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    • Knock out mice of FXR showed enhancing cholesterol metabolis

    2021-11-22

    Knock-out mice of FXR showed enhancing cholesterol metabolism in vivo and reducing serum levels of total cholesterol. Additionally, the results of FXR-deficiency mice revealed potential effects on the improvement of obesity and diabetes.19, 20 As a natural FXR ligand, guggulsterone possesses antagonistic activity in a coactivator assay and acts as an agonist on bile salt export pump expression in vivo. Treatment of guggulsterone reduced hepatic cholesterol in wild-type mice fed a high-cholesterol diet, suggesting that the cholesterol-lowering activity would arise from the inhibition of FXR by guggulsterone. With respect to the pharmacological profile of synthetic antagonists, some studies examined the anti-dyslipidemic effects have been performed in mice or primates.23, 24, 25, 26 Thus, the synthetic and low-molecular weight FXR antagonists would be a breakthrough in existing metabolic dysfunctions. In a series of nonsteroidal FXR ligands disclosed to date, several ligands share many types of heteroaromatic rings,17, 21, 28, 29, 30, 31, 32, 33 which are a selective scaffold in drug discoveries for protease or kinase ligands based on biological activity.34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 We also have an in-house library containing heteroaromatic compounds assembled by previous medicinal chemistry programs. As the starting point of our efforts on discovering new FXR ligands, a variety of building blocks, as exemplified by BB-1 and BB-242, 43, 44 (Fig. 1) in the library, were screened in a FXR time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay and a luciferase reporter assay. As a consequence, we identified a hit compound that is capable of inhibiting FXR activity. Subsequently, the optimization approach based on the structure of the hit compound provided novel chemotype to develop novel FXR antagonists.
    Chemistry Preparation of compounds BB-3-BB-6 has been reported in the literature. Experimental procedures for the synthesis and characterization (1H NMR and/or HR-MS) of 1–18 are available in Supplementary data (Schemes S1–S5). The synthesis of 19 is outlined in Scheme 1. The starting material, N2,4-dimethylbenzene-1,2-diamine, was coupled with (2S)-2-(tert-butoxycarbonylamino)-3-cyclohexyl-propanoic KN-62 to yield corresponding 19a. Immediate ring closure of 19a in CH3COOH gave the corresponding benzimidazole derivatives, 19b. The Boc group of 19b was removed and subsequent coupling with 2-(4-phenoxyanilino) acetic acid hydrochloride using HOAt and WSCI.HCl in DMF yielded 19c. Formation of the hydantoin was achieved by 4-nitrophenol chloroformate and 1M TBAF in THF to yield 19.
    Biology
    Results and discussion Our BB-1 and BB-2 derivatives42, 43, 44 in the compound archive and the existing representative FXR ligands have some of shared structures; namely, the fused heteroaromatic ring as the scaffold and appropriately functional moieties extending from the scaffold (Fig. 1). In our compound library, BB-3-BB-6 were found to have affinity with LBD of FXR (Table 1). Compound BB-3 showed the binding affinity approximately 4.3 times higher than that of (Z)-gugglusterone. The binding activity of BB-4-BB-6 was weaker than that of (Z)-gugglusterone, but was retained an IC50 of 66.6–174.5μM. The results implied that these structures possessing a chiral linker between the hydantoin and benzimidazole scaffold would be tolerated in LBD of FXR. An antagonistic activity against FXR of these compounds was measured in the luciferase reporter assay. Compound BB-4 proved to have the most potent antagonistic activity against FXR in our compound library (IC50=10.1μM). The results listed in Table 1 suggested that our unique structures could be employed to explore the novel features of the chemotype for FXR antagonists. We started by keeping the scaffold and modified region A of BB-4 (upper side of Fig. 2) to obtain structure-activity relationship (SAR) information and improve the antagonistic activity against FXR. We first focused on optimizing region A to determine the effect of chiral linkers and 1–3 were prepared and compared with BB-4-BB-6. (Table 2) As with BB-4-BB-6, compounds 1–3 also displayed binding activity to a varying degree. A hydrophobic moiety as the linker (1 and 2) was more active than BB-4 in the binding and luciferase reporter assays. The opposite chirality to 1 and 2 was apt to cause the loss of the antagonistic activity relative to that of 1 and 2 (1 vs. BB-6 and 2 vs. 3). On the basis of these results, we next focused on the hydrophobic moiety at R1 and 4–7 were prepared (Table 2). The potency of these compounds in both assays is much more likely to be related to bulkiness of substituents at R1. Indeed, the R1 group in 7 showed the highest antagonism against FXR among the compounds listed in Table 2 (IC50=24.4μM for the binding assay and IC50=2.0μM for the cell assay).