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Natural brassinosteroids possess a 22R, 23R configuration that appears essential for biological activity. It is, therefore, interesting to elucidate if the activity of brassinosteroids with a short side chain depends on the C22 configuration. Herein, we describe the synthesis of new brassinosteroids analogs with 24-norcholane type of side chain and R configuration at C22. The initial reaction is the dihydroxylation of a terminal olefin that leads to S/R epimers. Three different methods were tested in order to evaluate the obtained S/R ratio and the reaction yields. The results indicate that Upjohn dihydroxylation is the most selective reaction giving a 1.0:0.24 S/R ratio, whereas a Sharpless reaction leads to a mixture of 1.0:0.90 S/R with 95% yield. Using the latter mixture and following a previous reported method, benzoylated derivatives and both S and R brassinosteroids analogs were synthesized. All synthesized compounds were completely characterized by NMR spectroscopy, and HRMS of new compounds are also given. In conclusion, a synthetic route for preparation of new analogs of brassinosteroids of 24-norcholane type and R configuration at C22 were described. It is expected that this will help to elucidate if a configuration at C22 is a structural requirement for hormonal growth activity in plants.

Reactions of tetracyanoethylated pregnenolone with ammonia and amines in anhydrous medium at reduced temperature afforded new ionic pregnenolone derivatives, ammonium (aminium) 23,24,24-tricyano- 3β-hydroxy-20-oxo-21-nor-17β-cholane-5,21-dien-24-ides.

Brassinosteroids are polyhydroxysteroids that are involved in different plants’ biological functions, such as growth, development and resistance to biotic and external stresses. Because of its low abundance in plants, much effort has been dedicated to the synthesis and characterization of brassinosteroids analogs. Herein, we report the synthesis of brassinosteroid 24-nor-5β-cholane type analogs with 23-benzoate function and 22,23-benzoate groups. The synthesis was accomplished with high reaction yields in a four-step synthesis route and using hyodeoxycholic acid as starting material. All synthesized analogs were tested using the rice lamina inclination test to assess their growth-promoting activity and compare it with those obtained for brassinolide, which was used as a positive control. The results indicate that the diasteroisomeric mixture of monobenzoylated derivatives exhibit the highest activity at the lowest tested concentrations (1 × 10−8 and 1 × 10−7 M), being even more active than brassinolide. Therefore, a simple synthetic procedure with high reaction yields that use a very accessible starting material provides brassinosteroid synthetic analogs with promising effects on plant growth. This exploratory study suggests that brassinosteroid analogs with similar chemical structures could be a good alternative to natural brassinosteroids.

Cu- and Pd-catalyzed arylation of aminocholanes has been described for the first time. While this Cu-catalyzed protocol provides high yields in reactions of aminocholanes with iodoarenes, Pd catalysis was found to be preferable for the reactions of aminocholanes with dichloroanthraquinones. UV–vis titration of bis(cholanylamino)anthraquinones with a series of cations demonstrated their high binding affinity to Cu2+, Al3+, and Cr3+.

The G-protein coupled receptor of bile acids GPBAR1 is a bile acid receptor that plays an important role in regulating innate immunity, glucose homeostasis and energy expenditure representing an interesting target for the treatment of metabolic and degenerative diseases. A selective control of its activity over the other bile acid-activated receptors is desirable to reduce unwanted effects due to activation of multiple downstream signals regulated by diverse receptors. Here, we report the design and the synthesis of a small series of bile acid derivatives with their side chains decorated with an epoxide ring. We demonstrate that all the compounds selectively activate GPBAR1 in cell-based assays and regulate the expression of pro-glucagon, a canonical GPBAR1 targeted gene in an intestinal endocrine cell line, while have no effect on FXR, LXRα and LXRβ. Finally, we elucidate the binding mode of the most potent compound of this family through molecular modeling studies. Our study contributes to increase the arsenal of bile acid derivatives serving as GPBAR1 modulators, widening the chemical space that can be exploited in drug design.

Steroids are natural compounds with several important applications in many fields of research, such as medicinal chemistry, pharmacology, supramolecular chemistry and nanotechnology.In particular, bile acids such as lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) have been considered quite useful as starting points for a rich and different set of medicinal chemistry activities. Besides, the discovery of bioactive ingredients from plants and fungi is always the main target in medicinal chemistry. The lanostane-type triterpenoid 3b-hydroxylanosta-8,24-diene-21-oic acid (Trametenolic acid, TMA) was the main bioactive component of Gloeophyllum odoratum, which was reported to possess widely bioactivities, including tumor cell anti-proliferation effects (for example, human HL-60 leukemia, human KB epidermoid carcinoma, murine L1210 leukemia cells, Caski, HT-3, T-24, etc.), inhibition of enzyme activity (human thrombin, bovine trypsin and so on).Nevertheless, trametenolic acid was scarcely investigated as antimicrobial agent. Structurally, bile acids (LCA and UDCA) and trametenolic acid are similar since they may be regarded as consisting of two components, a rigid steroid nucleus and an aliphatic side chain possessing a carboxyl group. On the basis of these considerations, six new compounds bearing a guanidine moiety in their side chain were synthesized using LCA, UDCA and TMA as starting materials. The parent bile acids, TMA and their resulting derivatives were evaluated for antimicrobial activity against S. aureus, B. subtilis and M. smegmatis. The derivative 3a-hydroxy-23-guanidino-5b-cholane showed the best activity, with MIC values of 12.5 μM against S. aureus, 5 μM against B. subtilis and 50 μM against M. smegmatis. The cytotoxic activity of bile acids, trametenolic acid and derivatives was also evaluated against HT-29 cell line

As a cellular bile acid sensor, farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) participate in maintaining bile acid, lipid, and glucose homeostasis. To date, several selective and dual agonists have been developed as promising pharmacological approach to metabolic disorders, with most of them possessing an acidic conjugable function that might compromise their pharmacokinetic distribution. Here, guided by docking calculations, nonacidic 6-ethyl cholane derivatives have been prepared. In vitro pharmacological characterization resulted in the identification of bile acid receptor modulators with improved pharmacokinetic properties.

The copper-catalyzed azide–alkyne cycloaddition reaction was used for the preparation of a series of bis- and tris-3- and 24-5β-cholanetriazolyl derivatives of phosphorus acids, which contained anion-binding triazolium sites and hydrophobic cholane residues. The influence of the location of the triazolium moiety (in 3α-, 3β-position) on fluoride-ion complexation was investigated. The anion-binding properties of tris(triazolium) ligands were studied for a series of inorganic and organic anions and high complexation constants were observed with fluoride, hydrosulfate, and benzoate anions.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chin)]; (b) mPEG molecular weight (2 kDa mPEG and 5 kDa mPEG(114)); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG(114))(2)-DSPE confers the highest stealth properties to liposomes (similar to 31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG(114)-Chol had 3.2- and similar to 2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG(114))(2)-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG(114)-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG(114)-DSPE was lower than that obtained with liposomes decorated with linear mPEG(114)-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.

Large conductance, voltage- and Ca2+-gated K+ (BKCa) channels play a critical role in smooth muscle contractility and thus represent an emerging therapeutic target for drug development to treat vascular disease, gastrointestinal, bladder and uterine disorders. Several compounds are known to target the ubiquitously expressed BKCa channel-forming α subunit. In contrast, just a few are known to target the BKCa modulatory β1 subunit, which is highly expressed in smooth muscle and scarce in most other tissues. Lack of available high-resolution structural data makes structure-based pharmacophore modeling of β1 subunit-dependent BKCa channel activators a major challenge. Following recent discoveries of novel BKCa channel activators that act via β1 subunit recognition, we performed ligand-based pharmacophore modeling that led to the successful creation and fine-tuning of a pharmacophore over several generations. Initial models were developed using physiologically active cholane steroids (bile acids) as template. However, as more compounds that act on BKCa β1 have been discovered, our model has been refined to improve accuracy. Database searching with our best-performing model has uncovered several novel compounds as candidate BKCa β1 subunit ligands. Eight of the identified compounds were experimentally screened and two proved to be activators of recombinant BKCa β1 complexes. One of these activators, sobetirome, differs substantially in structure from any previously reported activator.

Reactions of 3-oxo derivatives of cholic acid esters with acetone cyanohydrin and trimethylsilyl cyanide lead to exclusive or predominant formation of the corresponding 3-cyano-3-hydroxy and 3-cyano-3-trimethylsiloxy derivatives with axial orientation of the cyano group. Reduction of the cyano group in the addition products gives 3-aminomethyl derivatives which can be used to obtain pincer ligands containing two cholane fragments.

Star block copolymers composed of hydrophilic poly(ethylene glycol) (PEG) as an inner block and tuneable poly(allyl glycidyl ether) (PAGE) as an outer block were made by sequential anionic polymerization on a cholane core. The 4-armed CA(EG n - b -AGE m ) 4 star block copolymers show dual thermo-responsive behavior in aqueous solutions, where the two cloud points (CPs) vary in the range of 10–34 °C for the first CP and 16−39 °C for the second CP, depending on the length of the PEG and PAGE segments. 1 H NMR spectroscopy, dynamic light scattering and atomic force microscopy all indicate a two-step aggregation process of flower-like micelles, where the micelles form through the association of the hydrophobic PAGE blocks and the bile acid core. The allyl groups of the star copolymers were further functionalized by radical thiol-ene coupling to introduce NH 2 or COOH groups in the outer block. As a result, star-shaped block copolymers with dual pH- and thermo-sensitivity were obtained with different aggregation characteristics corresponding to the pK a of the functionalized blocks. Interestingly, the deprotonated NH 2 -bearing polymers showed dual thermo-responsive characteristics different from their allyl group-bearing precursor.

Tweezer-type ionophores containing C3-carbamoylpropanamidoacetoxy and C12-dithiocarbamoyl groups on a 7-deoxycholic amide or cholane derivative were designed and synthesized. A representative 1H NMR titration study indicated that newly synthesized ionophores form 1:1 complexes with the Ca2 + ion. © 2012 Wiley Periodicals, Inc. Heteroatom Chem 23:187–194, 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/hc.21002

Proton and carbon resonances in NMR spectra of a number of derivatives of 3β-hydroxy-5α-cholanes, zymosterol synthesis intermediates, have been completely assigned using 2D NMR spectroscopy methods. The stereochemistry of the chiral centers and the structures of the molecules have been confirmed.

Palladium-catalyzed reactions of 24-(haloaryloxy)cholanes and 3,24-bis(haloaryloxy)cholanes with excess oxadiamines gave the corresponding mono- and bis(oxadiamino)-substituted cholanes which were subjected to palladium-catalyzed arylation with 1,3-dibromobenzene, 2,6-dibromopyridine, 1,8-dichloroanthracene, 9-bromoanthracene, 1-chloroanthracene, and 1-chloroanthraquinone. The results of these reactions were found to strongly depend on the haloarene nature. The arylation with 1,3-dibromobenzene and 2,6-dibromopyridine led to the formation of new macrocyclic compounds containing one cholane, one arene, and two oxadiamine fragments.

Four bile acid-triazine conjugates: N2′,N2′-dimethyl-6′-(3α-hydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (lithocholyl triazine, 4a), N2′,N2′-dimethyl-6′-(3α,7α-dihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (chenodeoxycholyl triazine, 4b), N2′,N2′-dimethyl-6′6′-(3α,12α-dihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (deoxycholyl triazine) (4c), and N2′,N2′-dimethyl-6′-(3α,7α,12α-trihydroxy-5β-24-norcholyl)-1′,3′,5′-triazine-2′,4′-diamine (cholyl triazine) (4d) have been prepared and characterized by liquid and solid state NMR. An improved synthetic method produced better yields and an easier purification procedure for 4d than reported in the literature. Single crystal structure of 4a is reported: empirical formula C28H47N5O, monoclinic P21 space group with unit cell dimensions, a 18.7135(5) A, b 7.4510(2) A, c 19.3073(5) A, β 95.7290(10)°, volume 2678.65(12) A3.

A series of 3-polyaminochenodeoxycholic acid derivatives were synthesized and their in vitro antimicrobial and antifungal activities were assessed. The antimicrobial activity was evaluated against Gram-positive and Gram-negative bacteria, and the antifungal activity was assessed against two strains Candida albicans (ATCC MYA-1003) and Aspergillus fumigatus (ATCC 16424). The introduction of an amine group to steroid was accomplished by reductive amination of 3-oxosteroid 9 with Boc-spermidine and Boc-spermine, in the presence of NaBH(OAc)3. This afforded a high yield of 3-polyaminosteroids of 10–13. The 3β-sperminyl-5β-cholane 6 showed the highest antimicrobial activity against Streptococcus pyogenes 308A, Staphylococcus aureus 503, Escherichia coli DC2 and Pseudomonas aeruginosa 9027 with a MIC value of 3.13 μg/mL.

By Mitsunobu reaction from 3,24-cholanediol and 2-hydroxy-8-chloroquinoline 24-(8-chloroquinolinyl-2-oxy)cholan-3-ol and 3,24-di(8-chloroquinolinyl-2-oxy)cholane were synthesized. These compounds were brought into reactions of palladium-catalyzed amination with propanediamine, oxaalkane diamines, and N,N′-bis(3-aminopropyl)ethylenediamine to obtain macrocycles of various structures, linear mono- and bis(steroid) derivatives of trioxaalkane diamine, and also cholane bis(oxaalkandiamine) derivative. The dependence was demonstrated of macrocyles and cyclooligomers yield on the polyamine nature. 4-Hydroxy-7-chloroquinoline afforded only 24-(7-chloroquinolinyl-4-oxy)cholan-3-ol.

Tweezer-type ionophores containing C3-bipyridyl and C12-dithiocarbamoyl groups, or C3-bithiophenyl with C12-dithiocarbamoyl groups on a 7-deoxycholic amide or cholane derivatives were designed and synthesized. Potentiometric evaluation of the PVC membranes containing those deoxycholic amides/cholanes linked with unsymmetrically substituted tweezer-type bipyridyl or bithiophenyl with dithiocarbamoyl moieties proved their good affinity and selectivity for silver(I) ion. Especially, ionophores with bithiophenyl moiety on the 3α-position and diphenylaminothioxomethylthioacetyloxy group on 12α-position of cholan-24-amide or cholane reveal the most excellent result with a theoretical slope value of 59 mV/decade.

A diastereomeric mixture (55:45) of 3-oxo-23,24-dinorchol-4-en-22-hydroxy acids (4A and 4B) was synthesized from 3-oxo-23,24-dinorchol-4-en-22-al (1) in 3 steps. Similarly, 3-oxo-5β-cholane-24-hydroxy acid (9) was synthesized from 5β-cholane-3α,24-diol (5) in 4 steps. Full spectroscopic data for these compounds are presented.

3,24-Bis(6-chloropyridin-2-yloxy)cholane, obtained from 3,24-cholandiol via a Mitsunobu reaction, was successfully used for the synthesis of a variety of polyazamacrocycles using Pd-catalyzed amination reaction with linear polyamines. The competing formation of cyclodimers and other cyclic oligomers was found to be strongly dependent on the nature of the polyamines employed.

TGR5 (Gpbar-1, M-Bar) is a class A G-protein coupled bile acid-sensing receptor predominately expressed in brain, liver and gastrointestinal tract, and a promising drug target for the treatment of metabolic disorders. Due to the lack of a crystal structure of TGR5, the development of TGR5 agonists has been guided by ligand-based approaches so far. Three binding mode models of bile acid derivatives have been presented recently. However, they differ from one another in terms of overall orientation or with respect to the location and interactions of the cholane scaffold, or cannot explain all results from mutagenesis experiments. Here, we present an extended binding mode model based on an iterative and integrated computational and biological approach. An alignment of 68 TGR5 agonists based on this binding mode leads to a significant and good structure-based 3D QSAR model, which constitutes the most comprehensive structure-based 3D-QSAR study of TGR5 agonists undertaken so far and suggests that the binding mode model is a close representation of the "true" binding mode. The binding mode model is further substantiated in that effects predicted for eight mutations in the binding site agree with experimental analyses on the impact of these TGR5 variants on receptor activity. In the binding mode, the hydrophobic cholane scaffold of taurolithocholate orients towards the interior of the orthosteric binding site such that rings A and B are in contact with TM5 and TM6, the taurine side chain orients towards the extracellular opening of the binding site and forms a salt bridge with R79(EL1), and the 3-hydroxyl group forms hydrogen bonds with E169(5.44) and Y240(6.51). The binding mode thus differs in important aspects from the ones recently presented. These results are highly relevant for the development of novel, more potent agonists of TGR5 and should be a valuable starting point for the development of TGR5 antagonists, which could show antiproliferative effects in tumor cells.

Starting from readily available lithocholic acid, a number of new cholane derivatives have been prepared consisting of the same or different electron‐donor groups at the 3‐ and 24‐positions. Yields ranged from good to excellent. The donor groups were chosen on the basis of their known ability to coordinate to transition‐metal ions and include pyridyl, 1,3‐dithiolanyl, phenylthioether, and oxime ether groups.

Aqueous gels derived from three cationic 24-nor 3,12-dihydroxy cholane (DC) derivatives with N-methyl-2-pyrrolidinone (NMP), N-methylmorpholine (NMM), and 1,4-diazabicyclo[2.2.2]octane (DABCO) at the side chain positions have been exhaustively characterized by small-angle neutron-scattering experiments. Although the molecular structures differ slightly by the heterocycle grafted to the steroid core, the derived gels exhibit a range of structural behaviors at the nanoscale that depart from those observed with simple deoxycholate systems. The NMM-DC aggregates are ribbons with a bimolecular thickness of t = 37 A and an anisotropy of the section b/a approximately 0.1. DABCO-DC exhibits a remarkable transition from ribbons (t = 29.5 A, b/a = 0.18) to thicker cylindrical fibers (R approximately 59 A), involving four original ribbons, upon a concentration increase. The NMP-DC system forms thick cylindrical fibers (R approximately 68 A) with steroid molecules organized in a specific morphology. Bilayered or interdigited structures are formed and favored by the presence of multiple polar interaction centers in the DC molecules. Secondary aggregation mechanisms are invoked in the formation of bundles having a lower cross-sectional anisotropic symmetry and exhibiting Bragg peaks corresponding to molecular length periodicities. The relations between the structural information and the rheological properties are discussed.

Tweezer-type and non-tweezer-type ionophores containing dithiocarbamoyl groups on a 7-deoxycholic amide or cholane derivatives were designed and synthesized. Potentiometric evaluation of the poly(vinyl chloride) (PVC) membranes containing those deoxycholic amides/cholanes linked with tweezer-type dithiocarbamoyl moieties showed excellent affinity and selectivity to silver(I) ion over alkali, alkaline earth and other transition metal cations. On the other hand, deoxycholic amides/cholanes substituted with one dithiocarbamoyl group, i.e., non-tweezer-type ionophores, resulted in relatively poor potentiometric sensitivity and detection limits. The enhanced potentiometric properties of newly synthesized tweezer-type dithiocarbamoyl containing ionophores have been further improved by employing silver ion complexing reagent in the internal reference solution, which resulted in greatly reduced detection limit ( approximately 100ppt) for the electrodes based on them.

The convenient synthesis of seven dinorcholane and 5β-cholane derivatives, 3β-acetoxy-23,24-dinorchol-5-en-22-oyl methoxy anhydride (2), 3β-acetoxy-23,24-dinorchol-5-en-22-oyl chloride (3), 3β-acetoxy-23,24-dinorchol-5-en-22-ol (4), 3α-acetoxy-5β-cholan-24-oic acid (6), 3α-acetoxy-5β-cholan-24-oic acid ethyl ester (7), 3-oxo-5β-cholan-24-oic acid (8) and 3-oxo-5β-cholan-24-oic acid ethyl ester (9) have been described. Full spectroscopic data for these compounds are presented.

Background & Aims In cholestatic syndromes, body accumulation of bile acids is thought to cause itching. However, the mechanisms supporting this effect remain elusive. Recently, GPBAR1 (TGR5) a G-protein coupled receptor has been shown to mediate itching caused by intradermal administration of DCA and LCA. 6α-ethyl-3α, 7α-dihydroxy-24-nor-5β-cholan-23-ol (BAR502) is a non-bile acid dual ligand for FXR and GPBAR1. Methods Cholestasis was induced in wild type and GPBAR1-/- mice by administration of α-naphthyl-isothiocyanate (ANIT) or 17α-ethynylestradiol. Results. In naive mice skin application of DCA, TLCA, 6-ECDCA, oleanolic and betulinic acid induces a GPBAR1 dependent pruritogenic response that could be desensitized by re-challenging the mice with the same GPBAR1 agonist. In wild type and GPBAR1-/- mice cholestasis induced by ANIT fails to induce spontaneous itching and abrogates scratching behavior caused by intradermal administration of DCA. In this model, co-treatment with BAR502 increases survival, attenuates serum alkaline phosphatase levels and robustly modulates the liver expression of canonical FXR target genes including OSTα, BSEP, SHP and MDR1, without inducing pruritus. Betulinic acid, a selective GPBAR1 ligand, failed to rescue wild type and GPBAR1-/- mice from ANIT cholestasis but did not induced itching. In the 17α-ethynylestradiol model BAR502 attenuates cholestasis and reshapes bile acid pool without inducing itching. Conclusions The itching response to intradermal injection of GPBAR1 agonists desensitizes rapidly and is deactivated in models of cholestasis, explain the lack of correlation between bile acids levels and itching severity in cholestatic syndromes. In models of non-obstructive cholestasis, BAR502 attenuates liver injury without causing itching.

Calcium/voltage-gated, large conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4, LTB4, LTC4, LTD4, and LTE4) are inflammatory lipid mediators. We performed patch clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory β1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nm-10 μm to either leaflet of cell-free membranes at a wide range of [Ca(2+)]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50 = 1 nm; Emax reached at 10 nm), with physiological [Ca(2+)]i and voltages favoring this activation. Homomeric cbv1 or cbv1-β2 channels were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK β1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate β1 subunit-containing channels when β1 carried T169A, A176S, or K179I within the docking site. Co-application of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, probably preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on a channel accessory subunit. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK β1. Moreover, the identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of β1-specific BK channel activators.

Nuclear and G-protein coupled receptors are considered major targets for drug discovery. FXR and GP-BAR1, two bile acid-activated receptors, have gained increasing consideration as druggable receptors. Because endogenous bile acids often target both receptor families, the development of selective ligands has been proven difficult, exposing patients to side effects linked to an unwanted activation of one of the two receptors. In the present study, we describe a novel library of semisynthetic bile acid derivatives obtained by modifications on the cholane scaffold. The pharmacological characterization of this library led to the discovery of 7α-hydroxy-5β-cholan-24-sulfate (7), 6β-ethyl-3α,7β-dihydroxy-5β-cholan-24-ol (EUDCOH, 26), and 6α-ethyl-3α, 7α-dihydroxy-24-nor-5β-cholan-23-ol (NorECDCOH, 30) as novel ligands for FXR and GP-BAR1 that might hold utility in the treatment of FXR and GP-BAR1 mediated disorders.

The 7-coordinate complex [Fe(qpy)(MeCN)2](ClO4)2 (1, qpy = 2,2':6',2″:6″,2''':6''',2''''-quinquepyridine) is a highly active nonheme iron catalyst for intra- and intermolecular amination of C(sp(3))-H bonds. This complex effectively catalyzes the amination of limiting amounts of not only benzylic and allylic C(sp(3))-H bonds of hydrocarbons but also the C(sp(3))-H bonds of cyclic alkanes and cycloalkane/linear alkane moieties in sulfamate esters, such as those derived from menthane and steroids cholane and androstane, using PhI═NR or "PhI(OAc)2 + H2NR" [R = Ts (p-toluenesulfonyl), Ns (p-nitrobenzenesulfonyl)] as nitrogen source, with the amination products isolated in up to 93% yield. Iron imide/nitrene intermediates [Fe(qpy)(NR)(X)](n+) (CX, X = NR, solvent, or anion) are proposed in these amination reactions on the basis of experimental studies including ESI-MS analysis, crossover experiments, Hammett plots, and correlation with C-H bond dissociation energies and with support by DFT calculations. Species consistent with the formulations of [Fe(qpy)(NTs)2](2+) (CNTs) and [Fe(qpy)(NTs)](2+) (C) were detected by high-resolution ESI-MS analysis of the reaction mixture of 1 with PhI═NTs (4 equiv). DFT calculations revealed that the reaction barriers for H-atom abstraction of cyclohexane by the ground state of 7-coordinate CNTs and ground state of C are 15.3 and 14.2 kcal/mol, respectively, in line with the observed high activity of 1 in catalyzing the C-H amination of alkanes under mild conditions.

BK channels regulate many physiological processes and thus constitute an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel-forming α subunit and cannot selectively target a particular tissue. Here, we performed a structure-activity relationship study of lithocholic acid (LCA), a cholane that was previously shown to activate BK channels via a steroid site in the accessory BK β1 subunit (Bukiya et al., 2011). This protein is highly abundant in smooth muscle but scarce in most other tissues (Brenner et al., 2000). Modifications to the LCA lateral chain length and functional group yielded a total of 14 compounds. Bath application of these LCA analogs (1-300 μM) to the cytosolic side of Xenopus oocyte membrane patches co-expressing BK channel-forming cbv1 and β1 subunits demonstrates that channel activation (EC50∼50 μM) requires the combination of a critical lateral chain length (optimal at C24), rather small volume of its functional group (65-92 A3), and net negative charge (−0.1 to −0.9 [e]). Thus, analogs having cyano or nitro groups substituting for the carboxyl at C24 in the LCA molecule constitute two novel BK channel activators that satisfy the aforementioned criteria. Data provide detailed structural information that helps us to better understand ligand recognition by the cholane steroid site present in the BK β1 protein, and will be useful to advance a pharmacophore in search of β1 subunit-selective BK channel activators. These compounds are expected to evoke smooth muscle relaxation, which would be beneficial in the pharmacotherapy of prevalent human disorders associated with increased smooth muscle contraction, such as systemic hypertension, cerebral or coronary vasospasm, bronchial asthma, bladder hyperactivity, and erectile dysfunction.Support: R01-HL104631; R37-AA011560 (AMD).

This paper describes a method for the chemical synthesis of 7α,12α-dihydroxy-4-cholesten-3-one (1a) and its biological precursor, 7α-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3α-hydroxy group of the steroidal A/B-ring to the desired 4-en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man.

Les populations de langues cholanes ont eu longtemps une situation centrale, tant géographiquement que culturellement, au sein de l’aire maya. Le paysage actuel présente une situation bien différente : avec une répartition géographique devenue périphérique et une démographie réduite, deux des trois langues cholanes « survivantes » sont déjà classées parmi les langues en danger par l’Unesco. L’objectif de cet article est de rendre compte des principales phases dans l’évolution des frontières spatiales des populations cholanes à travers leurs histoires et de donner quelques clefs pour comprendre leurs mouvements. Une telle étude aurait certes pu se contenter de données historiques, mais des considérations d’ordres ethnologique et linguistique permettent d’affiner les paramètres nécessaires à la caractérisation de ces frontières et d’apprécier les interrelations de ces populations entre elles et avec l’« extérieur ».Chol-language populations long held a central place in the Maya area, both geographically and culturally. The present landscape presents a quite different situation: with a geographic distribution that has become peripheral and a reduced demography, two of the three “surviving” Chol languages have already been classified as endangered by Unesco. The aim of this article is describe the principal phases of the evolution of the spatial boundaries of Chol populations through their stories, and to provide a few keys to understanding their movements. A study of this kind could certainly have been limited to historical data, but ethnological and linguistic observations make it possible to refine the parameters needed for the characterization of these boundaries, and to appreciate the interrelations these populations maintain with each other and with the “outer” world.

Les populations de langues cholanes ont eu longtemps une situation centrale, tant géographiquement que culturellement, au sein de l’aire maya. Le paysage actuel présente une situation bien différente : avec une répartition géographique devenue périphérique et une démographie réduite, deux des trois langues cholanes « survivantes » sont déjà classées parmi les langues en danger par l’Unesco. L’objectif de cet article est de rendre compte des principales phases dans l’évolution des frontières spatiales des populations cholanes à travers leurs histoires et de donner quelques clefs pour comprendre leurs mouvements. Une telle étude aurait certes pu se contenter de données historiques, mais des considérations d’ordres ethnologique et linguistique permettent d’affiner les paramètres nécessaires à la caractérisation de ces frontières et d’apprécier les interrelations de ces populations entre elles et avec l’« extérieur ». Chol-language populations long held a central place in the Maya area, both geographically and culturally. The present landscape presents a quite different situation: with a geographic distribution that has become peripheral and a reduced demography, two of the three “surviving” Chol languages have already been classified as endangered by Unesco. The aim of this article is describe the principal phases of the evolution of the spatial boundaries of Chol populations through their stories, and to provide a few keys to understanding their movements. A study of this kind could certainly have been limited to historical data, but ethnological and linguistic observations make it possible to refine the parameters needed for the characterization of these boundaries, and to appreciate the interrelations these populations maintain with each other and with the “outer” world.

PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5β-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5 ± 1.8 to 60.2 ± 2.4 μM as the PEG molecular weight increased. Similarly, light scattering analysis showed that the micelle size increased from 15.8 ± 4.9 to 23.2 ± 11.1 nm with the PEG molecular weight. Gel permeation studies showed that the polymer bioconjugates associate with recombinant human granulocyte colony stimulating factor (rh-G-CSF) to form supramolecular nanocomposites according to multi-modal association profiles. The protein loadings obtained with PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane were 7.4 ± 1.1, 2.7 ± 0.3 and 2.1 ± 0.4% (protein/polymer, w/w %), respectively. Scatchard and Klotz analyses showed that the protein/polymer affinity constant increased and that the number of PEG-cholane molecules associated to rh-G-CSF decreased as the PEG molecular weight increased. Isothermal titration calorimetry confirmed the protein/polymer multi-modal association. Circular dichroism analyses showed that the polymer association alters the secondary structure of the protein. Nevertheless, in vitro studies performed with NFS-60 cells showed that the polymer interaction does not impair the biological activity of the cytokine. In vivo studies performed by intravenous and subcutaneous administrations of rh-G-CSF to rats showed that the association with PEG(5 kDa)-cholane prolongs the body exposure of the protein. After subcutaneous administration, the protein t(max) values obtained with rh-G-CSF and 1:14 and 1:21 rh-G-CSF/PEG(5 kDa)-cholane (w/w ratio) nanocomplexes were 2, 8 and 24h, respectively. The 1:21 (w/w) rh-G-CSF/PEG(5kDa)-cholane formulation resulted in 149% relative bioavailability, and the pharmacokinetic behaviour was similar to that obtained with an equivalent protein dose of rh-G-CSF chemically conjugated with one linear 20-kDa PEG. A single administration of a 1.5mg/kg dose of a 1:21 (w/w) rh-G-CSF/PEG(5 kDa)-cholane formulation induced a high production of white blood cells for 96 h.

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2× increase in vascular myocyte BK activity (EC 50 = 45 μM), an action that requires β1 but not other (β2–β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK β1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit–specific pharmacology of BK channels.

The human apical sodium-dependent bile acid transporter (hASBT) may serve as a prodrug target for oral drug absorption. Synthetic, biological, NMR, and computational approaches identified the structure-activity relationships of mono- and dianionic bile acid conjugates for hASBT binding. Experimental data combined with a conformationally sampled pharmacophore/QSAR modeling approach (CSP-SAR) predicted that dianionic substituents with intramolecular hydrogen bonding between hydroxyls on the cholane skeleton and the acid group on the conjugate's aromatic ring increased conjugate hydrophobicity and improved binding affinity. Notably, the model predicted the presence of a conformational molecular switch, where shifting the carboxylate substituent on an aromatic ring by a single position controlled binding affinity. Model validation was performed by effectively shifting the spatial location of the carboxylate by inserting a methylene adjacent to the aromatic ring, resulting in the predicted alteration in binding affinity. This work illustrates conformation as a determinant of ligand physiochemical properties and ligand binding affinity to a biological transporter.

This report describes the chemical synthesis of a new bile acid analogue, namely, sodium 3 alpha, 7 alpha-dihydroxy-25-homo-5 beta-cholane-25-sulfonate from homochenodeoxycholic acid. The structure of the new compound was assigned by proton magnetic resonance and infrared spectrometry. Its metabolism was studied in the hamster in comparison with sodium 3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholane-23-sulfonate and sodium taurochenodeoxycholate. After intraduodenal administration of the 3H-labeled analogues into bile fistula hamsters, both sulfonates were absorbed from the intestine and nearly 80% of the radioactivity was secreted into bile within 8 h. Intra-ileal administration revealed that these compounds resembled taurochenodeoxycholate in that they were much more rapidly absorbed from the ileum than from the proximal small intestine: more than 85% of the radioactivity was recovered in bile within 1 h. After intravenous infusion the sulfonates were efficiently extracted by the liver at rates similar to that of sodium taurochenodeoxycholate. Chromatographic analysis of the bile showed that, regardless of the route of administration, most (> 95%) of the sulfonates were not biotransformed and they became major biliary bile acids. Sodium 3 alpha, 7 alpha-dihydroxy-25-homo-5 beta-cholane-25-sulfonate and, to a lesser extent, sodium 3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholane-23-sulfonate induced cholestasis at infusion rates at which sodium taurochenodeoxycholate produced choleresis.

A series of hydroxycholan-24-amines was synthesized; the carboxyl group of starting unconjugated bile acids was transformed into a basic moiety [NH 2 , NHCH 3 , N(CH 3 ) 2 , or NHCH 2 C 6 H 5 ] at C-24. Solubilities, acidities, partition coefficients, and critical micellar concentrations were measured and compared with those of the corresponding bile acids. Because the steroid nucleus in the amines is the same as that in the bile acids, most of the physical-chemical properties of the two compound classes were similar. The amines were more soluble than the corresponding acids; solubilities depended mainly on the number of steroid hydroxyls and, to a lesser extent, on the side chain. Amines are strong bases in water, whereas unconjugated bile acids can be classified as weak acids. N -Benzylamino derivatives have higher log P (P is partition coefficient) values, as a consequence of the bulky hydrophobic substituent; the log P values were almost the same for the amines and the bile acids and depended on the steroid hydroxyls. Amines can self-aggregate at an acidic pH and form cationic micelles; the critical micellar concentrations of amines were of the same order of magnitude as those of bile acids. The introduction of a basic function in the side chain of the cholane moiety increased the antimicrobial activity toward most gram-positive strains.

A trimer of cholic acid was prepared via a simple isocyanate linkage. Fluorescence and UV-vis spectroscopic studies showed that the same cholane trimer can form either a hydrophobic cavity in aqueous solutions or a hydrophilic cavity in an apolar solvent using pyrene and a derivative of pyrene as probes. The dynamic inversion of the cavities was demonstrated for the first time by a simple change of the polarity of the solvent media by adjusting from an aqueous buffer to tetrahydrofuran and vice versa. The NMR study of a dimer model compound of cholic acid indicated a restricted conformation exist in the solvent mixture of chloroform and methanol.

By Mitsunobu reaction from 3,24-cholanediol and 2-hydroxy-8-chloroquinoline 24-(8-chloroquinolinyl-2-oxy)cholan-3-ol and 3,24-di(8-chloroquinolinyl-2-oxy)cholane were synthesized. These compounds were brought into reactions of palladium-catalyzed amination with propanediamine, oxaalkane diamines, and N,N′-bis(3-aminopropyl)ethylenediamine to obtain macrocycles of various structures, linear mono- and bis(steroid) derivatives of trioxaalkane diamine, and also cholane bis(oxaalkandiamine) derivative. The dependence was demonstrated of macrocyles and cyclooligomers yield on the polyamine nature. 4-Hydroxy-7-chloroquinoline afforded only 24-(7-chloroquinolinyl-4-oxy)cholan-3-ol.

3,24-Bis(6-chloropyrdin-2-yloxy)cholane prepared from cholane-3,24-diol by the Mitsunobu reaction was successfully used to synthesize various polyaza macrocycles via palladium-catalyzed amination with linear polyamines. The contribution of side formation of cyclic oligomers was found to depend on the polyamine nature.

Purple membrane bacteriorhodopsin can be easily solubilized by Triton X-100 and other detergents, but not by deoxycholate. In order to understand this behavior, we have examined the effects of a variety of surfactants. We show that detergents containing the cholane ring (cholate, taurocholate, 3[(3-cholamidopropyl)diethyl-ammonio]propanesulfonic acid...) are virtually unable to solubilize native bacteriorhodopsin. However, when the protein is reconstituted in dimyristoyl phosphatidylcholine and solubilization is assayed at a temperature such that bacteriorhodopsin is in the form of monomers, solubilization by cholane detergents does occur. We propose that steric factors prevent access of the rigid planar surfactant molecules to the hydrophobic protein regions. These are perhaps located in the monomer-monomer interface, whose solvation by surfactants is essential for solubilization to occur. We note that the capacity of some detergents to solubilize bacteriorhodopsin is always associated within the same range of surfactant concentrations with bleaching (partial or total) of the protein chromophore. The detergent-induced bleaching is at least partially reversible, suggesting that free retinal remains associated to some membrane components. While some surfactant molecules remain tightly bound to the membrane protein, cholane detergents can be completely removed from bacteriorhodopsin. Our results indicate that a structure-function relationship exists for detergents applied to the solubilization of bacteriorhodopsin.

A simple and convenient route to 3 alpha- and 3 beta-amino-5 beta-cholan-24-oic acids was developed via Leuckart-Wallach amination reduction and subsequent acid hydrolysis. Two epimeric formylamino derivatives were produced (alpha and beta), approximately in a 1:1 ratio, as determined by 13C nuclear magnetic resonance spectroscopy. The two isomers were separated by making use of their different solubilities in ethyl ether. The absolute configuration of the two amino acids was assigned by comparison with authentic reference samples.

Physiological and pharmacological agents that activate large conductance, voltage-, and calcium-gated potassium (BK) channels located in the smooth muscle are effective vasodilators. Thus, activators of smooth muscle BK channels may be potential therapeutic tools to treat cardiovascular disease associated with vasoconstriction and/or impaired dilation, such as cerebrovascular spasm and constriction. We previously showed that lithocholic acid (LC) and other cholane derivatives activated smooth muscle BK channels and, thus, caused endothelium-independent cerebral artery dilation. However, clinical use of these cholane derivatives could be limited by the actions of these steroids, such as elevation of intracellular calcium and induction of apoptosis. Using LC as template, we designed and synthesized a series of hydroxy-alkynoic acids and corresponding methyl esters, as putative, non-steroid BK channel activators. Indeed, the newly synthesized compounds effectively and reversibly activated rat cerebrovascular myocyte BK channel at concentrations similar to those found effective with LC. Among all the novel compounds tested, C-10 hydroxy-alkynoic acid methyl ester appears to be the most effective activator of vascular myocyte BK channels.

This report describes the chemical synthesis of two new bile salt analogues, namely sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-sulfonate and sodium 3 alpha,7 beta-dihydroxy-5 beta-cholane-24-sulfonate from chenodeoxycholic acid and ursodeoxycholic acid, respectively. Each common bile acid was converted into the corresponding 5 beta-cholane-3,7,24-triol by treatment with ethyl chloroformate in the presence of triethylamine followed by sodium borohydride reduction. Reaction of the cholanetriol with p-toluenesulfonyl chloride at 4 degrees C afforded the partially tosylated product, 24-p-toluenesulfoxy-5 beta-cholane-3,7-diol, which was then treated with sodium iodide to produce 24-iodo-5 beta-cholane-3,7-diol. The 24-iodide was refluxed with sodium sulfite in aqueous ethanol to give the desired sulfonate analogue of the naturally occurring bile acid, chenodeoxycholic acid or ursodeoxycholic acid.

The present study was undertaken to determine the inhibitory effects of cholane compounds, ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) on the development of morphine-induced tolerance and physical dependence, and also to determine the hepatic glutathione contents. UDCA and CDCA inhibited the development of morphine-induced tolerance and physical dependence significantly. UDCA inhibited the hepatic glutathione decrease induced by morphine multiple injections, while this effect was not observed in CDCA treated mice. It was throught that the inhibitiory effects of hepatic glutathione decrease in morphine-treated mice by UDCA and CDCA showed a tendency of inhibitory effects of development of morphine tolerance and dependence.