Extending SAR of bile acids as FXR ligands: Discovery of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine

doi:10.1016/j.bmc.2011.03.004

Bioorganic & Medicinal Chemistry

Volume 19, Issue 8, 15 April 2011, Pages 2650-2658

https://doi.org/10.1016/j.bmc.2011.03.004 Get rights and content

Abstract

Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine was synthesized and evaluated for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR revealed that the 6α-ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with high binding affinity and efficacy, representing a promising lead candidate for further optimization. In addition, docking studies provide new insights into the molecular basis governing the partial and full agonist activity at FXR.

Graphical abstract

Introduction

The farnesoid-X receptor (FXR, also known as BAR; NR1H4) belongs to the superfamily of intracellular ligand-activated transcription factors that are involved in many physiological, developmental and toxicological processes.¹ Although initially characterized as a metabolic nuclear receptor activated by farnesol,² since 1999 FXR is considered a key nuclear receptor for bile acids (BAs), with chenodeoxycholic acid (CDCA, 1, Fig. 1) being its most potent endogenous agonist.³ FXR is abundantly expressed in tissues exposed to BAs, such as liver, intestine, kidneys, and in the ileal epithelium along the intestinal tract, where BAs are mainly absorbed.2, 4

Two FXR genes exist in lower mammalians including rodents, rabbits and dogs, that encode for α and β types of receptor. While FXRβ is a pseudogene in humans and primates,⁵ the expression of FXRα gene further originates four isoforms (FXRα1-4) as a result of alternative splicing and different promoters.⁶ Although the physiological importance of each isoform is still unclear, it is known that the four isoforms of FXRα exhibit diverse DNA binding and coregulator recruitment.⁷ Over the past decade, a number of evidences have shown the key role of FXR in regulating different metabolic pathways, including the homeostasis of BAs, lipids and glucose,¹ as well as in liver regeneration.⁸

The regulation of BA metabolism, for instance, occurs at three complementary levels of action: (i) decreasing the BA synthetic pathway; (ii) supporting the bile flow in the enterohepatic circulation; (iii) increasing the clearance of toxic biliary constituents. At the first level, FXR negatively regulates the BA synthetic pathway through the down-regulation of the rate-limiting enzyme CYP7A1, and the repression of CYP8B1, another key enzyme in BA synthesis.⁹ Interestingly, the repression of CYP7A1 occurs through the activation of FXR in the liver and intestine (Gut-Liver Axis) via the induction of the small heterodimer partner (SHP) and the activation of the fibroblast growth factor (mouse FGF15/human FGF19) signaling pathway.¹⁰ In addition, FXR inhibits HMG-CoA reductase and down-regulates lanosterol 14α-demethylase which are both important in the synthesis of cholesterol, the endogenous precursor of BAs.¹¹ At the second level of action, FXR supports bile flow in the enterohepatic circulation through the induction of genes involved in BA secretion from liver, such as the bile salt export pump (BSEP) and the multidrug resistant-associated protein 2 (MRP2); the down-regulation of genes involved in the import of BAs into the liver, that include the organic anion transporting polypeptide-2 and -8 (OATP-2 and -8) and the sodium-dependent taurocholate co-transporting protein (NTCP); the regulation of genes involved in the transport of BAs in the intestine such as the intestinal bile acid binding protein (IBABP), the apical sodium-dependent bile acid cotransporter (ASBT) and the organic solute transporters-α and -β (OST-α and -β).¹² Finally, FXR increases the clearance of toxic biliary constituents trough the regulation of additional genes, including the dehydroepiandrosterone sulfotransferase (SULT2A1), the bile acid N-acetyltransferase (BAT), and the bile acid CoA synthetase (BACS).^1d

The unraveling of the physiological functions of FXR has disclosed unprecedented opportunities for this receptor as drug target in different therapeutic areas such as liver diseases, metabolic syndrome and, more recently, hepatocarcinogenesis.¹ In this scenario, a number of molecules have been discovered, including both non-steroidal and steroidal compounds that were able to bind and interact with the ligand binding domain (LBD) of FXR in different ways, leading to ligand and promoter selectivity for the FXR-mediated gene transcription.1(e), 1(f) While non-steroidal compounds have been identified using high-throughput screenings of chemical libraries eventually filtered for lead-like and/or drug-like properties,1(a), 1(b), 1(f), 13 the identification of FXR steroidal modulators has mostly relied on a design strategy based on extensive modifications of the BA body and side chain.¹⁴ Embracing the latter approach, we have developed thorough structure activity relationships of BA derivatives as FXR ligands. These studies unveiled a striking increase of agonist activity at the receptor when introducing an ethyl group in the C₆ alpha position of CDCA (1), thereby disclosing 6-ECDCA (INT-747, 2) as the most potent and orally bioavailable agonist for FXR.^14e 6-ECDCA (2) is being advanced in clinical studies for non-alcoholic steatohepatitis (NASH), and recent results of phase II clinical studies for primary biliary cirrhosis (PBC) show a significant reduction of primary endpoint biomarkers of the clinical status and progression of the disease.

While significant progress has been made towards the discovery of potent agonists, great efforts are still needed for the development of selective FXR modulators (referred to as ‘selective bile acid receptor modulators’, SBARMs), compounds able to regulate individual or a cluster of FXR target genes.1(f), 1(g) These peculiar FXR modulators will offer the possibility to reduce the pleiotropism of the receptor’s action and to explore novel therapeutic opportunities which cannot be reacted by the use of FXR activators. In pursing this aim, we have reported that the substitution of the carboxylic tail of CDCA (1) with diverse substituted carbamate moieties was able to affect the agonist potency of BAs at FXR, leading to a broad range of efficacy in both cell-free FRET and cell-based luciferase assays (Fig. 2).14(b), 14(c) Docking experiments suggested for the first time that the LBD of FXR contains two contiguous pockets: the steroid binding pocket and the ‘back door’ pocket.^14b The former, in particular, accommodates the BA body and C₂₄ side chain, whereas the latter lodges the extended side chain substitution. Although the significance of this observation and its possible extension to other members of the nuclear receptor superfamily deserves further investigation, we demonstrated that it is possible to achieve full agonism, partial agonism, or antagonism by modifying a part of the molecule that is not directly interacting with any of the LBD elements known to affect coactivator binding.

With the aim of shedding light on the existence of any cross-talk between the steroid binding pocket and the ‘back door’ pocket responsible for the transactivation properties of FXR, and continuing our efforts in the identification of FXR selective modulators, in this study we report the synthesis, biological evaluations and molecular modeling studies of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine (4) (Fig. 2). We were, indeed, attracted by the interesting biological properties of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-24-nor-5β-cholan-23-amine (3), a carbamate BA derivative 10-fold more potent than CDCA (1) but endowed with partial agonism in FRET-assay (EC₅₀ = 0.62 μM, efficacy = 63%).^14b We focused our attention, in particular, on the incorporation of the crucial 6α-ethyl group in the scaffold of 3 as key structural element to improve the potency of the resulting compound.

Section snippets

Chemistry

The synthesis of 4 is outlined in Scheme 1.14(b), 16(d) Treatment of methyl 7-keto-deoxycholate 5 with LDA in THF at −78 °C followed by reaction of the enolate thus formed with trimethylchlorosilane (TMSCl) afforded the corresponding silyl enolether 6, in nearly quantitative yield. Aldol-type addition of intermediate 6 with acetaldehyde in the presence of BF₃·OEt₂ at −60 °C in CH₂Cl₂ gave the desired methyl 3α-hydroxy-6-ethylidene-7-keto-5β-cholan-24-oate (7) (Z:E, 85:15) in 80% yield.

Discussion

While substitution of the carboxylic group of CDCA (1) by an amino group preserved both the potency and efficacy of the parent derivative 1, carbamoylation of the distal C₂₄-amino derivative with a cinnamyl functionality gave the corresponding carbamate derivative 3, which displayed the profile of a partial FXR agonist (Table 1).14(b), 14(c) Further insertion of an ethyl group in position C6α resulted in 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine (4), which was

General methods

Chemical reagents and solvents were obtained from commercial sources. When necessary, solvents were dried and purified by standard methods. Melting points were determined with an electrothermal apparatus and are uncorrected. NMR spectra were recorded on a Bruker AC 200 or 400 MHz spectrometer and the chemical shifts are reported in parts per million (ppm). The abbreviations used are as follows: s, singlet; br s, broad singlet; d, doublet; dd, double doublet; m, multiplet. IR spectra were

Acknowledgments

This work was supported by Intercept Pharmaceuticals (New York, NY). Thanks are due also to Erregierre (Bergamo, Italy) for the gift of bile acids as starting materials.

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Extending SAR of bile acids as FXR ligands: Discovery of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholan-23-amine

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Discussion

General methods

Acknowledgments

Mol. Cell Biol.

Hepatology

Mol. Endocrinol.

Steroids

J. Med. Chem.

J. Med. Chem.

J. Lipid Res.

J. Med. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Med. Chem.

J. Med. Chem.

J. Med. Chem.

Exp. Opin. Ther. Pat.

Curr. Med. Chem.

Nat. Rev. Drug Disc.

Physiol. Rev.

Trends Biochem. Sci.

Exp. Opin. Ther. Pat.

J. Med. Chem.

Cell

Science

Science

Mol. Cell