Solvent-free synthesis of 6ß-phenylamino-cholestan-3ß,5α-diol and (25R)-6ß-phenylaminospirostan-3ß,5α-diol as potential antiproliferative agents.

In this paper is described a synthetic route to 6ß-phenylamino-cholestan-3ß,5α-diol and (25R)-6ß-phenylaminospirostan-3ß,5α-diol, starting from cholesterol and diosgenin, respectively. The products were obtained in two steps by epoxidation followed by aminolysis, through an environmentally friendly and solvent-free method mediated by SZ (sulfated zirconia) as catalyst. The use of SZ allows chemo- and regioselective ring opening of the 5,6α-epoxide during the aminolysis reaction eliminating the required separation of the epoxide mixture. The products obtained were spectroscopically characterized by 1H, PENDANT 13C NMR and HETCOR experiments, and complemented with FTIR-ATR and HRMS. The antiproliferative effect of the ß-aminoalcohols was evaluated on MCF-7 cells after 48h of incubation, by MTT and CVS assays. These methodologies showed that both compounds have antiproliferative activity, being more active the cholesterol analogue. Additionally, the cell images obtained by Harris' Hematoxylin and Eosin (H&E) staining protocol, evidenced formation of apoptotic bodies due to the presence of the obtained ß-aminoalcohols in a dose-dependent manner.

Improvement of 5,6α-epoxycholesterol, 5,6ß-epoxycholesterol, cholestane-3ß,5α,6ß-triol and 6-oxo-cholestan-3ß,5α-diol recovery for quantification by GC/MS.

5,6α-epoxycholesterol (5,6α-EC) and 5,6ß-epoxycholesterol (5,6ß-EC) are oxysterols involved in the anticancer pharmacology of the widely used antitumor drug tamoxifen. They are both metabolized into cholestane-3ß,5α,6ß-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH) enzyme, and CT is metabolized by an as-yet uncharacterized enzyme into 6-oxo-cholestan-3ß,5α-diol (OCDO). A recent feasibility study showed that the 5,6-ECs may represent surrogate markers of tamoxifen activity in breast cancer patients undergoing endocrine therapy, thus there is a growing interest in their accurate quantification. These oxysterols are usually quantified by gas-liquid chromatography coupled to mass spectrometry (GC/MS), using an isotope dilution methodology with the corresponding deuterated oxysterol. This method is considered to be relative quantitative since all of the standards used are deuterated oxysterols, however it is not known whether the preparation of each oxysterol is affected in the same way by the extraction, pre-purification by solid phase extraction (SPE) and trimethylsilylation steps, particularly when using biological samples that contain many other reactive compounds. Thus, in this study we investigated the yield of the 5,6-ECs, CT and OCDO recovery from patient serum samples at different stages of their work-up and trimethylsilylation prior to GC/MS analysis, using [14C]-labeled analogs to follow these oxysterols at each step. We measured a 40 to 60% loss of material for the 5,6-ECs and OCDO, however we also describe the conditions that improved their recovery. Our data also show that the use of deuterated 5,6α-EC, 5,6ß-EC, CT and OCDO is an absolute requirement for their accurate quantification.

Naturally occurring marine steroid 24-methylenecholestane-3ß,5α,6ß,19-tetraol functions as a novel neuroprotectant.

Steroids have been shown to have multiple effects on the nervous system including neuroprotective activities, and they have the potential to be used for the treatment of neurodegenerative diseases. In this current study, we tested the hypothesis that the marine steroid 24-methylenecholestane-3ß,5α,6ß,19-tetraol (Tetrol) has a neuroprotective effect. (1) We synthesized Tetrol through a multiple step reaction starting from hyodeoxycholic acid (HDCA). (2) We then evaluated the neuroprotective effect of Tetrol with a glutamate-induced neuronal injury model in vitro. Tetrol concentration dependently increased the survival rate of cerebellar granule neurons challenged with toxic concentration of glutamate. Consistently, Tetrol significantly decreased glutamate-induced lactate dehydrogenase (LDH) release with a threshold concentration of 2.5 µM. (3) We further evaluated the neuroprotective effect of Tetrol in a middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in rat. Tetrol, at a dose of 12 mg/kg, significantly decreased MCAO-induced infarction volume by ∼50%. (4) Finally, we probed the mechanism and found that Tetrol concentration dependently attenuated N-methyl-d-aspartate (NMDA)-induced intracellular calcium ([Ca(2+)]i) increase with an IC50 of 7.8±0.62 µM, and inhibited NMDA currents in cortical neurons with an IC50 of 10.28±0.71 µM. Taken together, we have synthesized and characterized Tetrol as a novel neuroprotectant through negative modulation of NMDA receptors.

An improved synthesis of [26-(2)H3]castasterone.

Commercially available epicastasterone has been employed as a starting material for the preparation of [26-(2)H3 ]castasterone. The chemical synthesis has been realized in 13 chemical steps and 4.6% total yield. The target compound is intended to be used as internal standard for the quantitative analysis of brassinosteroids.

Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstane-3α,17ß-diol derivatives.

Aminosteroid derivatives represent a new family of compounds with promising antiproliferative activity over different cancer cell lines. Among all the aminosteroid derivatives synthesised in our laboratory, we have identified E-37P as one of the more potent when tested in vitro. Unfortunately, the pharmacokinetic properties of E-37P decrease its effectiveness when tested in vivo. To improve the bioavailability and increase the efficiency of aminosteroid E-37P, two series of analog compounds were synthesised by classic chemical synthesis, they were then characterized, and the concentration that inhibits 50% of cell proliferation (IC50) was determined on different cell lines. RM-133, a 5α-androstane-3α,17ß-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2ß and an ethinyl at position 17α, showed very good antiproliferative activity among the five cancer cell lines studied (IC50=0.1, 0.1, 0.1, 2.0 and 1.1 µM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively). Moreover, the plasmatic concentration of RM-133 at 3h, when injected subcutaneously in rats, was 2.3-fold higher than that of E-37P (151 vs 64.8 ng/mL). Furthermore, RM-133 weakly inhibited the two representative liver enzymes, CYP3A4 and CYP2D6, indicating a very low risk of drug-drug interactions. The cytotoxicity of RM-133 against normal cells was tested on peripheral blood lymphocytes (PBL) obtained from different donors and previously activated with phytohemagglutinin-L. PBL responded differently to treatment with RM-133, we observed a stimulation of cell proliferation and/or cytotoxicity in a dose-dependent manner. Based on these results, additional studies are currently underway to evaluate the selectivity of our lead compound against normal cell lines in a more detailed fashion.

Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain.

Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological activity in plant and animal systems. The cytotoxic activity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard.

Determination of C-23 configuration in (20R)-23-hydroxycholestane side chain of steroid compounds by 1H and 13C NMR spectroscopy.

Epimeric (20R,23R)- and (20R,23S)-23-hydroxycholestane steroids were synthesized. Their structures were elucidated by extensive 1H and 13C NMR spectroscopy and application of the Mosher's method. All proton and carbon signals of the side chains were assigned. Based on these assignments spectral data allow the determination of the C-23 stereochemistry of (20R)-23-hydroxycholestane side chains of the new natural steroids by comparison with spectra of the obtained model compounds. As a result, the C-23 configuration of two steroid compounds from the starfishes Lethasterias nanimensis chelifera and Lethasterias fusca was established.

Inhibitory effect of oxygenated cholestan-3ß-ol derivatives on the growth of Mycobacterium tuberculosis.

A variety of cholestan-3ß-ol derivatives, which are oxygenated at different positions of the steroid ring system, were prepared and tested for their inhibition of the Mycobacterium tuberculosis H37Rv strain. Several compounds showed significant antitubercular activities with MIC90 values in the range 4-8 µM and low or non-detectable toxicity against mammalian cells.

In-vitro archaeacidal activity of biocides against human-associated archaea.

BACKGROUND: Several methanogenic archaea have been detected in the human intestinal microbiota. These intestinal archaea may contaminate medical devices such as colonoscopes. However, no biocide activity has been reported among these human-associated archaea. METHODOLOGY: The minimal archaeacidal concentration (MAC) of peracetic acid, chlorhexidine, squalamine and twelve parent synthetic derivatives reported in this study was determined against five human-associated methanogenic archaea including Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arboriphilicus, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis and two environmental methanogens Methanobacterium beijingense and Methanosaeta concilii by using a serial dilution technique in Hungates tubes. PRINCIPAL FINDINGS: MAC of squalamine derivative S1 was 0.05 mg/L against M. smithii strains, M. oralis, M. arboriphilicus, M. concilii and M. beijingense whereas MAC of squalamine and derivatives S2-S12 varied from 0.5 to 5 mg/L. For M. stadtmanae and M. luminyensis, MAC of derivative S1 was 0.1 mg/L and varied from 1 to ≥ 10 mg/L for squalamine and its parent derivatives S2-S12. Under the same experimental conditions, chlorhexidine and peracetic acid lead to a MAC of 0.2 and 1.5 mg/L, respectively against all tested archaea. CONCLUSIONS/SIGNIFICANCE: Squalamine derivative S1 exhibited a 10-200 higher archaeacidal activity than other tested squalamine derivatives, on the majority of human-associated archaea. As previously reported and due to their week corrosivity and their wide spectrum of antibacterial and antifungal properties, squalamine and more precisely derivative S1 appear as promising compounds to be further tested for the decontamination of medical devices contaminated by human-associated archaea.

Towards squalamine mimics: synthesis and antibacterial activities of head-to-tail dimeric sterol-polyamine conjugates.

Four dimeric sterol-polyamine conjugates have been synthesized from the homo- and hetero-connection of monomeric sterol-polyamine analogs in a head-to-tail manner. These dimeric conjugates show strong antibacterial activity against a broad spectrum of Gram-positive bacteria, whereas their corresponding activities against Gram-negative bacteria are relatively moderate. Though no significant difference was observed in the activities of these conjugates, cholic acid-containing dimeric conjugates generally exhibit higher activities than the corresponding deoxycholic acid-derived analogs. This is in contrast to the finding that a monomeric deoxycholic acid-spermine conjugate was more active than the corresponding cholic acid-derived analog.

Modification in the side chain of solomonsterol A: discovery of cholestan disulfate as a potent pregnane-X-receptor agonist.

Seven synthetic analogues of the PXR (pregnane-X-receptor) potent natural agonist solomonsterol A were prepared by total synthesis. Their activity toward PXR was assessed by transactivation and RT-PCR assays. The study discloses cholestan disulfate (8) as a new, simplified agonist of PXR. By in vitro studies on hepatic cells we have demonstrated that this compound is a potent PXR agonist and functional characterization in human macrophages and hepatic stellate cells provided evidence that cholestan disulfate (8) has the ability to modulate the immune response triggered by bacterial endotoxin as well as to counter-activate hepatic stellate cell activation induced by thrombin. Because inhibition of immune-driven circuits might have relevance in the treatment of inflammation and liver fibrosis, the present data support the development of cholestan disulfate (8) in preclinical models of inflammatory diseases.

Synthesis of new 3,20-bispolyaminosteroid squalamine analogues and evaluation of their antimicrobial activities.

3,20-Amino- and polyaminosteroid analogues of squalamine and trodusquemine were synthesized involving a stereoselective titanium reductive amination reaction in high chemical yields in numerous cases. These derivatives were evaluated for their in vitro antimicrobial properties against references and clinical bacterial strains exhibiting minimum inhibitory concentrations of 2.5-40 µg/mL. The mechanism of action of these derivatives was determined using bioluminescence for ATP efflux measurements and fluorescence methods for membrane depolarization assays.

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 C(27) 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 C(24)allo-cholic acid performate with NaBH(4)/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I(2)/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 C(18) column and their structural characteristics, particularly the chiral center at C-25, delineated using (1)H and (13)C 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 the enantiomer of the oxysterol-antagonist LY295427.

Cellular cholesterol homeostasis is regulated by oxygenated cholesterol metabolites called oxysterols. While the importance of oxysterols in the acute regulation of cholesterol homeostasis is known, the precise molecular mechanisms through which oxysterols exert their effects remain to be elucidated. LY295427 (1) is a known antagonist of the cholesterol-homeostatic effects of 25-hydroxycholesterol (25-HC), a biologically active oxysterol. In order to examine the mechanism of action of this antagonism, and to further explore recent evidence suggesting that the membrane effects of 25-HC contribute to acute cholesterol regulation, we synthesized the enantiomer of LY295427 (ent-LY295427). ent-LY295427 (2) will serve as a unique probe to provide insight into the role of transcription-independent mechanisms in regulation of cholesterol homeostasis.

Synthesis of 26-hydroxy-22-oxocholestanic frameworks from diosgenin and hecogenin and their in vitro antiproliferative and apoptotic activity on human cervical cancer CaSki cells.

Certain steroidal compounds have demonstrated an antiproliferative effect against several tumor cell lines; however, their complete role on cancer cells is not currently established. Herein, we report the synthesis and evaluation of two new 26-hydroxy-22-oxocholestanic steroids on cervical cancer CaSki cells. The title compounds were prepared from diosgenin and hecogenin in excellent yields. We determined their effect on cell proliferation, cell cycle, and cell death. The cytotoxic effect of the title compounds on CaSki and human lymphocytes was also evaluated, indicating that the main cell death process is not necrosis; the null effect on lymphocytes implies that they are not cytotoxic. The observation of apoptotic bodies as well as the increase in the expression of active caspase-3 along with the fragmentation of DNA confirmed that such new cholestanic frameworks induced apoptosis in tumor cells. Significantly, their antiproliferative activity on tumor cells did not affect the proliferative potential of normal fibroblasts from cervix and peripheral blood lymphocytes. The title compounds show selective antitumor activity and therefore serve as promising lead candidates for further optimization.

Preparation and synthetic application of partially protected brassinosteroids.

Preparation of partially protected brassinosteroids is achieved through the reaction of the source material (24-epicastasterone and 24-epibrassinolide) with diol-specific reagents (2,2-dimethoxypropane and methylboronic acid). The obtained products were shown to be useful synthetic intermediates for further preparation of minor representatives of this class of natural phytohormones (such as 3,24-diepicastasterone and 3-dehydro-24-epibrassinolide).

Synthesis of new alkylaminooxysterols with potent cell differentiating activities: identification of leads for the treatment of cancer and neurodegenerative diseases.

We describe here the syntheses and the biological properties of new alkylaminooxysterols. Compounds were synthesized through the trans-diaxial aminolysis of 5,6-alpha-epoxysterols with various natural amines including histamine, putrescine, spermidine, or spermine. The regioselective synthesis of these 16 new 5alpha-hydroxyl-6beta-aminoalkylsterols is presented. Compounds were first screened for dendrite outgrowth and cytotoxicity in vitro, and two leads were selected and further characterized. 5alpha-Hydroxy-6beta-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3beta-ol, called dendrogenin A, induced growth control, differentiation, and the death of tumor cell lines representative of various cancers including metastatic melanoma and breast cancer. 5alpha-Hydroxy-6beta-[3-(4-aminobutylamino)propylamino]cholest-7-en-3beta-ol, called dendrogenin B, induced neurite outgrowth on various cell lines, neuronal differentiation in pluripotent cells, and survival of normal neurones at nanomolar concentrations. In summary, we report that two new alkylaminooxysterols, dendrogenin A and dendrogenin B, are the first members of a class of compounds that induce cell differentiation at nanomolar concentrations and represent promising new leads for the treatment of cancer or neurodegenerative diseases.

Synthesis and cytotoxic analysis of some disodium 3beta,6beta-dihydroxysterol disulfates.

Disodium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH(4) in the presence of NiCl(2) to produce cholest-3beta,6beta-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na(+)) yielded the target steroid 1. Disodium 24-ethyl-3beta,6beta-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3beta,6beta-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

[Synthesis of brassinolide and its biosynthetic precursors using methyl-3-hydroxy-2-methylpropionate].

Formal synthesis of plant hormones that belong to the group of 24alpha-methylbrassinosteroids, including brassinolide and its biosynthetic precursors with one hydroxyl group in their side chain, was performed. Stereochemistry of a methyl group at the C24 atom was provided by the choice of the desired enanthiomer of methyl-3-hydroxy-2-methylpropionate and by the sequence of its conversions into the chiral intermediate that was necessary for the formation of the C23-C28 fragment of the side chain. The (22R,23R)-diol group was introduced by the Sharpless asymmetric dihydroxylation of the intermediate delta22-steroids, the products of consecutive reactions of attachment of a low-molecular sulfone to the steroid C22-aldehyde, acetylation, and reductive desulfurization of the intermediate beta-acetoxysulfones. Reduction of 22-acetoxy-23,25-disulfones was shown to proceed with the preservation of the functional group at atom C22.

4Alpha-bromo-5alpha-cholestan-3beta-ol and nor-5alpha-cholestan-3beta-ol derivatives-stereoselective synthesis and hormonal activity in Caenorhabditis elegans.

We describe the stereoselective synthesis of 4alpha-bromo-5alpha-cholestan-3beta-ol, 21-nor-5alpha-cholestan-3beta-ol, 27-nor-5alpha-cholestan-3beta-ol and 21,27-bisnor-5alpha-cholestan-3beta-ol. In order to clarify the in vivo metabolism of cholesterol, these compounds have been used for feeding experiments in Caenorhabditis elegans. Our preliminary results provide important insights into the metabolism of cholesterol in worms.