopropylidene-d-apiose [3-C-(hydroxymethyl)-1,2-O-isopropylidene-β-l-threofuranose] (1). Acid-catalyzed methanolysis of 4 caused migration of the isopropylidene group and the formation of methyl 4-acetamido-4-deoxy-3-C-(hydroxymethyl)-2,3-O-isopropylidene-β-d-erythrofuranoside (8) in 25% yield. The major product (45%) from the acetolysis of 4 was also a pyrrolidine derivative, namely, 4-acetamido-3-C-(acetoxymethyl)-1-O-acetyl-4-deoxy-2,3-O-isopropylidene-β-d-erythrofuranose (10). Acetolysis of 5 removed the isopropylidene group and gave four acetylated pyrrolidines (isomeric at C-1 and C-2). Conditions which resulted in minimal epimerization at C-2 were established, and the major isomers 12 and 13 were isolated in reasonable yields. ¹H- and ¹³C-n.m.r. data for equilibrium solutions of the pyrrolidines, and for intermediates 1-5, are given.     

Lytic Enzymes toward Aniline-assimilating Rhodococcus erythro-polis AN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN-13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH₄)₂S0₄ fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and 11.0. The molecular weights of C-4 and C-5 were 26,000 and that of C-2 was 36,000, as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-13 than C-4 and C-5.     

Chemical syntheses of 5α-cholesta-6,8(14)-dien-3β-ol-15-one and related 15-oxygenated sterols

Hydroboration of 5α-cholesta-8,14-dien-3β-ol (I) gave 5α-cholest-8-en-3β,15α-diol (IV) in 89% yield. 5α-Cholest-7-en-3β,15α-diol (V) was prepared in 91% yield by hydroboration of 5α-cholesta-7,14-dien-3β-ol (II). Hydroboration of 27:63 mixture of I and II gave IV and V in 18% and 70% yields, respectively. 5α-Cholest-8-en-15α-ol-3-one and 5α-cholest-7-en-15α-ol-3-one were prepared in high yields from IV and V, respectively, by either selective oxidation with silver carbonate-celite or by enzymatic oxidation using cholesterol oxidase. 7α,8α-Epoxy-5α-cholestan-3β,15α-diol (VIII) was prepared in 93% yield by treatment of V with m-chloroperbenzoic acid. 5α-Cholest-8(14)-en-7α-ol-3,15-dione (IX) was prepared in 56% yield by oxidation of VIII with pyridinium chlorochromate followed by treatment of the crude product with acid. Compound IX was also obtained in 72% yield by selective chemical oxidation of 5α-cholest-8(14)-en-3β,7α,15α-triol. 5α-Cholesta-6,8(14)-dien-3,15-dione (X) was prepared in 89% yield by treatment of IX with p-toluenesulfonic acid under controlled conditions. Reduction of X with lithium tri-tert-butoxyaluminum hydride under controlled conditions gave 5α-cholesta-6,8(14)-dien-3β-ol-15-one in 84% yield.     

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method—precursors for the synthesis of labeled conjugates

Isotopic labeling of the C-6 of a model glycosphingolipid (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-β-d-galactopyranoside, GalCAda, is described. Oxidation of (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri-O-benzoyl-β-d-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled d-GalCAda, with a cumulative yield of 35%.