Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5ß-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5ß-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 µM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5ß-Cholane and 23-Benzoate Function in the Side Chain.

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-5ß-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.

Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs.

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.

Introduction of Nonacidic Side Chains on 6-Ethylcholane Scaffolds in the Identification of Potent Bile Acid Receptor Agonists with Improved Pharmacokinetic Properties.

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.

Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors.

Bile acids exert genomic and nongenomic effects by interacting with membrane G-protein-coupled receptors, including the bile acid receptor GP-BAR1, and nuclear receptors, such as the farnesoid X receptor (FXR). These receptors regulate overlapping metabolic functions; thus, GP-BAR1/FXR dual agonists, by enhancing the biological response, represent an innovative strategy for the treatment of enteroendocrine disorders. Here, we report the design, total synthesis, and in vitro/in vivo pharmacological evaluation of a new generation of dual bile acid receptor agonists, with the most potent compound, 19, showing promising pharmacological profiles. We show that compound 19 activates GP-BAR1, FXR, and FXR regulated genes in the liver, increases the intracellular concentration of cAMP, and stimulates the release of the potent insulinotropic hormone GLP-1, resulting in a promising drug candidate for the treatment of metabolic disorders. We also elucidate the binding mode of the most potent dual agonists in the two receptors through a series of computations providing the molecular basis for dual GP-BAR1/FXR agonism.

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

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

A concise synthesis and antimicrobial activities of 3-polyamino-23,24-bisnorcholanes as steroid-polyamine conjugates.

A series of steroid-polyamine conjugates were synthesized and evaluated for their antimicrobial activity. This study was focused on the effect of stereochemistry at the C-3 and C-5 of steroids and types of polyamine at C-3 on activity against various human pathogens. All the conjugates exhibited strong antimicrobial activities against Gram-positive strains. Compound 18 was found to be the most potent in these series with a MIC value as low as 1 µg/mL against the bacterium Staphylococcus aureus ATCC6538P.

Total synthesis and pharmacological characterization of solomonsterol A, a potent marine pregnane-X-receptor agonist endowed with anti-inflammatory activity.

Recently, we reported the identification of a novel class of pregnane-X-receptor (PXR) agonists, solomonsterols A and B, isolated from the marine sponge Theonella swinhoei. Preliminary pharmacological studies demonstrated that these natural compounds are potential leads for the treatment of human disorders characterized by dysregulation of innate immunity. In this article, we describe the first total synthesis of solomonsterol A and its in vivo characterization in animal models of colitis. Using transgenic mice expressing the human PXR, we found that administration of synthetic solomonsterol A effectively protects against development of clinical signs and symptoms of colitis and reduced the generation of TNFα, a signature cytokine for this disorder. In addition, we have provided the first evidence that solomonsterol A might act by triggering the expression of TGFß and IL-10, potent counter-regulatory cytokines in inflammatory bowel diseases (IBD). Finally, we have shown that solomonsterol A inhibits NF-κB activation by a PXR dependent mechanism. In summary, solomonsterol A is a marine PXR agonist that holds promise in the treatment of inflammation-driven immune dysfunction in clinical settings.

Synthesis of 12-oxa, 12-aza and 12-thia cholanetriols.

An efficient synthesis of 12-hetero steroids was achieved via a Baeyer-Villiger oxidation and a photolysis as the key steps. We set out to describe in this paper the first synthesis of 12-aza steroids. The characteristic (1)H and (13)C NMR spectroscopic features of the synthesized compounds are reported.

Synthesis and spectroscopic data analyses of 5beta-cholane derivatives.

Facile synthesis and spectroscopic data analyses of three 5beta-cholane derivatives, 3alpha-tosyloxy-5beta-cholan-24-ol (3), 5beta-cholan-24-ol (4) and 5beta-cholan-24-yl tosylate (5), are described.

3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate: synthesis and suitability for the study of cholate transport.

In order to facilitate the study of transport processes of unconjugated C-24 bile salts, simple syntheses of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate (norcholansulfonate) and 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-[7 beta 5H] cholan-23-sulfonate were devised. The hydrophilic-hydrophobic properties of norcholansulfonate, as determined by its chromatographic behavior as well as by its partition between l-octanol and water, are more similar to those of cholyltaurine than to those of cholate. Self-association of norcholansulfonate in phosphate buffer, pH 7.4, with an ionic strength of 150 mM begins at a concentration of about 1 mM, comparable to that of cholyltaurine and cholate, as determined by spectral changes in fluorescence emissions of {N-[7-(4-nitrobenzo-2-oxa-1, 3-diazol)]-7b-amino-3a, 12a-dihydroxy-5b-cholan-24 - oyl}-2'-aminoethanesulfonate (7 beta-NBD-NCT). The apparent CMC value obtained from solubilization of the dye Orange OT, 8.5 mM, is comparable to that of cholytaurine. 7.5 mM, and lower than that of cholate, 9.5 mM. Norcholansulfonate is readily taken up by rat liver and completely excreted unmetabolized into bile with about the same secretion maximum (Tm) as cholyltaurine. Biliary excretion of norcholansulfonate is inhibited by cholyltaurine, and, vice versa, norcholansulfonate inhibits cholyltaurine secretion. Concerning metabolism and excretion, norcholansulfonate with the sulfonate group in the position where cholate has the carboxylate group should behave as an appropriate cholate analogue in mediated transport processes.

Potential bile acid metabolites. 19. The epimeric 3 alpha,6,7 beta-trihydroxy- and 3 alpha,6,7 beta,12 alpha-tetrahydroxy-5 alpha-cholanoic acids.

Syntheses by a new procedure of the known 3 alpha,6 alpha,7 beta- and 3 alpha,6 beta,7 beta-trihydroxy-5 alpha-cholanoic acids, and of the once-reported analog 3 alpha,6 alpha,7 beta,12 alpha-, as well as the new 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 alpha-cholanoic acids, are described. Key intermediates of the syntheses are the 6-oxo-7 beta-ols of the respective 5 alpha-cholanoic acids (and their methyl esters) prepared by allomerization at C-5 of appropriate 6-bromo-7-oxo derivatives of the corresponding 5 beta-acids. Successful reduction of the 6,7-ketols to the desired products depended on the proper choice of reagents, either Zn(BH4)2 or Li/NH3/MeOH.

Basic cholane derivatives. XI: Comparison between acid and basic derivatives.

A series of hydroxycholan-24-amines was synthesized; the carboxyl group of starting unconjugated bile acids was transformed into a basic moiety [-NH2, -NHCH3, -N(CH3)2, or -NHCH2C6H5] 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.

Antimicrobial activity of basic cholane derivatives. Part IX.

Twenty new compounds derived from deoxycholic acid have been synthesized. They contain two basic functions: at C-24 (benzylamino, morpholino, diethanolamino, N,N-diethylethylenediamino, N-methylpiperazino) and at beta-C-3 (amino, methylamino, ethylamino, benzylamino). The compounds showed interesting antimicrobial activity, as expressed in terms of the low M.I.C. values (0.9-31-micrograms/ml) against five Gram(+) and four Gram(-) strains, two fungi and one yeast. The compounds inhibit the production of a fluorescent pigment in Pseudomonas aeruginosa: this result suggests that the ability to cross the bacterium cell membrane is the first step of activity. A discussion in terms of structure-activity relationship is reported.

Antimicrobial activity of basic cholane derivatives, VIII.

Two series of new compounds derived from deoxycholic acid have been synthesized: 3,12-dioxo-5 beta-cholan-24-N-substituted amides and their 3 beta-amino and 3 beta-N-alkylamino derivatives. The first series of five compounds 1-5 carries at C-24 the residue of benzylamine, morpholine, diethanolamine, N,N-diethyl-ethylenediamine, and N-methylpiperazine. The second series of twenty compounds 1A-D - 5A-D was prepared by means of reductive amination starting from the compounds of the first series. This reaction proved to be regioselective and stereospecific: it attacks only C-3 of the steroid moiety and introduces the following axial beta-oriented substituents: amino, methylamino, ethylamino, and benzylamino. The compounds of the first series showed moderate scattered antimicrobial activity; while introduction of the 3 beta-amino and 3 beta-N-alkylamino residue greatly increased activity towards Gram (+) strains; even yeast and fungi appear to be sensitive towards this last series of compounds. The results have been discussed with respect to the nature of the substituents both at C-3 and C-24, the highest activity being associated to the hydrophobicity of these residues.

Potential tissue-imaging agents: 23-(trimethyl [117mSn]stannyl)-24-nor-5 alpha-cholan-3 beta-ol.

Tin-117m-labeled 23-(trimethylstannyl)-24-nor-5 alpha-cholan-3 beta-ol (2) has been prepared by reaction of trimethyl [117mSn]tin lithium with 3 beta-acetoxy-23-bromo-24-nor-5 alpha-cholane (1). Tin-117m (2) shows pronounced adrenal uptake (2.5% injected dose) in female rats 1 day after injection. Furthermore, the adrenal to liver (9.1:1) and adrenal to blood (33.7:1) ratios are high after this period. The absorbed radiation dose values from [117mSn]2 to human organs have also been estimated by using rat tissue distribution and excretion data. [117mSn]2 is the first reported tissue-specific organic radiopharmaceutical labeled with this nuclide and may have potential as an adrenal imaging agent.

The synthesis of methyl 3 alpha, 7 alpha-diacetoxy-11-oxo-5 beta-cholan-24-oate.

Reaction of methyl-3 alpha-7 alpha-diacetoxy-11 alpha-bromo-12-oxo-5 beta-cholan-24-oate with sodium borohydride in pyridine solution containing sodium acetate gave the corresponding 11 beta, 12 beta-epoxide in 65% yield. The epoxy-ring was opened with hydrobromic or hydroiodic acid to give the corresponding 12 alpha-halo-11 beta-alcohols, which were converted to the halo-ketones and finally to methyl 3 alpha,7 alpha-diacetoxy-11-oxo-5 beta-cholan-24-oate.