Synthesis and Biological Investigation of Bile Acid-Paclitaxel Hybrids.

Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.

Physiology and Physical Chemistry of Bile Acids.

Bile acids (BAs) are facial amphiphiles synthesized in the body of all vertebrates. They undergo the enterohepatic circulation: they are produced in the liver, stored in the gallbladder, released in the intestine, taken into the bloodstream and lastly re-absorbed in the liver. During this pathway, BAs are modified in their molecular structure by the action of enzymes and bacteria. Such transformations allow them to acquire the chemical-physical properties needed for fulling several activities including metabolic regulation, antimicrobial functions and solubilization of lipids in digestion. The versatility of BAs in the physiological functions has inspired their use in many bio-applications, making them important tools for active molecule delivery, metabolic disease treatments and emulsification processes in food and drug industries. Moreover, moving over the borders of the biological field, BAs have been largely investigated as building blocks for the construction of supramolecular aggregates having peculiar structural, mechanical, chemical and optical properties. The review starts with a biological analysis of the BAs functions before progressively switching to a general overview of BAs in pharmacology and medicine applications. Lastly the focus moves to the BAs use in material science.

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates.

Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities. The category and magnitude of bile salt metabolism can have positive or negative effects on the host. A key modification is bile salt hydrolysis, which is a prerequisite for all additional microbial transformations. We have synthesized five different fluorogenic bile salts for simple and continuous reporting of hydrolysis in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced primary bile salts, but some microbially modified secondary bile salts also display significant turnover.

Facial Amphiphilicity-Induced Polymer Nanostructures for Antimicrobial Applications.

New antimicrobial agents are needed to address ever-increasing antimicrobial resistance and a growing epidemic of infections caused by multidrug resistant pathogens. We design nanostructured antimicrobial copolymers containing multicyclic natural products that bear facial amphiphilicity. Bile acid based macromolecular architectures of these nanostructures can interact preferentially with bacterial membranes. Incorporation of polyethylene glycol into the copolymers not only improved the colloidal stability of nanostructures but also increased the biocompatibility. This study investigated the effects of facial amphiphilicity, polymer architectures, and self-assembled nanostructures on antimicrobial activity. Advanced nanostructures such as spheres, vesicles, and rod-shaped aggregates are formed in water from the facial amphiphilic cationic copolymers via supramolecular interactions. These aggregates were particularly interactive toward Gram-positive and Gram-negative bacterial cell membranes and showed low hemolysis against mammalian cells.

Overcoming environmental problems of biocides: Synthetic bile acid derivatives as a sustainable alternative.

Marine biofouling represents a global economic and ecological challenge. Some marine organisms produce bioactive metabolites, such as steroids, that inhibit the settlement and growth of fouling organisms. The aim of this work was to explore bile acids as a new scaffold with antifouling (AF) activity by using chemical synthesis to produce a series of bile acid derivatives with optimized AF performance and understand their structure-activity relationships. Seven bile acid derivatives were successfully synthesized in moderate to high yields, and their structures were elucidated through spectroscopic methods. Their AF activities were tested against both macro- and microfouling communities. The most potent bile acid against the settlement of Mytilus galloprovincialis larvae was the methyl ester derivative of cholic acid (10), which showed an EC50 of 3.7 µM and an LC50/EC50 > 50 (LC50 > 200 µM) in AF effectiveness vs toxicity studies. Two derivatives of deoxycholic acid (5 and 7) potently inhibited the growth of biofilm-forming marine bacteria with EC50 values < 10 µM, and five bile acids (1, 5, and 7-9) potently inhibited the growth of diatoms, showing EC50 values between 3 and 10 µM. Promising AF profiles were achieved with some of the synthesized bile acids by combining antimacrofouling and antimicrofouling activities. Initial studies on the incorporation of one of these promising bile acid derivatives in polymeric coatings, such as a marine paint, demonstrated the ability of these compounds to generate coatings with antimacrofouling activity.

C-12 vs C-3 substituted bile salts: An example of the effects of substituent position and orientation on the self-assembly of steroid surfactant isomers.

Biomolecule derivatives are transversally used in nanotechnology. Deciphering their aggregation behavior is a crucial issue for the rational design of functional materials. To this end, it is necessary to build libraries of selectively functionalized analogues and infer general rules. In this work we enrich the highly applicative oriented collection of steroid derivatives, by reporting a rare example of C-12 selectively modified bile salt. While nature often exploits such position to encode functions, it is unusual and not trivial to prepare similar analogues in the laboratory. The introduction of a tert-butyl phenyl residue at C-12 provided a molecule with a self-assembly that remarkably switched from rigid pole-like structures to twisted ribbons at a biologically relevant critical temperature (∼25 °C). The system was characterized by microscopy and spectroscopy techniques and compared with the C-3 functionalized analogue. The twisted ribbons generate samples with a gel texture and a viscoelastic response. The parallel analysis of the two systems suggested that the observed thermoresponsive self-assemblies occur at similar critical temperatures and are probably dictated by the nature of the substituent, but involve aggregates with different structures depending on position and orientation of the substituent. This study highlights the self-assembly properties of two appealing thermoresponsive systems. Moreover, it adds fundamental insights hereto missing in the investigations of the relation between self-assembly and structure of synthetic steroids, which are valuable for the rational design of steroidal amphiphiles.

A continuous fluorescence assay for simple quantification of bile salt hydrolase activity in the gut microbiome.

The microbiota of the mammalian gut plays a dynamic role in controlling host physiology. The effect of gut microbiota activity on host health is particularly evident in the case of bile homeostasis. Bile is produced by the host and is modified by the gut microbiota, which impacts the net hydrophobicity of the total bile acid pool, and also modulates host signaling pathways. A key mechanism by which the microbiota modify bile is through deconjugation of bile salts through bile salt hydrolase (BSH) enzymatic activity, which is postulated to be a prerequisite for all further microbial metabolism. BSH activity in the gut is largely considered to be beneficial for the host, and genes encoding BSHs are found in the genomes of many taxa found in over-the-counter probiotics. Despite the therapeutic relevance of this enzyme, there is no sensitive and simple assay for continuous monitoring of BSH activity, and there are no non-destructive means of characterizing its activity in whole cell or microbial community samples. Herein, we describe a continuous fluorescence assay that can be used for characterization of BSH activity with purified protein, cell lysates, whole cells, and in human gut microbiome samples. The method is a "turn-on" reporter strategy, which employs synthetic substrates that yield a fluorescent product upon BSH-dependent turnover. This assay is used to show the first in vivo characterization of BSH activity. We also demonstrate continuous, non-destructive quantification of BSH activity in a human fecal microbiome sample containing recombinant BSH.

Discovery of ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureidyl derivatives as selective non-steroidal agonists of the G-protein coupled bile acid receptor-1.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRß and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Solvent-Free Synthesis of 2-Alkylbenzothiazoles and Bile Acid Derivatives Containing Benzothiazole Ring by Using Active Carbon/Silica Gel and Microwave.

A highly efficient and simple method for the synthesis of 2-alkylbenzothiazoles through the condensation of 2-aminothiophenol and aliphatic aldehydes in the presence of active carbon/silica gel is described. The reaction proceeded under solvent-free and microwave irradiation to afford 2-alkylbenzothiazoles in high yields. This method was extended to the synthesis of bile acid derivatives containing a benzothiazole ring and obtained the desired products in high yields. The anti-inflammatory activity and cytotoxicity of the newly synthesized benzothiazole derivatives of bile acid were tested; the results showed that anti-inflammatory activities of all tested compounds tested were higher than that of standard drugs, such as indomethacin.

Clickable conjugates of bile acids and nucleosides: Synthesis, characterization, in vitro anticancer and antituberculosis studies.

A series of clickable bile acid-nucleosides conjugates linked directly or via amino acid linker were synthesized, and characterized by spectroscopic techniques such as 1H NMR, 13C NMR, FT-IR, HRMS and HPLC. The synthesized compounds 6a-p were screened for their in vitro anticancer property against a panel of three cancer cell lines (PC-3, MCF-7, IMR-32). In addition, the synthesized derivatives were also tested for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC 27294 strain). Among the screened compounds, cholic acid-uridine clicked conjugate (6c), and cholic acid-uridine clicked conjugate liked via phenylalanine moiety (6m) were found to be most active against MCF-7 and IMR-32 exhibiting an IC50 value of 8.084 and 8.71 µM, respectively. The antimycobacterial study of the synthesized conjugates revealed all the conjugates to be active with MIC values in the range of 4.09-15.41 µM. Deoxycholic acid-adenosine clicked conjugate (6b) showed most promising antituberculosis property with MIC value of 4.09 µM. Most of the synthesized conjugates were found to be safe at 50 µM against normal human embryonic kidney (HEK 293 T) cell line.

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC50 value of 1.8 µM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.

Evaluating Hepatobiliary Transport with 18F-Labeled Bile Acids: The Effect of Radiolabel Position and Bile Acid Structure on Radiosynthesis and In Vitro and In Vivo Performance.

Introduction: An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET-) imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods: A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7ß-[18F]FCA; 12ß-[18F]FCA); a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3ß-[18F]FGCA and 3ß-[18F]FCDCA, resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n = 3 per group): hepatobiliary transport was monitored and compared to a reference tracer, namely, 3ß-[18F]FCA. Results: Compounds 3α-[18F]FCA, 3ß-[18F]FGCA, and 3ß-[18F]FCDCA were synthesized in moderate radiochemical yields (4-10% n.d.c.) and high radiochemical purity (>99%); 7ß-[18F]FCA and 12ß-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3ß-FGCA, and 3ß-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3ß-[18F]FGCA, and 3ß-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3ß-[18F]FCA epimers. Conjugation of 3ß-[18F]FCA with glycine had no significant effect in vivo. Compound 3ß-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion: A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as PET-biomarkers for hepatobiliary transport of bile acids.

Novel Semisynthetic Derivatives of Bile Acids as Effective Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

An Important task in the treatment of oncological and neurodegenerative diseases is the search for new inhibitors of DNA repair system enzymes. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of the DNA repair system enzymes involved in the removal of DNA damages caused by topoisomerase I inhibitors. Thus, reducing the activity of Tdp1 can increase the effectiveness of currently used anticancer drugs. We describe here a new class of semisynthetic small molecule Tdp1 inhibitors based on the bile acid scaffold that were originally identified by virtual screening. The influence of functional groups of bile acids (hydroxy and acetoxy groups in the steroid framework and amide fragment in the side chain) on inhibitory activity was investigated. In vitro studies demonstrate the ability of the semisynthetic derivatives to effectively inhibit Tdp1 with IC50 up to 0.29 µM. Furthermore, an excellent fit is realized for the ligands when docked into the active site of the Tdp1 enzyme.

Chemical Synthesis of Rare Natural Bile Acids: 11α-Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids.

A method for the preparation of 11α-hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α-mesyloxy group of the methyl esters of 3α-acetate-12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △11 -3α-acetoxy and △11 -3α,7α-diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α-hydroxylation of the △11 -3α,24-diol and △11 -3α,7α,24-triol intermediates with B2 H6 /tetrahydrofuran (THF); and (4) selective oxidation at C-24 of the resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the corresponding C-24 carboxylic acids with NaClO2 catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5ß-cholan-24-oic acid and 3α,7α,11α-trihydroxy-5ß-cholan-24-oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.

Synthesis and evaluation of bile acid amides of [Formula: see text]-cyanostilbenes as anticancer agents.

A series of amino-substituted [Formula: see text]-cyanostilbene derivatives and their bile acid (cholic and deoxycholic acid) amides were designed and synthesized. A comparative study on the anticancer and antibacterial activity evaluation on the synthesized analogs was carried against the human osteosarcoma (HOS) cancer cell line, and two gram -ve (E. coli and S. typhi) and two gram [Formula: see text]ve (B. subtilis and S. aureus) bacterial strains. All the cholic acid [Formula: see text]-cyanostilbene amides showed an [Formula: see text] in the range 2-13 [Formula: see text] against human osteosarcoma cells (HOS) with the most active analog (6g) possessing an [Formula: see text] of [Formula: see text]. One of the amino-substituted [Formula: see text]-cyanostilbene, 4e, was found to possess an [Formula: see text] of [Formula: see text]. An increase in the number of cells at the sub-[Formula: see text] phase of the cell was observed in the in vitro cell cycle analysis of two most active compounds in the series (4e, 6g) suggesting a clear indication toward induction of apoptotic cascade. With respect to antibacterial screening, amino-substituted [Formula: see text]-cyanostilbenes were found to be more active than their corresponding bile acid amides. The synthesized compounds were also subjected to in silico study to predict their physiochemical properties and drug-likeness score.

Polymeric bile acid sequestrants: Review of design, in vitro binding activities, and hypocholesterolemic effects.

Polymeric bile acid sequestrants (BAS) have recently attracted much attention as lipid-lowering agents. These non-absorbable materials specifically bind bile acids (BAs) in the intestine, preventing bile acid (BA) reabsorption into the blood through enterohepatic circulation. Therefore, it is important to understand the structure-property relationships between the polymer sequestrant and its ability to bind specific BAs molecules. In this review, we describe pleiotropic effects of bile acids, and we focus on BAS with various molecular architectures that result in different mechanisms of BA sequestration. Here, we present 1) amphiphilic polymers based on poly(meth)acrylates, poly(meth)acrylamides, polyalkylamines and polyallylamines containing quaternary ammonium groups, 2) cyclodextrins, and 3) BAS prepared via molecular imprinting methods. The synthetic approaches leading to individual BAS preparation, as well as results of their in vitro BA binding activities and in vivo lipid-lowering activities, are discussed.

Synthesis and anticancer activity of bile acid dendrimers with triazole as bridging unit through click chemistry.

Triazole-based novel dendrimers with bile acid surface groups have been synthesized through click chemistry by divergent approach and characterized by spectral data. All the dendrimers exhibit excellent anticancer activity. Higher-generation dendrimers exhibit better anticancer activity than the lower-generation dendrimers.

Synthesis and Biological Evaluation of Bile Acid Analogues Inhibitory to Clostridium difficile Spore Germination.

Standard antibiotic-based strategies for the treatment of Clostridium difficile infections disrupt indigenous microbiota and commonly fail to eradicate bacterial spores, two key factors that allow recurrence of infection. As an alternative approach to controlling C. difficile infection, a series of bile acid derivatives have been prepared that inhibit taurocholate-induced spore germination. These analogues have been evaluated in a highly virulent NAP1 strain using optical density and phase-contrast microscopy assays. Heterocycle substitutions at C24 were well-tolerated and several tetrazole-containing derivatives were highly potent inhibitors in both assays, with complete inhibition of spore germination observed at 10-25 µM. To limit intestinal absorption, C7-sulfated analogues designed to avoid active and passive transport pathways were prepared. One of these derivatives, compound 21b, was found to be a potent inhibitor of C. difficile spore germination and poorly permeable in a Caco-2 model of intestinal epithelial absorption, suggesting that it is likely to be gut-restricted.

Improved synthesis of glycine, taurine and sulfate conjugated bile acids as reference compounds and internal standards for ESI-MS/MS urinary profiling of inborn errors of bile acid synthesis.

Bile acid synthesis defects are rare genetic disorders characterized by a failure to produce normal bile acids (BAs), and by an accumulation of unusual and intermediary cholanoids. Measurements of cholanoids in urine samples by mass spectrometry are a gold standard for the diagnosis of these diseases. In this work improved methods for the chemical synthesis of 30 BAs conjugated with glycine, taurine and sulfate were developed. Diethyl phosphorocyanidate (DEPC) and diphenyl phosphoryl azide (DPPA) were used as coupling reagents for glycine and taurine conjugation. Sulfated BAs were obtained by sulfur trioxide-triethylamine complex (SO3-TEA) as sulfating agent and thereafter conjugated with glycine and taurine. All products were characterized by NMR, IR spectroscopy and high resolution mass spectrometry (HRMS). The use of these compounds as internal standards allows an improved accuracy of both identification and quantification of urinary bile acids.

Bile Salt Stabilized Vesicles (Bilosomes): A Novel Nano-Pharmaceutical Design for Oral Delivery of Proteins and Peptides.

With the advent of novel vesicular drug delivery systems especially bilosomes, for large molecular weight proteins and peptides, their oral administration seems a viable approach. These nano-vesicles have shown promising results for the effective delivery of insulin and other therapeutics, perhaps due to their structural composition. The present review has elaborated the biopharmaceutical challenges for the oral delivery of therapeutic proteins and peptides as well as presented a novel approach to deliver the essential macromolecules through oral route as bilosomes. The extensive search has been presented related to the formulation, evaluation and in vivo performance of bilosomes. Some of the crucial findings related to bilosomes have corroborated them superior to other colloidal carriers. The successful drug delivery through bilosomes requires significant justifications related to their interaction with the biological membranes. The other aspects such as absolute absorption, safety and toxicity of bilosome drug delivery should also be equally considered.