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.

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.

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.

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.

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.

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.

Navigation in bile acid chemical space: discovery of novel FXR and GPBAR1 ligands.

Bile acids are signaling molecules interacting with nuclear receptors and membrane G-protein-coupled receptors. Among these receptors, the farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) have gained increasing consideration as druggable receptors and their exogenous dual regulation represents an attractive strategy in the treatment of enterohepatic and metabolic disorders. However, the therapeutic use of dual modulators could be associated to severe side effects and therefore the discovery of selective GPBAR1 and FXR agonists is an essential step in the medicinal chemistry optimization of bile acid scaffold. In this study, a new series of 6-ethylcholane derivatives modified on the tetracyclic core and on the side chain has been designed and synthesized and their in vitro activities on FXR and GPBAR1 were assayed. This speculation resulted in the identification of compound 7 as a potent and selective GPBAR1 agonist and of several derivatives showing potent dual agonistic activity.

Synthesis, characterization and biological evaluation of bile acid-aromatic/heteroaromatic amides linked via amino acids as anti-cancer agents.

A series of bile acid (Cholic acid and Deoxycholic acid) aryl/heteroaryl amides linked via α-amino acid were synthesized and tested against 3 human cancer cell-lines (HT29, MDAMB231, U87MG) and 1 human normal cell line (HEK293T). Some of the conjugates showed promising results to be new anticancer agents with good in vitro results. More specifically, Cholic acid derivatives 6a (1.35 µM), 6c (1.41 µM) and 6m (4.52 µM) possessing phenyl, benzothiazole and 4-methylphenyl groups showed fairly good activity against the breast cancer cell line with respect to Cisplatin (7.21 µM) and comparable with respect to Doxorubicin (1 µM), while 6e (2.49µM), 6i (2.46 µM) and 6m (1.62 µM) showed better activity against glioblastoma cancer cell line with respect to both Cisplatin (2.60 µM) and Doxorubicin (3.78 µM) drugs used as standards. Greater than 65% of the compounds were found to be safer on human normal cell line.

Mutational mapping of the transmembrane binding site of the G-protein coupled receptor TGR5 and binding mode prediction of TGR5 agonists.

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.

Design and synthesis of 11α-substituted bile acid derivatives as potential anti-tuberculosis agents.

We have synthesized a series of novel 11α-triazoyl bile acid derivatives. In addition, we also have synthesized N-alkyl and N-acyl derivatives of C-11 amino bile acid esters. All the compounds were evaluated for the inhibitory activity against Mycobacterium tuberculosis H37Ra (MTB) at 30 µg/mL level. Four lead compounds (2b, 3, 7 and 8) were further confirmed from their dose dependent effect against MTB. These compounds were found to be active against Dormant and active stage MTB under both in vitro as well as within THP1 host macrophages. The most promising compound 2b showed strong antitubercular activities against MTB under in vitro and ex vivo (IC90 value of ∼3 µg/mL) conditions and almost insignificant cytotoxicity up to 100 µg/mL against THP-1, A549 and PANC-1 human cancer cell lines. Inactivity of all these compounds against Gram positive and Gram negative bacteria indicates their specificity. Molecular docking studies of these compounds into the active site of DprE1 enzyme revealed a similar binding mode to native ligands in the crystal structure thereby helping to establish a structural basis of inhibition of MTB. The synthesized compounds were analyzed for ADME properties and showed potential to develop good oral drug candidates. Our results clearly indicate the identification of some novel, selective and specific inhibitors against MTB that can be explored further for potential antitubercular drug.

Synthesis and Evaluation of Bile Acid-Ribavirin Conjugates as Prodrugs to Target the Liver.

Ribavirin is used to treat hepatitis C but causes serious hemolytic anemia. The objective of the study was to develop a ribavirin prodrug to achieve liver-specific drug delivery and to reduce its off-target effect in red blood cells (RBCs). The approach aimed to target the human sodium taurocholate cotransporting polypeptide (NTCP), which is a bile acid transporter predominately expressed in the liver. Six prodrugs with ribavirin conjugation at C-3 or C-24 of the bile acids were synthesized. In vitro uptake studies indicated that all six prodrugs were NTCP substrates. Metabolic studies in vitro indicated that ribavirin-l-Val-glycochenodeoxycholic acid (GCDCA) was able to release ribavirin in the mouse liver S9 fraction. Additionally, in vitro studies showed that ribavirin in RBC was reduced by 16.7-fold from prodrug compared with parent drug incubation. Moreover, almost no prodrug was present in RBC. In vivo study in mice also showed that ribavirin-l-Val-GCDCA could provide almost the same ribavirin exposure in the liver as ribavirin administration, but with about 1.8-fold less exposure of ribavirin in RBC, plasma, and kidney. Overall, the study suggested that ribavirin-l-Val-GCDCA has the potential to achieve ribavirin-specific liver delivery.

Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines.

We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116 whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity and that the α-hydroxyl group was found to be more beneficial at the 7-position in steroid skeleton.

Synthesis of atypical bile acids for use as investigative tools for the genetic defect of 3ß-hydroxy-Δ(5)-C27-steroid oxidoreductase deficiency.

Deficiency of 3ß-hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7), an enzyme catalyzing the second step in the pathway for bile acid synthesis, leads to a complete lack of the primary bile acids, cholic and chenodeoxycholic acids, and the accumulation of 3ß,7α-dihydroxy- and 3ß,7α,12α-trihydroxy-Δ(5)-cholenoic acids. Patients affected by this autosomal recessive genetic defect develop cholestatic liver disease that is clinically responsive to primary bile acid therapy. Reference standards of these compounds are needed to facilitate diagnosis and to accurately quantify biochemical responses to therapy. Described are a novel synthesis of atypical bile acids that characterize the HSD3B7 deficiency and their effect on bile acid-activated nuclear receptors, target genes and cytochromes involved in bile acid homeostasis and detoxification. The failure of 3ß-hydroxy-Δ(5)-cholenoic acids to function as FXR, PXR and CAR agonists and to exert hepatoprotective actions explains the mechanism for progressive cholestatic liver disease in patients with HSD3B7 deficiency.

Synthesis and haemolytic activity of novel salts made of nicotine alkaloids and bile acids.

A series of novel salts made of nicotine alkaloids and bile acids were synthesized and their haemolytic activity was examined in vitro using human erythrocytes. All compounds were characterized by spectroscopic methods. The novel salts show membrane-perturbing properties inducing the erythrocyte shape alterations and haemolysis in dose-dependent manner. Nicotine decreases the membrane interacting potential of bile acids in the novel compounds. The presence of sulfur or selenium atom in the nicotine molecule affects the haemolytic activity of its novel salts depending on the hydrophobicity of bile acids.

Design and characterization of a novel fluorinated magnetic resonance imaging agent for functional analysis of bile Acid transporter activity.

PURPOSE: To synthesize a trifluorinated bile acid that can be used for (19)F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and (19)F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice. METHODS: Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150 mg/kg or 300 mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t = 5 h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50 mg/kg CA-lys-TFA. RESULTS: CA-lys-TFA was a potent substrate of hASBT (Kt = 39.4 µM, normalized Vmax = 0.853) and hNTCP (Kt = 8.99 µM, normalized Vmax = 0.281). (19)F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4-7 h. CONCLUSIONS: These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction.

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.

Shortcut access to peptidosteroid conjugates: building blocks for solid-phase bile acid scaffold decoration by convergent ligation.

We present three versatile solid-supported scaffold building blocks based on the (deoxy)cholic acid framework and decorated with handles for further derivatization by modern ligation techniques such as click chemistry, Staudinger ligation or native chemical ligation. Straightforward procedures are presented for the synthesis and analysis of the steroid constructs. These building blocks offer a new, facile and shorter access route to bile acid-peptide conjugates on solid-phase with emphasis on heterodipodal conjugates with defined spatial arrangements. As such, we provide versatile new synthons to the toolbox for bile acid decoration.

Bile acid toxicity structure-activity relationships: correlations between cell viability and lipophilicity in a panel of new and known bile acids using an oesophageal cell line (HET-1A).

The molecular mechanisms and interactions underlying bile acid cytotoxicity are important to understand for intestinal and hepatic disease treatment and prevention and the design of bile acid-based therapeutics. Bile acid lipophilicity is believed to be an important cytotoxicity determinant but the relationship is not well characterized. In this study we prepared new azido and other lipophilic BAs and altogether assembled a panel of 37 BAs with good dispersion in lipophilicity as reflected in RPTLC RMw. The MTT cell viability assay was used to assess cytotoxicity over 24 h in the HET-1A cell line (oesophageal). RMw values inversely correlated with cell viability for the whole set (r2=0.6) but this became more significant when non-acid compounds were excluded (r2=0.82, n=29). The association in more homologous subgroups was stronger still (r2>0.96). None of the polar compounds were cytotoxic at 500 microM, however, not all lipophilic BAs were cytotoxic. Notably, apart from the UDCA primary amide, lipophilic neutral derivatives of UDCA were not cytotoxic. Finally, CDCA, DCA and LagoDCA were prominent outliers being more toxic than predicted by RMw. In a hepatic carcinoma line, lipophilicity did not correlate with toxicity except for the common naturally occurring bile acids and their conjugates. There were other significant differences in toxicity between the two cell lines that suggest a possible basis for selective cytotoxicity. The study shows: (i) azido substitution in BAs imparts lipophilicity and toxicity depending on orientation and ionizability; (ii) there is an inverse correlation between RMw and toxicity that has good predictive value in homologous sets; (iii) lipophilicity is a necessary but apparently not sufficient characteristic for BA cytocidal activity to which it appears to be indirectly related.