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

Abstract

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, LXRβ 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.

Figure 1

Compound F from VS and derivatives 1–14 identified in this study as a new chemotype of GPBAR1 agonists.

Figure 2

Preparation of compound F from VS, its simplified derivatives 1–4 and optimized GPBAR1 agonists 5–14. Reagents and conditions: (a) NH₂OH·HCl, K₂CO₃, CH₃OH reflux, quantitative yield; (b) (3R)-hydroxy-Boc-L-proline, DIPEA, HBTU in DMF, 80 °C, 60%; (c) MsCl, TEA, −10 °C, 2 h, quantitative yield; (d) NaN₃ in DMSO, 150 °C, 4 h, 75%; (e) NH₄Cl, Zn, CH₃OH:H₂O (1:0.1), 61%; (f) 4-(trifluoromethyl)phenyl isocyanate, CH₂Cl₂, 0 °C, 84%; (g) TFA, 2 h, quantitative yield; (h) HCHO, HCOOH, 80–90 °C overnight, 56%; (i) R₂NCO, CH₂Cl₂, 0 °C, 74%.

Figure 3

(A) Dose response curves of compounds 9 and 10 (concentrations ranging from 1 to 50 µM) in luciferase reporter assay. (B) Real-time PCR analysis of mRNA expression of Pro-glucagon in GLUTag cells treated with compounds 9 and 10 at 10 μM, and TLCA used as a positive control (10 μM). Values are normalized to GAPDH and are expressed relative to those of not treated cells (NT) which are arbitrarily settled to 1. The relative mRNA expression is expressed as 2^(−ΔΔCt). (C) Specificity of compounds 9 and 10 at 10 μM dose in luciferase reporter assays versus PPARα and PPARγ using Gemfibrozil (GEM, 10 μM) and Rosiglitazone (ROSI, 100 nM) as positive controls respectively. (D) Specificity of compounds 9 and 10 at 10 μM in luciferase reporter assays versus LXRα and LXRβ using GW3965 (GW, 10 μM) as positive control. (E) Specificity of compounds 9 and 10 at 10 μM in luciferase reporter assays versus FXR using CDCA (10 μM) as positive control; (F) Specificity of compounds 9 and 10 at 10 μM in luciferase reporter assays versus PXR using Rifaximin (Rifax, 10 μM) as positive control.

Figure 4

(A) Binding mode of 10 (yellow sticks) to GPBAR1 (gray cartoon). Amino acids essential for ligand binding are depicted as sticks. Polar contacts are shown as dashed black lines. ECL1, residues 145–156 in ECL2, ECL3 and nonpolar hydrogens are undisplayed for clarity. (B) Interatomic distances (mean ± S.E.M.) representative of the main ligand/receptor contacts observed along the last 100 ns of MD simulations: (I) pyrrolidinyl ring (N)/Glu169 (Cδ), cyan bar; (II) pyrrolidinyl ring (N)/Asn93 (Cγ), orange bar; (III) phenyl (centroid)/Tyr240 (centroid of the phenolic ring), green bar; (IV) ureidic moiety (C)/Tyr (OH), gray bar; (V) cyclohexyl (centroid)/Leu266 (Cγ), yellow bar.