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Steroid hormones are essential for the biological processes of eukaryotic organisms. The steroid endocrine system of C. elegans, which includes dafachronic acids (DA) and the nuclear receptor ceDAF-12, provides a simple model for exploring the role of steroid hormone signaling pathways in animals. In this study, we show for the first time the feasibility of designing synthetic steroids that can modulate different physiological processes, such as development, reproduction and ageing, in relation to ceDAF-12. Our results not only confirm the conclusions derived from genetic studies linking these processes but also provide new chemical tools to selectively manipulate them, as we found that different compounds produce different phenotypic results. The structures of these compounds are much more diverse than those of endogenous hormones and analogues previously described by other researchers, allowing further development of the chemical modulation of the steroid endocrine system in C. elegans and related nematodes., (© 2024 Wiley-VCH GmbH.)

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER + breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3β-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Every cell in vertebrates possesses the machinery to synthesise cholesterol and to metabolise it. The major route of cholesterol metabolism is conversion to bile acids. Bile acids themselves are interesting molecules being ligands to nuclear and G protein-coupled receptors, but perhaps the intermediates in the bile acid biosynthesis pathways are even more interesting and equally important. Here, we discuss the biological activity of the different intermediates generated in the various bile acid biosynthesis pathways. We put forward the hypothesis that the acidic pathway of bile acid biosynthesis has primary evolved to generate signalling molecules and its utilisation by hepatocytes provides an added bonus of producing bile acids to aid absorption of lipids in the intestine., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that transcriptionally regulates lipid metabolism and inflammation; therefore, PPARα agonists are promising agents to treat dyslipidemia and metabolic disorders. PPARα full agonists, such as fibrates, are effective anti-hypertriglyceride agents, but their use is limited by adverse side effects. Hence, the aim of this study was to identify small molecules that can activate PPARα while minimizing the adverse effects. Antrodia cinnamomea, a rare medical mushroom, has been used widely in Asian countries for the treatment of various diseases, including liver diseases. Antcin B, H and K (antcins) and ergostatrien-3β-ol (EK100) are bioactive compounds isolated from A. cinnamomea with anti-inflammatory actions. Antcins, ergostane-type triterpenoids, contain the polar head with carboxylate group and the sterol-based body. Here, we showed at the first time that sterol-based compounds, antcins, but not EK100, activate PPARα in a cell-based transactivation study. The in silico docking studies presented several significant molecular interactions of antcins, including Tyr314, and His440 in the ligand-binding domain of PPARα, and these interactions are required for helix 12 (H12) stabilization. We propose that PPARα activation activity of antcins is related to their binding mode which requires conventional H12 stabilization, and that antcins can be developed as safe selective PPARα modulators., (Copyright © 2018. Published by Elsevier Taiwan LLC.)

Antrodia camphorata, a well-known and highly valued edible medicinal mushroom with intriguing activities like liver protection, has been traditionally used for the treatment of alcoholic liver disease. A. camphorata shows highly medicinal and commercial values with the demand far exceeds the available supply. Thus, the petri-dish cultured A. camphorata (PDCA) is expected to develope as a substitute. In this paper, nineteen triterpenes were isolated from PDCA, and thirteen of them were the unique anthroic acids in A. camphorata, including the main content antcin K, which suggested that PDCA produced a large array of the same anthroic acids as the wild one. Furthermore, no obvious acute toxicity was found suggesting the edible safety of PDCA. In mice alcohol-induced liver injury model, triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) had been reduced by the PDCA powder as well as the main content antcin K, which indicated that the PDCA could protect alcoholic liver injury in mice model and antcin K could be the effective component responsible for the hepatoprotective activities of PDCA against alcoholic liver diseases., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography - mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In the present paper we describe the optimization and the application of a chromatographic method suitable to get all four diastereoisomers of C24-C25 cyclopropyl dafachronic acid derivatives in sufficient amount for their biological appraisal towards the nuclear hormone receptor transcription factor DAF-12. A preliminary column screening of six anion exchange type Cinchona alkaloid-based chiral stationary phases (CSPs) allowed to identify the one with a quinine scaffold and carrying a O-9-(3,5-bis(trifluoromethyl)phenyl) moiety at C9 position as the best CSP. Few modifications of the experimental conditions revealed that a content of 18mM acetic acid used as counterion and displacer in acetonitrile and a column temperature fixed at 35°C were optimal for the simultaneous discrimination of all four diastereoisomers with a 1.0mL/min flow rate. With such conditions, transoid (S,S) and (R,R) diastereoisomers were resolved with R S >1.4. With non chiral reversed-phase columns, neither the cisoid nor the transoid diastereoisomers could be resolved. This way, ca. 1.0mg of each stereoisomer was isolated with a diastereomeric purity >98%, suitable for the following biological tests. The indirect stereochemical assignments of the four diastereoisomers, and hence the corresponding chromatographic elution order (24R,25R)<(24S,25S)<(24R,25S)<(24S,25R) were made in an analogy manner on the basis of the resolution of fully assigned and structurally very similar ursodeoxycholic acid derivatives. As support of this indirect way of assigning the absolute configuration of the C24 and C25 chiral centre a molecular modeling procedure based on dynamic simulation was successfully applied., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography - mass spectrometry with multistage fragmentation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Sterols are major cell membrane lipids, and in many organisms they are modified with glucose to generate sterylglucosides. Glucosylation dramatically changes the functional properties of sterols. The formation of sterylglucosides from sterols in plants, fungi, and bacteria uses UDP-glucose as a glucose donor. By contrast, sterylglucoside biosynthesis in mammals is catalyzed by the transglucosylation activity of glucocerebrosidases, with glucosylceramide acting as the glucose donor. Recent success in isolation and structural determination of sterylglucosides in the vertebrate central nervous system shows that transglucosylation also occurs in vivo. These analyses also revealed that sterylglucoside aglycons are composed of several cholesterol-related metabolites, including a plant-type sitosteryl., Scope of Review: In this review, we discuss the biological functions and metabolism of sterylglucosides. We also summarize new findings from studies on the metabolism of vertebrate sterylglucosides and review the circumstances underlying the recent discovery of sterylglucosides in vertebrate brain. Finally, we discuss the role of sterylglucosides in a variety of neurodegenerative disorders such as Gaucher disease and Parkinson's disease., Major Conclusions: The biological significance of UDP-glucose-independent sterol glucosylation is still unknown, but it is plausible that glucosylation may provide sterols with novel biological functions. Even though sterol glucosylation is a simple reaction, it can dramatically change the physical properties of sterols., General Significance: Sterylglucosides may play roles in various physiological processes and in the pathogenesis of different diseases. Arriving at a better understanding of them at the organ and cellular level may open up new approaches to developing therapeutics for a variety of diseases. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A new methodology to obtain C-25 and C-26 steroidal acids starting from pregnenolone is described. Construction of the side chain was achieved by applying the Mukaiyama aldol reaction with a non-hydrolytic work-up to isolate the trapped silyl enol ether with higher yields. Using this methodology we synthesized three new steroidal acids as potential ligands of DAF-12, Liver X and Glucocorticoid nuclear receptors and studied their activity in reporter gene assays. Our results show that replacement of the 21-CH 3 by a 20-keto group in the side chains of the cholestane scaffold of DAF-12 or Liver X receptors ligands causes the loss of the activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol ® ; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRβ expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3β,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The DAF-12 receptor in nematodes and the Liver X Receptor (LXR) in mammals are structurally related transcription factors that play key roles in determining the life span of the organism. Both types of receptors are activated by oxysterols, cholesterol metabolites with oxidized side chains. Restricting the movement of the oxysterol side chain to certain orientations may have profound effects in the activity profile, however this has not been explored so far. In a first attempt to obtain analogues of natural ligands of DAF-12 and LXR with restricted side chain mobility we introduced a 16,22-oxygen bridge in 26-hydroxycholesterol, a cholestenoic acid and a dafachronic acid (5-7). Diosgenin was used as starting material, the key step to obtain the 16,22 epoxy functionality was the one pot formation and reduction of a cyclic hemiketal via the oxocarbenium ion using sodium cyanoborohydride. All new compounds were characterized by NMR and mass spectrometry and assayed as ceDAF-12 or LXR ligands in transactivation cell-based assays. The dafachronic acid analogue 7 behaved as a ceDAF-12 agonist., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological functions. The possible roles of oxysterols in various neurological diseases makes the analysis of these metabolites in the central nervous system of particular interest. Here, we report the identification and quantification of a panel of twelve sterols in mouse cerebrospinal fluid (CSF) using liquid chromatography-mass spectrometry exploiting enzyme assisted derivatisation for sterol analysis technology. We found low levels of oxysterols and cholestenoic acids in CSF in the range of 5pg/mL-2.6ng/mL. As found in man, these concentrations are one to two orders of magnitude lower than in plasma., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Steroid hormones play significant roles in both worms and mammalians. (25S)-Δ(7)-Dafachronic acid (Δ(7)-DA, 1) is a member of the dafachronic acid hormonal series that regulates both development and lifespan of C. elegans. Despite its importance, effective tools for the illumination of its mode of action are lacking. Herein, we report an efficient synthesis of trideuterated Δ(7)-DA, [5,24,25-D3]-(25S)-Δ(7)-dafachronic acid ([D3]-Δ(7)-DA, 2), as a useful chemical tool for subsequent biological studies. Key steps for this bioinspired synthesis approach include site-selective aliphatic C-H oxidation mediated by methyl(trifluoromethyl)dioxirane (TFDO), and the iridium/phosphine-oxazoline-catalyzed late-stage asymmetric deuterium reduction., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 μm core-shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dafachronic acids (DAs) are 3-keto cholestenoic acids bearing a carboxylic acid moiety at the end of the steroid side chain. These compounds interact with the DAF-12 receptor, a ligand-dependent transcription factor that acts as a molecular switch mediating the choice between arrest at diapause or progression to reproductive development and adult lifespan in different nematodes. Recently, we reported that the 27-nor-Δ4-DA was able to directly activate DAF-12 in a transactivation cell-based luciferase assay and rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants. In the present paper, to investigate further the relationship between the structure of the steroid side chain and DAF-12 activity, we evaluated the in vitro and in vivo activity of Δ4-DA analogues with modified side chains using transactivation cell-based assays and daf-9(dh6) C. elegans mutants. Our results revealed that introduction of a 24,25-double bond on the cholestenoic acid side chain did not affect DAF-12 activity, whereas shortening the side chain lowered the activity. Most interestingly, the C24 alcohol 24-hydroxy-4-cholen-3-one (6) was an antagonist of the DAF-12 receptor both in vitro and in vivo.

Two new and six known steroidal glucosides were isolated from the tuber of Ophiopogon japonicus. The new steroidal glucosides were established as (20R,25R)-26-O-β-d-glucopyranosyl-3β,26-dihydroxycholest-5-en-16,22-dioxo-3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside (1) and 26-O-β-d-glucopyranosyl-(25R)-furost-5-en-3β,14α,17α,22α,26-pentaol-3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside (3) on the basis of spectroscopic data as well as chemical evidence.

For the first time, a successful chromatographic method based on the use of a novel triazole click quinidine (QD) derivative anion-exchange chiral stationary phase (CSP) is applied for the epimer separation of dafachronic acids (Δ(4)- and Δ(7)-DAs). The use of a polar-ionic eluent system consisting of 18mM AcOH in ACN furnished an excellent separation of both Δ(4)- (α=1.19, RS=2.52) and Δ(7)-DA (α=1.14, RS=2.06) C-25 epimer couples. The pool of data collected during the chromatographic analyses revealed the prominent role of anion-exchange interactions in governing the analyte (SA) retention and also indicated the occurrence of stereoselective H-bond contacts with the chiral selector (SO) substructural motifs. In any case, molecular modelling studies corroborate the need for sufficient spatial freedom for optimum binding between the SO and the SAs which seems less the case for the immobilised SO unit of the commercialized QD-based CSP., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.

Small-molecule ligands of nuclear hormone receptors (NHRs) govern the transcriptional regulation of metazoan development, cell differentiation, and metabolism. However, the physiological ligands of many NHRs remain poorly characterized, primarily due to lack of robust analytical techniques. Using comparative metabolomics, we identified endogenous steroids that act as ligands of the C. elegans NHR, DAF-12, a vitamin D and liver X receptor homolog regulating larval development, fat metabolism, and lifespan. The identified molecules feature unexpected chemical modifications and include only one of two DAF-12 ligands reported earlier, necessitating a revision of previously proposed ligand biosynthetic pathways. We further show that ligand profiles are regulated by a complex enzymatic network, including the Rieske oxygenase DAF-36, the short-chain dehydrogenase DHS-16, and the hydroxysteroid dehydrogenase HSD-1. Our results demonstrate the advantages of comparative metabolomics over traditional candidate-based approaches and provide a blueprint for the identification of ligands for other C. elegans and mammalian NHRs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A new steroidal alkaloid, (20S,22R,24R)-24-ethyl-3-oxocholest-4-en-22-amino, named as nandsterine (1), together with 10 known alkaloids, palmatine (2), O-methylbulbocapnine (3), nantenine (4), dehydronantenine (5), glaucine (6), didehydroglaucine (7), dehydrocorydaline (8), jatrorrhizine (9), magnoflorine (10) and berberine (11), was isolated from the fruit of Nandina domestica Thunb. Their structures were elucidated by using spectroscopic methods as well as by comparing with the published data. Compound 1 was a new class of steroidal alkaloid isolated from the family Berberidaceae, meanwhile compounds 2, 3, 6-8 and 10 were obtained from N. domestica for the first time. Compound 1 exhibited cytotoxicity against HL-60 cells (human leukaemia) with IC50 values of 52.1 μM.

Two variants of the Claisen rearrangement were evaluated for a stereoselective construction of a C-25 stereogenic center in cholestenoic acids based on 1,4-chirality transfer. Johnson orthoester Claisen rearrangement of (22R)- and (22S)-propargyl enol ethers proceeded in a highly stereoselective manner to give (25R)- and (25S)-isomeric allenes. The stereochemical outcome of the Ireland-Claisen rearrangement of 22-allylic alcohols was dependent on the configuration of the C-22 hydroxyl group and the geometry of the enol ether. The latter could be controlled by the solvent (THF or a mixture of THF/HMPA) chosen for the generation of silyl enolate., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Molecular dynamics simulations of bilayer systems consisting of varying proportions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), and intrinsically fluorescent Chol analogues dehydroergosterol (DHE) or cholestatrienol (CTL) were carried out to study in detail the extent to which these fluorescent probes mimic Chol's behavior (location, orientation, dynamics) in membranes as well as their effect on host bilayer structure and dynamics (namely their ability to induce membrane ordering in comparison with Chol). Control properties of POPC and POPC/Chol bilayers agree well with published experimental and simulation work. Both probes and Chol share similar structural and dynamical properties within the bilayers. Additionally, the fluorescent sterols induce membrane ordering to a similar (slightly lower) extent to that of Chol. These findings combined demonstrate that the two studied fluorescent sterols are adequate analogues of Chol, and may be used with advantage over side-chain labeled sterols. The small structural differences between the three studied sterols are responsible for the slight variations in the calculated properties, with CTL presenting a more similar behavior to Chol (correlating with its larger structural similarity to Chol) compared to DHE.

27-Nor-Δ(4)-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20β-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ(4)-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid-protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid-protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The transverse motion of molecules from one leaflet to the other of a lipid bilayer, or flip-flop, represents a putative mechanism for their transmembrane transport and may contribute to the asymmetric distribution of components in biomembranes. However, a clear understanding of this process is still missing. The scarce knowledge derives from the difficulty of experimental determination. Because of its slow rate on the molecular time scale, flip-flop is challenging also for computational techniques. Here, we report a study of the passive transbilayer diffusion of steroids, based on a kinetic model derived from the analysis of their free energy surface, as a function of their position and orientation in the bilayer. An implicit membrane description is used, where the anisotropy and the nonuniformity of the bilayer environment are taken into account in terms of the gradients of density, dielectric permittivity, acyl chain order parameters, and lateral pressure. The flip-flop rates are determined by solving the Master Equation that governs the time evolution of the system, with transition rates between free energy minima evaluated according to the Kramers theory. Considering various steroids (cholesterol, lanosterol, ketosterone, 5-cholestene, 25-hydroxycholesterol, and testosterone), we can discuss how differences in molecular shape and polarity affect the pathway and the rate of flip-flop in a liquid crystalline 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayer, at low steroid concentration. We predict time scales ranging from microseconds to milliseconds, strongly affected by the presence of polar substituents and by their position in the molecular skeleton.

A structure for the ligand binding domain (LBD) of the DAF-12 receptor from Caenorhabditis elegans was obtained from the X-ray crystal structure of the receptor LBD from Strongyloides stercoralis bound to (25R)-Δ(7)-dafachronic acid (DA) (pdb:3GYU). The model was constructed in the presence of the ligand using a combination of Modeller, Autodock, and molecular dynamics (MD) programs, and then its dynamical behavior was studied by MD. A strong ligand binding mode (LBM) was found, with the three arginines in the ligand binding pocket (LBP) contacting the C-26 carboxylate group of the DA. The quality of the ceDAF-12 model was then evaluated by constructing several ligand systems for which the experimental activity is known. Thus, the dynamical behavior of the ceDAF-12 complex with the more active (25S)-Δ(7)-DA showed two distinct binding modes, one of them being energetically more favorable compared with the 25R isomer. Then the effect of the Arg564Cys and Arg598Met mutations on the (25R)-Δ(7)-DA binding was analyzed. The MD simulations showed that in the first case the complex was unstable, consistent with the lack of transactivation activity of (25R)-Δ(7)-DA in this mutant. Instead, in the case of the Arg598Met mutant, known to produce a partial loss of activity, our model predicted smaller effects on the LBM with a more stable MD trajectory. The model also showed that removal of the C-25 methyl does not impede the simultaneous strong interaction of the carboxylate with the three arginines, predicting that 27-nor-DAs are putative ceDAF-12 ligands., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: To investigate the use of nano self-assemblies formed by polyallylamine (PAA) modified with 5 or 10% mole fluorenylmethoxy carbonyl (Fmoc(5)/(10)), dimethylamino-1-naphthalenesulfonyl (Dansyl(5)/(10)) and 5% mole cholesteryl group (Ch(5)) for oral hydrophobic drug delivery., Methods: Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. Particle size and morphology of drug-loaded self-assemblies were determined using photon correlation spectroscopy and transmission electron microscopy. Solubilising capacity, in vitro drug release and formulation stability were analysed by HPLC, and in vitro biocompatibility studies (haemolysis and cytotoxicity) were carried out on bovine erythrocytes and Caco-2 cells, respectively. Dansyl(10) and Ch(5) griseofulvin formulations were administered intra-gastrically to rats, and drug plasma levels were analysed by HPLC., Results: Drug-encapsulated self-assemblies typically have hydrodynamic size of 300-400 nm. Dansyl(10) exhibited universal drug solubiliser property and had significantly improved prednisolone, griseofulvin and propofol solubility by 145, 557 and 224-fold, respectively. Fmoc polymers resulted in modest drug solubility improvement. These polymers were non-haemolytic, did not enhance cytotoxicity compared to unmodified PAA, and demonstrated significant increase in griseofulvin plasma concentration compared to griseofulvin in water after oral administration., Conclusions: Ch(5) and Dansyl(10) showed promising potential as nano-carriers for oral hydrophobic drug delivery.

Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.

In a recent letter (EPL 97, 36006 (2012)), we have shown that the Leslie thermomechanical coupling cannot alone explain the Lehmann effect (namely the rotation of cholesteric droplets when they are subjected to a temperature gradient). This result was obtained by measuring in a compensated cholesteric mixture the "Lehmann coefficient" as a function of temperature both below and at the transition to the isotropic liquid. In this article, we detail these experiments and present new ones performed with other compensated mixtures and a diluted cholesteric mixture. The new results confirm the macroscopic origin of the Lehmann effect, in contrast to the Leslie thermomechanical effect that is clearly of microscopic origin.

3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol % sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterols. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol % cholesterol), the average lifetime of trans-parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average lifetime of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial.

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The gum resin of Commiphora wightii [(Hook. ex Stocks) Engl.] is an ayurvedic medicine for the treatment of arthritis, inflammation, obesity, lipid disorders, and cardiovascular diseases and is known as guggul. Morphologically, it is not easy to distinguish guggul from closely related gum resins of other plants. Reliability of the commercially available guggul is critical due to the high risk of adulteration. To check authenticity, a commercial guggul sample was investigated for its chemical markers and 17 metabolites were identified, including three new, 20(S),21-epoxy-3-oxocholest-4-ene (1), 8 β-hydroxy-3,20-dioxopregn-4,6-diene (2), and 5-(13' Z-nonadecenyl)resorcinol (17) from the ethyl acetate soluble part. During the current study, compounds 14- 17 were identified as constituents of Mangifera indica gum, as an adulterant in the commercial guggul sample. This discovery highlighted the common malpractices in the trade of medicinal raw material in the developing world. The structures of the compounds were deduced by the spectroscopic technique and chemical methods, as well as by comparison with the reported data. The structure of 20(S),21-epoxy-3-oxocholest-4-ene (1) was also unambiguously deduced by single-crystal X-ray diffraction technique., (© Georg Thieme Verlag KG Stuttgart · New York.)

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi, Indonesia, together with two known cholic-acid-type compounds, 3-oxochol-1,4-dien-24-oic acid (3) and 3-oxochol-4-en-24-oic acid (4). The structures of these compounds were determined on the basis of their spectroscopic data. The absolute stereochemistry at C-25 of 2 was determined by comparative (1)H NMR study using chiral anisotropic reagents [(S)- and (R)-phenylglycine methyl esters]. This is the first to report compound 1 as a natural product.

The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-Delta(7)-dafachronic acid isomer as the most potent compound, but the (25S)-Delta(4)-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-Delta(7)-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC(50) values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Delta(4)-dafachronic acids, with EC(50) values of 23 and 66 nm, respectively. The (25S)- and (25R)-Delta(5)-isomers were much less potent, with EC(50) values approaching 1000 nm, and saturated 5alpha- and 5beta-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-Delta(7)-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Delta(7)-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25.

Derivatives of 3beta-amino-5-cholestene (3beta-cholesterylamine) are of substantial interest as cellular probes and have potential medicinal applications. However, existing syntheses of 3beta-amino-5-cholestene are of limited preparative utility. We report here a practical method for the stereoselective preparation of 3beta-amino-5-cholestene, 3beta-chloro-5-cholestene, 3beta-bromo-5-cholestene, and 3beta-iodo-5-cholestene from inexpensive cholesterol. A sequential i-steroid/retro-i-steroid rearrangement promoted by boron trifluoride etherate and trimethylsilyl azide converted cholest-5-en-3beta-ol methanesulfonate to 3beta-azido-cholest-5-ene with retention of configuration in 93% yield.

We report a stereoselective synthesis of the (25S)-cholestenoic-26-acids which are highly efficient ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans.

Steriodal heterocyclic systems namely cholest-5-en-3-oxazolo and thiazoloquinoxaline have been synthesized via the reaction of cholest-5-en-3-one semicarbazone/thiosemicarbazone with 2,3-dichloroquinoxaline at 80 degrees C in high yield. Cholest-5-en-3-one semicarbazone is obtained by the condensation of cholest-5-en-3-one with semicarbazide in the presence of AcONa in ethanol and cholest-5-en-one thiosemicarbazone is obtained by the condensation of cholest-5-en-3-one with thiosemicarbazide in ethanol in the presence of a few drops of HCl. The structures of these compounds were evident by elemental analysis, IR, 1H NMR and FAB mass spectral analysis. These synthesized compounds were investigated for antibacterial activity first by the disk-diffusion assay against two Gram-positive and two Gram-negative bacteria and then the minimum inhibitory concentration (MIC) of these compounds were determined and the results were compared with the standard drug Amoxicillin. The results showed that these compounds oxazolo/thiazoloquinoxaline are better antibacterial agents as compared to the standard drug Amoxicillin.

The success of targeting systems to alveolar macrophages critically depends on internalization into these cells for pharmacological intervention. Direct respiratory delivery via inhalation of mannose modified liposomal carriers to alveolar macrophages is of great interest. To evaluate the targeting efficiency to alveolar macrophages by intratracheal administration of mannosylated liposomes (Man-liposomes), Man-liposomes with various ratio of mannosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) as mannose receptor ligand were investigated with regard to their in vitro uptake in primary cultured alveolar macrophages and in vivo intratracheal administration in rats. The in vitro uptake of Man-liposomes took place in a concentration-dependent manner. The internalization of Man-liposomes with 7.5% (Man-7.5-liposomes) and 5.0% (Man-5.0-liposomes) Man-C4-Chol was considerably higher than that of Man-liposomes with 2.5% of Man-C4-Chol (Man-2.5-liposomes) and Bare-liposomes and significantly inhibited by an excess of mannan, suggesting mannose receptor-mediated endocytosis. After intratracheal administration of Man-7.5 and Man-5.0-liposomes in rats, a significantly high internalization and selective targeting to alveolar macrophages was observed. The enhanced cellular uptake in alveolar macrophages related to the mannose density of Man-liposomes was also confirmed both in vitro and in vivo confocal microscopy studies. These results demonstrate the efficient targeting to alveolar macrophages by the intratracheally administered Man-liposomes via mannose receptor-mediated endocytosis.

The petroleum ether, chloroform, ethyl acetate and methanol extracts of the whole plant of Oldenlandia herbacea were screened for in vitro antioxidant activity by using ABTS, DPPH, nitric oxide and hydrogen peroxide assay methods. The chloroform, ethyl acetate and methanol extracts showed potent antioxidant activity against ABTS free radicals. In the hydrogen peroxide method, the methanol and ethyl acetate extracts showed potent activity. Chemical investigation of the petroleum ether extract yielded a long chain hydrocarbon, 9,9-dimethyl hexacosane (1), and a steroid, 23-ethyl-cholest-23-en-3beta-ol (2).

Objective: To search for chemical constituents with structural diversity from Laurencia tristicha to supply for biological assay., Method: Compounds were isolated by means of column chromatography over normal phase silica gel and Sephadex LH-20, recrystallization and HPLC. Structures were identified by spectroscopic methods including 1D NMR, IR and MS. Cytotoxicities of the purified compounds were evaluated by MTT method., Result: Seven compounds were isolated from L. tristicha. Their structures were elucidated as cholesterol (1), cholesta- 5-en-3beta, 7alpha-diol (2), beta-stigmasterol (3), phytol (4), zeaxanthin (5), 4 -hydroxybenzaldehyde (6), indolyl-3-carbaldehyde (7). In the cytotoxic assay compound 2 was active against human cancer cell lines HCT-8, Bel-7402, BGc-823, A549 and HELA with IC50 values of 1.90, 2.02, 1.99, 6.52 and 1.20 microg x mL(-1), respectively. Compound 4 showed cytotoxicity against HCT-8 and HELA with IC50 value of 3.51 and 2.04 microg x mL(-1), and other compounds were inactive ( IC50 > 10 microg x mL(-1))., Conclusion: Compounds 1-7 were isolated from L. tristicha for the first time. In additon, compounds 2 and 4 were cytotoxic against several human cancer cell lines.

The effect of a large increase in mass and extension of substituents on the thermal rearrangement of a 1,2-divinylcyclobutane system has been investigated by the introduction of two 3-cholest-4-ene units. The ratio of two types of exit channel, fragmentation and stereomutation (Fr/St), from a widely accepted diradical intermediary (caldera) has increased significantly relative to the ratio in a simpler system. We believe this to be the first example of the influence of a ponderal effect on the ratio of two competing processes in a thermal rearrangement. The internal torsional rotations necessary prior to reclosure for the realization of stereomutation have been markedly depressed. One of the three stereoisomeric cyclobutanes reacts substantially more slowly than the other two. In its structure, determined by X-ray diffraction, the two cholestenyl wings are folded closely together within van der Waals radii. The slower reaction may reasonably be ascribed to a relative lowering of heat of formation in this ground-state conformation.

Seven sulfated polyhydroxysteroids were isolated from the Far East starfish Pteraster obscurus and the ophiura (snake star) Asteronyx loveni (collected in the Sea of Okhotsk) and characterized: disodium and sodium salts of (20R)-24-methyl-2beta-hydroxycholesta-5,24(28)-diene-3alpha,21-diyl disulfate, (20R)-5alpha-cholestane-3beta,21-diyl disulfate, (20R)-3beta-hydroxy-5alpha-cholestan-21-yl sulfate, (20R)-cholest-5-ene-3beta,21-diyl disulfate, (20R)-2beta-hydroxycholest-5-ene-3alpha,21-diyl disulfate, (20R)-cholest-5-en-3beta-yl sulfate, and (20R)-5alpha-cholestan-3beta-yl sulfate. The first four compounds turned out to be new, whereas the others were identical to the known compounds. Structures of the isolated steroids were identified by two-dimensional NMR spectroscopy and other physicochemical methods. The compounds isolated from starfish are structurally similar to typical ophiuroid metabolites, which support the opinion of some taxonomists that starfish and ophiuroids are phylogenetically related classes.

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

The chemoselectivity of rigid cyclic alpha,beta-unsaturated carbonyl group on the reducing agents was influenced by the ring size and steric factor. Cholesterol (cholest-5-en-3beta-ol) and dehydroepiandrosterone (DHEA) were oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione. They were reduced with NaBH(4), lithium tri-sec-butylborohydride (l-Selectride), LiAlH(4), 9-borabicyclo[3.3.1]nonane (9-BBN), lithium triethylborohydride (Super-hydride), and BH(3) x (CH(3))(2)S in various conditions, respectively. Reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by NaBH(4) (4 equiv.) produced 4,6-cholestadien-3beta-ol and 4,6-androstadiene-3beta,17beta-diol, respectively. Reduction by l-Selectride (12 equiv.) afforded 4,6-cholestadien-3alpha-ol and 4,6-androstadiene-3alpha,17beta-diol, chemoselectively. Reaction with Super-hydride (12 equiv.) produced 4,6-cholestadien-3-one and 3-oxo-4,6-androstadien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by 9-BBN (14 equiv.) produced 1,4,6-cholestatrien-3alpha-ol, but 1,4,6-androstatriene-3,17-dione was not reacted with 9-BBN in the reaction conditions. Reaction of LiAlH(4) (6 equiv.) formed 4,6-cholestadien-3beta-ol and 3-oxo-1,4,6-androstatrien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by BH(3) x (CH(3))(2)S (11 equiv.) gave cholestane as major compound and unlike reactivity of cholesterol, 1,4,6-androstatriene-3,17-dione by 8 equiv. of BH(3) x (CH(3))(2)S formed 3-oxo-1,4,6-androstatrien-17beta-ol. LiAlH(4) and BH(3) x (CH(3))(2)S showed relatively low chemoselectivity.

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).