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  • Encouraged by the observation obtained from region A


    Encouraged by the observation obtained from region A, we selected the structure of 1 as the next template to successively pursue the SAR on regions B-D of 1 (lower side of Fig. 2). Next, we turned attention to the external aromatic ring of 1 to verify the impact on the antagonistic activity and prepared 8–13 (Table 3). Compounds 8, 11 and 12 indicated increasing affinity with LBD of FXR compared to 1. 4-Phenoxy derivatives (8 and 9) retained potency for FXR in the cell assay. In contrast, replacement (10) and elimination (13) of an external phenyl ring failed to show a significant effect in both activities, suggesting that the phenoxy group at the 4-position on internal benzene would have be optimal in terms of the interaction with FXR. The results of the SAR study on the substituents on the scaffold (region C) are described in Table 4. Compound 14 decreased the affinity with LBD of FXR compared to 1. Introduction of a methyl group (15) marginally reduced the interaction with LBD of FXR (IC50=65.6μM) and revealed sub-micromolar potency against FXR in luciferase reporter assay (IC50=0.175μM). Likewise the modifications of region A and region C are suggested to exert an influence on the antagonism against FXR. Hydantoin in region D combined phenoxybenzene part and the scaffold was replaced by dimethylhydantoin (16), imidazolizinone (17) and diketopiperazine (18) (Table 5). Although 17 resulted in retention of the binding affinity and antagonistic activity, 16 and 18 led to the loss of activity in both assays. It is suggested that sterically bulky parts would not acceptable for region D. In order to confirm whether a combination of regions A and C can reverse the antagonist potency, TKI-258 australia 19 was prepared and evaluated as the first example in the combination study (Fig. 3). The compound with cyclohexyl moiety as the liker and the methyl group on the scaffold was tolerated in LBD of FXR (IC50=126.1μM) and also improved antagonism for FXR (IC50=1.2nM). There is a relationship between the regulation of SHP and CYP7A1 expression; in fact, chenodeoxycholic acid (CDCA), which is an endogenous ligand of FXR, represses CYP7A1 expression that is required to induce SHP expression. CDCA-activated SHP and CYP7A1 mRNA levels were measured to serve as surrogate markers to reflect the inactivation of FXR by 19 (Fig. 4). Compound 19 significantly decreased the expression levels of SHP and increased the expression levels of CYP7A1. These results suggested that 19 induced CYP7A1 induction via SHP suppression. Selectivity of 19 for FXR was examined using a reporter-gene transcriptional assay for human retinoid X receptor-α (RXR-α). (Fig. S1 in Supplementary data) No cross-reactivity was observed with RXR-α even at a concentration of 100μM. Thus, these findings on 19 give us new insights into the development of novel types of FXR ligands (Fig. S2 in Supplementary data).
    Conclusion We have discovered a new kind of nonsteroidal FXR antagonists using our compound library assembled by a medicinal chemistry approach in conjunction with chemical synthesis and in vitro assay. On the basis of the structure of hit compound BB-4, a total of 19 novel derivatives have been synthesized and evaluated in FXR TR-FRET binding and luciferase reporter assays. The SAR studies on regions A-D suggested that regions A and C significantly contribute to the interaction with LBD of FXR and the antagonism against FXR. Compound 19 possessing the promising motifs in regions A and C not only showed the binding affinity and the antagonistic capability against FXR, but also regulated mRNA expression level of FXR target genes, SHP and CYP7A1, while maintaining the selectivity for RXRα. Thus, appropriate structural optimization in the above regions can substantially alter antagonist potency.