22,23-Epoxides of Sitosterol and Related 7-Oxygenated Δ5-Sterols

(22S,23S)-22,23-Epoxysitosterol, (22R,23R)-22,23-epoxysitosterol, (22S, 23S)-22,23-epoxy-7-ketositosterol, (22R,23R)-22,23-epoxy-7-ketositosterol, (22S, 23S)-22,23-epoxy-7α-hydroxysitosterol, (22S,23S)-22,23-epoxy-7β-hydroxysitosterol, and (22R, 23R)-22,23-epoxy-7β-hydroxysitosterol were synthesized. Their ¹H and ¹³C NMR and the mass spectra of their trimethylsilyl derivatives were studied.     

Delta5-7-Ketosterols with modified side chain: the synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells

(22E)-3beta-Hydroxysitosta-5,22-dien-7-one, (22R, 23R)-3beta,22,23-trihydroxysitost-5-en-7-one, and (22R, 23R)-3beta-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3beta-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.     

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 cytotoxicity evaluation of 22,23-oxygenated stigmastane derivatives

Starting from (22E)-3alpha,5alpha-cyclo-6beta-methoxystigmast-22-ene eighteen derivatives of (22S,23S)-22,23-oxidostigmastane, (22R,23R)-22,23-oxidostigmastane, and (22R,23R)-22,23-dihydroxystigmastane were synthesized and screened for cytotoxicity in human hepatoma Hep G2 cells and human breast carcinoma MCF-7 cells using MTT assay. Four compounds of this series exhibited high cytotoxicity in both cells; three compounds were selectively toxic in MCF-7 cells, one compound was toxic in Hep G2 cells, rather than in MCF-7 cells; four compounds at low concentrations increased MTT test values over the control.     

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.     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

(22<Emphasis Type="Italic">R</Emphasis>,23<Emphasis Type="Italic">R</Emphasis>)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one: Improved synthesis and toxicity to MCF-7 cells

The chemical synthesis of (22R,23R)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one from (22A)-3β-acetoxy-5α-ergosta-7,14,22-triene was improved. The stages of obtaining and isomerization of (22A)-3β-acetoxy-14α15α-epoxy-5α-ergosta-7,22-diene were optimized. The introduction of (22R,23R)-epoxide cycle was carried out by alkaline treatment of intermediate (22S,23R)-3β,23-diacetoxy-22-iodo-5α-ergost-8(14)-en-15-one. In cells of human breast carcinoma MCF-7, (22R,23R)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one showed a high toxicity (TC₅₀ = 0.4±0.1 μM at 48-h incubation in serum-free medium).     

Synthesis and bioactivity evaluation of brassinosteroid analogs

Four new analogs of 28-homocastasterone have been synthesized and completely characterized for the first time from stigmasterol. (22R, 23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-stigmastan+ ++-6-one (17), (22R,23R,24S)-3beta-bromo-22,23-dihydroxy-5alpha-stigmast an-6-one (18), (22R,23R,24S)-3beta-acetoxy-5,22, 23-trihydroxy-5alpha-stigmastan-6-one (20), and (22R,23R, 24S)-3beta-bromo-5,22,23-trihydroxy-5alpha-stigmastan-6-one (21), were obtained through a synthetic route based on regioselective Delta(5) epoxidation. Compounds 17 and 18, bearing a 5alphaH moiety, were prepared through a reductive opening of the 5beta,6beta epoxy precursor, and compounds 20 and 21, analogs with a 5alphaOH moiety were obtained by hydrolytic opening of a mixture of 5alpha,6alpha and 5beta,6beta epoxy precursors. Known compounds 19 and 22 were also obtained following the described synthetic routes, respectively. The new compounds were tested with the traditional auxin-like bioassay for brassinosteroids with 19 and 22 as standards. All compounds were comparatively evaluated for their inhibitory effect on the replication of DNA (HSV-1) virus.     

Biosynthesis of 2,3-epoxybrassinosteroids in seedlings of Secale cereale

Two new brassinosteroids, (22R,23R,24S)-22,23-dihydroxy-24-methyl-5alpha-cholest-2-en-6-one (secasterol) and (22R,23R,24S)-22,23-dihydroxy-2alpha,3alpha-epoxy-24-methyl-5alpha-cholest-6-one (2,3-diepisecasterone) have been identified together with a known 2,3-epoxybrassinosteroid, secasterone, in seedlings of Secale cereale. Deuterated secasterol, teasterone, and typhasterol, upon administration to rye seedlings, were incorporated into secasterone and 2,3-diepisecasterone, indicating a biosynthetic route via teasterone/typhasterol to secasterol to 2,3-epoxybrassinosteroids.     

Tirucallane triterpenoids from Dysoxylum hainanense

Four tirucallane derivatives, 3beta,22S-dihydroxy-tirucalla-7,24-dien-23-one, 22,23-epoxy-tirucalla-7-ene-3beta,24,25-triol, 3beta,25-dihydroxy-tirucalla-7,23-diene, 23,26-dihydroxy-tirucalla-7,24-dien-3-one, together with two known triterpenoids, 24,25-epoxy-3beta,23-dihydroxy-7-tirucallene, tirucalla-7,24-diene-3beta,23-diol, were isolated from Dysoxylum hainanense. Their structures were elucidated on the basis of spectroscopic evidence.     

Δ5-7-Ketosterols with modified side chain: The synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells

(22E)-3β-Hydroxysitosta-5,22-dien-7-one, (22R,23R)-3β,22,23-trihydroxysitost-5-en-7-one, and (22R,23R)-3β-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3β-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.     

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.     

Synthesis and bioactivity of natural and C-3 fluorinated biosynthetic precursors of 28-homobrassinolide.

In this paper we describe the synthesis of two new fluorinated brassinosteroids: (22R,23R)-22,23-dihydroxy-3alpha-fluorostigmastan-6-one and (22R,23R)-22,23-dihydroxy-3beta-fluorostigmastan-6-one. Their bioactivities were evaluated in the rice lamina inclination test and compared with that of 28-homocastasterone, 28-homotyphasterol and 28-homoteasterone, possible biosynthetic precursors of 28-homobrassinolide. Results confirmed expected similarities between the biosynthesis of 24-ethylbrassinosteroids (named as the 28-homo series) and that described for 24-methylbrassinosteroids, and also indicated that these precursors might exhibit per se activities.     

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.     

The effects of (22S,23S)-and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-ones on lipid biosynthesis and metabolism of exogenous cholesterol in Hep G2 cells

Novel synthetic oxysterols (22S,23S)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (I) and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (II) efficiently inhibited cholesterol biosynthesis in human hepatoma Hep G2 cells during short-term incubation in a serum free medium (IC₅₀ values of 1.9 ± 0.2 and 0.6 ± 0.2 μ M, respectively). Cultivation of Hep G2 cells in the presence of 5 μM concentration of either (I) or (II) resulted in significant reduction of cholesterol biosynthesis (52% and 57% from control), and also changes in biosynthesis of fatty acids, triglycerides, and cholesteryl esters. Compounds (I) and (II) stimulated transformation of exogenous cholesterol to polar products secreted into the culture medium (156 % and 175% of control) as it that was shown in experiments in Hep G2 cells prelabeled with [³H]cholesterol.     

Regulation of cholesterol biosynthesis and metabolism in Hep G2 cells by Δ8(14)-15-ketoergostane derivatives

The comparative study of effects of 5α-cholest-8(14)-en-15-on-3β-ol (I), (22E)-5α-ergosta-8(14),22-dien-15-on-3β-ol (II), (22S,23S)-22,23-oxido-5α-ergost-8(14)-en-15-on-3β-ol (III), and (22R,23R)-22,23-oxido-5α-ergost-8(14)-en-15-on-3β-ol (IV) on HMG-CoA reductase, CYP27A1 and CYP3A4 genes expression in Hep G2 cells was performed. In the contrast to the 15-ketocholestane derivative (I), 15-ketoergostane derivatives (II–IV) decreased the HMG-CoA reductase mRNA level; (22R, 23R)-22,23-oxido-5α-ergost-8(14)-en-15-on-3β-ol (IV) significantly increased CYP3A4 mRNA level (320% from control). Ketosterol (II) was found to be a more potent inhibitor of cholesterol biosynthesis in Hep G2 cells during prolonged incubation, compared with ketosterol (I). The side chain conformation of compounds (I)–(IV) was evaluated by computational modeling; the correlation between biological activity of these compounds and conformational flexibility of their side chains was found. The results obtained indicate that Δ8(14)-15-ketoergostane derivatives may be used as a sterol biosynthesis and metabolism regulators in liver cells.     

Anti-herpetic and anti-inflammatory activities of two new synthetic 22,23-dihydroxylated stigmastane derivatives

Stromal keratitis resulting from ocular infection with Herpes simplex virus type 1 (HSV-1) is a common cause of blindness. This report investigates the antiviral and anti-inflammatory properties of two new synthetic stigmastane analogs in the experimental model of HSV-1-induced ocular disease in mice. (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (1) and (22S,23S)-22,23-dihydroxystigmasta-1,4-dien-3-one (2) exhibited anti-HSV-1 activity in vitro and ameliorated the signs of murine herpetic stromal keratitis (HSK), although none of the compounds showed antiviral activity in vivo. We discuss that the improvement of HSK could be due to an immunomodulatory effect of both compounds.     

Incorporation of oxyphytosterols in tissues of hamster

Oxyphytosterols (OPS) were fed to hamsters, at different concentrations, in order to observe their eventual incorporation into plasma, aorta, liver, kidneys and heart. The animals receiving the very high level (2500 ppm) presented 7beta-hydroxycampesterol, beta-epoxycampesterol, campestanetriol, 7-ketocampesterol, 7beta-hydroxysitosterol, beta-epoxysitosterol, sitostanetriol and 7-ketositosterol in all tissues. The same compounds were observed in the tissues of animals receiving 500 ppm of OPS in their diet, but with much lower levels. In hamsters fed 100 ppm of OPS, as well as in control animals, in most cases, the only observed OPS was sitostanetriol, which seems to be difficult to eliminate from the animal.     

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.