Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.

The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.

Design, synthesis and evaluation of side-chain hydroxylated derivatives of lithocholic acid as potent agonists of the vitamin D receptor (VDR).

A high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists.

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy Derivatives of Chenodeoxycholic and Lithocholic Acids.

The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is reported. For initiating the synthesis of the 9α-hydroxy derivative of chenodeoxycholic acid, cholic acid was used; for the synthesis of the 9α-hydroxy derivative of lithocholic acid, deoxycholic acid was used. The principal reactions involved were (1) decarbonylation of conjugated 12-oxo-Δ(9(11))-derivatives using in situ generated monochloroalane (AlH2Cl) prepared from LiAlH4 and AlCl3, (2) epoxidation of the deoxygenated Δ(9(11))-enes using m-chloroperbenzoic acid catalyzed by 4,4'-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Markovnikov 9α-hydroxylation of the Δ(9(11))-enes with AlH2Cl, and (4) selective oxidation of the primary hydroxyl group at C-24 in the resulting 3α,9α,24-triol and 3α,7α,9α,24-tetrol to the corresponding C-24 carboxylic acids using sodium chlorite (NaClO2) in the presence of a catalytic amount of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The (1)H- and (13)C-NMR spectra are reported. The 3α,7α,9α-trihydroxy-5ß-cholan-24-oic acid has been reported to be present in the bile of the Asian bear, and its 7-deoxy derivative is likely to be a bacterial metabolite. These bile acids are now available as authentic reference standards, permitting their identification in vertebrate bile acids.

Synthesis and biological evaluation of 4,4-dimethyl lithocholic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of both insulin and leptin signals. For years, inhibiting of PTP1B has been considered to be a potential therapeutics for treating Type 2 diabetes and obesity. Recently, we recognized lithocholic acid (LCA) as a natural inhibitor against PTP1B (IC(50)=12.74 µM) by a vertical screen for the first time. Further SAR research was carried out by synthesizing and evaluating a series of compounds bearing two methyls at C-4 position and a fused heterocycle to ring A. Among them, compound 14b achieved a PTP1B inhibitory activity about eightfold than LCA and a 14-fold selectivity over the homogenous enzyme TCPTP.

Synthesis and proteasome inhibition of lithocholic acid derivatives.

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC(50) = 1.9 µM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.

A novel sialyltransferase inhibitor AL10 suppresses invasion and metastasis of lung cancer cells by inhibiting integrin-mediated signaling.

Aberrant sialylation catalyzed by sialyltransferases (STs) is frequently found in cancer cells and is associated with increased cancer metastasis. However, ST inhibitors developed till now are not applicable for clinical use because of their poor cell permeability. In this study, a novel ST inhibitor AL10 derived from the lead compound lithocholic acid identified in our previous study is synthesized and the anti-cancer effect of this compound is studied. AL10 is cell-permeable and effectively attenuates total sialylation on cell surface. This inhibitor shows no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of alpha-2,3-ST-overexpressing A549 and CL1.5 human lung cells. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of various integrin molecules and attenuated activation of the integrin downstream signaling mediator focal adhesion kinase. More importantly, AL10 significantly suppresses experimental lung metastasis in vivo without affecting liver and kidney function of experimental animals as determined by serum biochemical assays. Taken together, AL10 is the first ST inhibitor, which exhibits potent anti-metastatic activity in vivo and may be useful for clinical cancer treatment.

Conjugation of bile esters to cellulose by olefin cross-metathesis: A strategy for accessing complex polysaccharide structures.

Bile salts tend to form micelles in aqueous media and can thereby contribute to drug solubilization; they also exhibit crystallization inhibition properties that can stabilize supersaturated drug solutions. Herein, we explore conjugation of bile salts with polysaccharides to create new, amphiphilic polysaccharide derivatives with intriguing properties, portending broad utility in various applications. We introduce efficient conjugation of cholesterol (as a model steroid), lithocholic acid, and deoxycholic acid by mild, modular olefin cross-metathesis reactions. These small molecules were first modified with an acrylate group from the A-ring hydroxyl, then reacted with cellulose derivatives bearing olefin-terminated metathesis "handles". Successful conjugation of bile acids has demonstrated chemoselective cross-metathesis with complex, polyfunctional structures, and large multi-ring systems. It also enabled an efficient, general pathway for polysaccharide-bile salt conjugates, which promise synergy for applications such as amorphous solid dispersion (ASD).

In-vitro and in-vivo difference in gene delivery by lithocholic acid-polyethyleneimine conjugate.

Polyethyleneimine (PEI) is widely used for the delivery of nucleic acids, but its clinical application is limited due to high cytotoxicity and instability in biological fluids. To overcome these challenges, linear PEI (2.5 kDa) was modified with lithocholic acid (LCA) to produce a LCA-PEI conjugate (lp), and its complex with plasmid DNA (pDNA) was covered with hyaluronic acid (HA). Ternary complexes of pDNA, lp, and HA ("DlpH") were prepared in different ratios and tested in cells and tumor-bearing mice for gene transfection efficiency. DlpH with a relatively high lp/pDNA ratio (Hi-DlpH) was more resistant to DNase and heparin treatment and showed more efficient gene transfection than DlpH with a lower lp/pDNA ratio (Lo-DlpH) in vitro. In contrast, Hi- and Lo-DlpH showed distinct transfection efficiency in vivo in a tumor-size dependent manner, where Hi-DlpH showed relatively high gene transfection in tumors of <300 mm3 but performed poorly in tumors of >500 mm3 and Lo-DlpH did the opposite. Tumor-associated macrophages, which increase with tumor growth and preferentially intercept Hi-DlpH, may account for the poor performance of Hi-DlpH in relatively large tumors. Accordingly, suggestions are made for future in vitro screening of new gene formulations to better predict their in vivo performances.

Computational study of new 1,2,3-triazole derivative of lithocholic acid: Structural aspects, non-linear optical properties and molecular docking studies as potential PTP 1B enzyme inhibitor.

We have reported synthesis of a novel 1,2,3-triazole conjugate of lithocholic acid by 1,3-dipolar cycloaddition reaction. The molecular properties such as geometry, conformations, bond lengths and dihedral angles were investigated theoretically. The bond order analysis was performed using Wiberg bond order (WBO), Fuzzy bond order (FBO) and Laplacian bond order (MBO) method. Electronic properties of molecule such as electrostatic surface potential analysis, frontier molecular orbital analysis, reduced density gradient, total density of states, and global chemical reactivity indices have been investigated. The nonlinear optical properties were also investigated. Total dipole moment, mean polarizability and hyperpolarizability were found to be much higher than standard urea molecule which suggests that it could act as potential NLO material. The molecular docking calculations are also performed to investigate its potential as PTP 1B enzyme inhibitor.

New lithocholic and chenodeoxycholic piperazinylcarboxamides with antiproliferative and pro-apoptotic effects on human cancer cell lines.

Six new synthetic bile acid derivatives were synthesized and tested in vitro against various human cancer cells (glioblastoma multiforme (GBM), multiple myeloma (KMS-11), and colonic carcinoma (HCT-116) cell lines. The best activity was obtained with compound IIIb on multiple myeloma cells (LD(50): 8.5+/-0.5 microM). This activity was associated with Mcl-1 and PARP-1 cleavage, inhibition of NFkappaB signaling, and DNA fragmentation, demonstrating an apoptotic cell death signaling pathway.

Lithocholate glucuronide is a cholestatic agent.

Lithocholic acid and its taurine, glycine, and sulfate derivatives are potent cholestatic agents. Lithocholate glucuronide is present in the plasma and urine of patients with cholestatic syndromes, but little is known of its metabolism, excretion, and cholestatic potential. [3 beta-3H]lithocholate 3-O-beta-D-glucuronide was synthesized, and chemical and radiochemical purity were established. The aqueous solubility of lithocholate glucuronide was determined and found to be greater than that of lithocholic acid or several of its derivatives. In the range of concentrations examined, calcium ions precipitated lithocholate glucuronide stoichiometrically. The material was administered to rats prepared with an external biliary fistula. When 17-25 micrograms quantities were administered, 89.1 +/- 4.5% (mean +/- SEM) of the radiolabel was secreted in bile within the first 20 h after administration, the major fraction being secreted in less than 20 min. Four-fifths of the radiolabeled material in bile was the administered unaltered parent compound, while a minor fraction consisted of a more polar derivative(s). We showed that increasing biliary concentrations of more polar derivatives were observed with milligram doses of [3H]lithocholate glucuronide, and with time after the administration of these loading doses. Milligram doses of [3H]lithocholate glucuronide resulted in partial or complete cholestasis. When induced cholestasis was partial, secretion in bile remained the primary excretory route (82.5-105.6% recovery in bile), while, when complete cholestasis was induced, wide tissue distribution of radiolabel was observed. Cholestasis developed rapidly during infusion of [3H]lithocholate glucuronide. Bile flow was diminished within 10-20 min of the start of an infusion of 0.05 mumol, 100 g-1 body weight, minute-1, administered concomitantly with an equimolar infusion of taurocholate. The results establish that lithocholate glucuronide exerts cholestatic effects comparable to those exerted by unconjugated lithocholic acid.

Synthesis, characterization, and receptor interaction profiles of enantiomeric bile acids.

Bile acids are endogenous steroid detergents with receptor-mediated physiologic actions including activation of the G-protein coupled receptor TGR5 and gene regulation mediated by nuclear receptors. In this study, we report the first synthesis of enantiomeric lithocholic acid (ent-LCA, ent-1) and chenodeoxycholic acid (ent-CDCA, ent-2) via ent-testosterone (3). ent-1 was synthesized in 21 total steps in 4.2% yield, whereas ent-2 was obtained in 23 total steps in 0.8% yield. Critical micelle concentrations of the enantiomeric bile acids were found to be identical to their natural counterparts. Furthermore, enantiomeric bile acids were also tested for their ability to modulate bile acid activated proteins: farnesoid X receptor, vitamin D receptor, pregnane X receptor, and TGR5. Interestingly, ent-1 and ent-2 showed differential interactions with these proteins as compared to their corresponding natural bile acids. These data highlight the potential for using enantioselectivity as a way to distinguish between receptor and nonreceptor-mediated functions of natural bile acids.

Metadynamics for Perspective Drug Design: Computationally Driven Synthesis of New Protein-Protein Interaction Inhibitors Targeting the EphA2 Receptor.

Metadynamics (META-D) is emerging as a powerful method for the computation of the multidimensional free-energy surface (FES) describing the protein-ligand binding process. Herein, the FES of unbinding of the antagonist N-(3α-hydroxy-5ß-cholan-24-oyl)-l-ß-homotryptophan (UniPR129) from its EphA2 receptor was reconstructed by META-D simulations. The characterization of the free-energy minima identified on this FES proposes a binding mode fully consistent with previously reported and new structure-activity relationship data. To validate this binding mode, new N-(3α-hydroxy-5ß-cholan-24-oyl)-l-ß-homotryptophan derivatives were designed, synthesized, and tested for their ability to displace ephrin-A1 from the EphA2 receptor. Among them, two antagonists, namely compounds 21 and 22, displayed high affinity versus the EphA2 receptor and resulted endowed with better physicochemical and pharmacokinetic properties than the parent compound. These findings highlight the importance of free-energy calculations in drug design, confirming that META-D simulations can be used to successfully design novel bioactive compounds.

The synthesis and antitumor activity of lithocholic acid and its derivatives.

In this paper, a new and concise synthetic route of lithocholic acid (LCA) using commercially available steroid source deoxycholic acid is reported. A series of amide derivatives of LCA were also synthesized and investigated for their activity against the growth of MCF-7 and MCF-7/ADR cells using the sulforhodamine B assay. For MCF-7, the most potent compound 20 showed a 20-fold higher antitumor activity than LCA. For MCF-7/ADR, the most potent compound 24 showed a 22-fold higher antitumor activity than LCA. The transwell migration assay of 20 was evaluated on MDA-MB-231 cells. The colony formation and apoptosis assays of 20 were performed on MCF-7 and MCF-7/ADR cell lines.

Lithocholic acid analogues, new and potent alpha-2,3-sialyltransferase inhibitors.

A new type of noncompetitive alpha-2,3-sialyltransferase inhibitor has been synthesized; we report the discovery, preparation and inhibitory activity of sixteen lithocholic acid analogues.

Biocompatible hydrogelators based on bile acid ethyl amides.

Four novel bile acid ethyl amides were synthetized using a well-known method. All the four compounds were characterized by IR, SEM, and X-ray crystal analyses. In addition, the cytotoxicity of the compounds was tested. Two of the prepared compounds formed organogels. Lithocholic acid derivative 1 formed hydrogels as 1% and 2% (w/v) in four different aqueous solutions. This is very intriguing regarding possible uses in biomedicine.

Synthesis of B- and C-ring-modified lithocholic acid analogues as potential sialyltransferase inhibitors.

In order to identify structural features of lithocholic acid (LCA) critical for inhibition of the enzyme sialyltransferase (ST) novel analogues with modifications of the skeleton (7-9, 16-18 and 20) were designed and synthesized. Methyl 3α-acetoxy-7-oxo-cholanate (1), methyl 3α-acetoxy-12-oxo-cholanate (2) and methyl 3α,7α-diacetoxy-12-oxo-cholanate (3) were subjected to Baeyer-Villiger oxidation to provide homolactones (7-9) or to the Beckmann rearrangement of the corresponding oximes to give homolactams (16-18). Both reactions proceed regio- and stereoselectively. Ring B homolog of lithocholic acid (20) was efficiently synthesized. Among these compounds, 7, 9 and 16 were found to have the significant activity, with IC50 values ⩽3µM against α-2,6-(N)-ST selectively, which are 5-fold lower than that of Lith-O-Asp. Given the reality that LCA and its analogue, Lith-O-Asp, have been revealed to improve inhibitory efficacy of ST and to have a wide range of antimetastatic activities in different human cancer cells, the up-to-date findings have noteworthy pharmacological significance as they open a promising path to the improvement of a prospective molecular targeted application of modified LCA analogues as agents for the treatment of cancer metastasis.

Synthesis and evaluation of vitamin D receptor-mediated activities of cholesterol and vitamin D metabolites.

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer.

Labeling of the nicotinic acetylcholine receptor by a photoactivatable steroid probe: effects of cholesterol and cholinergic ligands.

A photoactivatable steroid, p-azidophenacyl 3 alpha-hydroxy-5 beta-cholan-24- ate (APL), has been synthesized and used instead of cholesterol to functionally reconstitute purified acetylcholine receptor (AcChR) into vesicles made of asolectin phospholipids. Upon irradiation, the extent of AcChR photolabeling by APL is directly proportional to the amount of APL incorporated into the reconstituted vesicles and the maximum stoichiometry observed corresponds to approx. 50 mol of APL bound per mol of AcChR. Furthermore, all four subunits of the AcChR become labeled by APL and the observed labeling pattern resembles the 2:1:1:1 stoichiometry characteristic of these subunits within the AcChR complex. The presence of either cholesterol or neutral lipids from asolectin in the reconstituted bilayer decreases both, the incorporation of APl into the vesicles and the covalent labeling of the AcChR upon irradiation, without altering the stoichiometry of labeling in AcChR subunits stated above. This suggests that the potential interaction sites for the photoactivatable probe in the reconstituted AcChR are mostly those normally occupied by the natural neutral lipids. Carbamylcholine, a cholinergic agonist, also reduces the extent of APL photolabeling of the AcChR in a dose-dependent manner but, in contrast to the effects of cholesterol, the presence of carbamylcholine alters the stoichiometry of labeling in the AcChR subunits. This, along with the observation that such a decrease in the extent of APL photolabeling caused by carbamylcholine can be blocked by preincubation with alpha-bungarotoxin, suggest that AcChR desensitization induced by prolonged exposure to cholinergic agonists encompasses a rearrangement of transmembrane portions of the AcChR protein, which can be sensed by the photoactivatable probe. Conversely, presence of (+)-tubocurarine, a competitive cholinergic antagonist, has no effects on altering either the extent of APL photolabeling of the AcChR or the distribution of the labeling among AcChR subunits.