Side-Chain Modified Ergosterol and Stigmasterol Derivatives as Liver X Receptor Agonists.

A series of stigmasterol and ergosterol derivatives, characterized by the presence of oxygenated functions at C-22 and/or C-23 positions, were designed as potential liver X receptor (LXR) agonists. The absolute configuration of the newly created chiral centers was definitively assigned for all the corresponding compounds. Among the 16 synthesized compounds, 21, 27, and 28 were found to be selective LXRα agonists, whereas 20, 22, and 25 showed good selectivity for the LXRß isoform. In particular, 25 showed the same degree of potency as 22R-HC (3) at LXRß, while it was virtually inactive at LXRα (EC50 = 14.51 µM). Interestingly, 13, 19, 20, and 25 showed to be LXR target gene-selective modulators, by strongly inducing the expression of ABCA1, while poorly or not activating the lipogenic genes SREBP1 and SCD1 or FASN, respectively.

Synthetic stigmasterol derivatives inhibit capillary tube formation, herpetic corneal neovascularization and tumor induced angiogenesis: Antiangiogenic stigmasterol derivatives.

Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice. Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin. Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events.

An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system.

Spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, were synthesized from commercially available stigmasterol by a four steps reactions. In addition, the effects of these phytosterols on cell growth and mitochondrial activity were evaluated on 158N murine oligodendrocytes, C6 rat glioma cells, and SK-N-BE human neuronal cells with the crystal violet test and the MTT test, respectively. The effects of spinasterol and schottenol were compared with 7-ketocholesterol (7KC) and ferulic acid, which is also present in argan and cactus pear seed oil. Whatever the cells considered, dose dependent cytotoxic effects of 7KC were observed whereas no or slight effects of ferulic acid were found. With spinasterol and schottenol, no or slight effects on cell growth were detected. With spinasterol, reduced mitochondrial activities (30-50%) were found on 158N and C6 cells; no effect was found on SK-N-BE. With schottenol, reduced mitochondrial activity were revealed on 158N (50%) and C6 (10-20%) cells; no effect was found on SK-N-BE. Altogether, these data suggest that spinasterol and schottenol can modulate mitochondrial activity and might therefore influence cell metabolism.

Stereochemical assignment of C-24 and C-25 of amarasterone A, a putative biosynthetic intermediate of cyasterone.

A C29 phytoecdysteroid named amarasterone A (1) has been isolated from Cyathula capitata (Amaranthaceae), Leuzea carthamoides (Asteraceae), and Microsorum scolopendria (Polypodiaceae). We recently isolated amarasterone A from C. officinalis. Amarasterone A has been postulated as a biosynthetic intermediate of cyasterone in Cyathula sp. The stereochemistry at the C-24 and C-25 positions of these amarasterone A samples was investigated by comparing the NMR spectroscopic data with those of stereodefined model compounds, (24R,25S)-, (24R,25R)-, (24S,25S)-, and (24S,25R)-isomers of (20R,22R)-3ß-methoxystigmast-5-ene-20,22,26-triol (2a-d), which were synthesized in the present study. Amarasterone A isolated from Cyathula officinalis was determined to be the (24R,25S)-isomer (1a), while amarasterone A from L. carthamoides was found to be the (24R,25R)-isomer (1b). Amarasterone A from M. scolopendria was found to be a mixture of 1a and 1b. The biosynthesis of cyasterone in Cyathula sp. is discussed on the basis of the identical C-24 configuration of sitosterol and amarasterone A.

Biological activities of Schottenol and Spinasterol, two natural phytosterols present in argan oil and in cactus pear seed oil, on murine miroglial BV2 cells.

The objective of this study was to evaluate the biological activities of the major phytosterols present in argan oil (AO) and in cactus seed oil (CSO) in BV2 microglial cells. Accordingly, we first determined the sterol composition of AO and CSO, showing the presence of Schottenol and Spinasterol as major sterols in AO. While in CSO, in addition to these two sterols, we found mainly another sterol, the Sitosterol. The chemical synthesis of Schottenol and Spinasterol was performed. Our results showed that these two phytosterols, as well as sterol extracts from AO or CSO, are not toxic to microglial BV2 cells. However, treatments by these phytosterols impact the mitochondrial membrane potential. Furthermore, both Schottenol and Spinasterol can modulate the gene expression of two nuclear receptors, liver X receptor (LXR)-α and LXRß, their target genes ABCA1 and ABCG1. Nonetheless, only Schottenol exhibited a differential activation vis-à-vis the nuclear receptor LXRß. Thus Schottenol and Spinasterol can be considered as new LXR agonists, which may play protective roles by the modulation of cholesterol metabolism.

Polyamine conjugates of stigmasterol.

Three new polyamine conjugates with stigmasterol [(3ß,22E)-stigmasta-5,22-dien-3-ol] were synthesized and subjected to basic antimicrobial and cytotoxic tests. The conjugate derived from spermine, (3ß,22E)-stigmasta-5,22-dien-3-yl 4(12-amino-4,9-diaza-dodecylamino)-4-oxobutanoate (5c), displayed considerable antimicrobial activity on Staphylococcus aureus at low concentration (50 µg mL(-1)). The cytotoxic activity was tested on cells of human T-lymfoblastic leukemia (IC(50)=35.8 ± 10.3 µM (5c) and IC(50)=35.9 ± 5.7 µM (5b)) and normal human fibroblasts (IC(50)=38.0 ± 2.8 µM (5c) and IC(50)=45.5 ± 1.9 µM (5b)). Conjugate 5a displayed no activity in both tests.

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.

Synthesis of dehydro-oogoniol, a female-activating hormone of Achlya: the progesterone route.

The structure of dehydro-oogoniol (3 beta,11 alpha,15 beta,29-tetrahydroxystigmasta-5,24(28)(E)-dien-7-one), a female-activating hormone of the water mold Achlya, has been confirmed by synthesis. The starting material was progesterone, which was converted to the 11 alpha, 15 beta-dihydroxy derivative by microbiological hydroxylation with Aspergillus giganteus (ATCC 10059). The side chain was constructed in a stepwise manner by means of Wittig and Horner-Emmons reactions, and the C-7 ketone was then introduced by allylic oxidation. The biological activity of the synthetic compound was the same as that of the natural hormone.

Fucosterol epoxide lyase of insects: synthesis of labeled substrates and development of a partition assay.

Fucosterol epoxide labeled with tritium in the C-29 methyl has been synthesized and employed in the development of a partition assay which allows the rapid determination of fucosterol epoxide lyase activity in vitro in homogenates of insect tissues. An independent synthesis of [24-14C]fucosterol epoxide provided a control substrate to evaluate nondealkylative transfer of labeled steroid to the aqueous layer during the enzyme assay. The diastereomeric 24R,28R- and 24S,28S-[29-3H]fucosterol epoxides were obtained via HPLC separation of their benzoate esters. Homogenates of the midgut tissue of larval tobacco hornworms (Manduca sexta) were examined at pH 5 to 9 in several buffer systems, and at temperatures of 7 to 67 degrees C in phosphate buffer. Optimal activity was found using pH 7.4, 76 mM phosphate buffer at 37 degrees C. The 24R,28R diastereomer of fucosterol epoxide was metabolized at a rate at least 100 times that of the 24S,28S isomer by this enzyme system.