We designed and synthesized 27 new amide and dipeptide derivatives containing a substituted phenylalanine as negative allosteric modulators (NAMs) for the beta-2 adrenergic receptor (β2AR). These analogs aimed to improve the activity of our lead compound, Cmpd-15, by introducing variations in three key regions: the meta-bromobenzyl methylbenzamide (S1), para-formamidophenylalanine (S2), and 1-cyclohexyl-1-phenylacetyl (S3) groups. The synthesis involved the Pd-catalyzed β-C(sp3)-H arylation of N-acetylglycine with 1-iodo-4-substituent-benzenes as the key step. GloSensor cAMP accumulation assay revealed that six analogs (A1, C5, C6, C13, C15 and C17) surpass Cmpd-15 in β2AR allosteric function. This highlights the crucial role of the S1 region (meta-bromobenzyl methylbenzamide) in β2AR allostery while suggesting potential replaceability of the S2 region (para-formamidophenylalanine). These findings serve as a valuable springboard for further optimizing Cmpd-15, potentially leading to smaller, more active, and more stable β2AR-targeting NAMs.
In this study, we designed and synthesized 12 novel non-steroidal cholic acid (CA) analogues as potential Takeda G protein coupled receptor 5 (TGR5) allosteric agonists based on scaffold hopping strategy. Biological results showed that all final compounds were able to activate the receptor both in human TGR5 (hTGR5) and mouse TGR5 (mTGR5) by Glosensor cAMP accumulation assay. Remarkably, lactone A1 and lactam A10 displayed 48- and 24-fold higher potencies than that of CA in hTGR5 cAMP accumulation, respectively. Furthermore, target compounds exhibited higher selectivity for cAMP formation compared to β-arrestin2 recruitment. Interestingly, target compounds A1–10 positively modulated the functional activity and potency of lithocholic acid (LCA) in TGR5, indicating that A1–10 are TGR5 allosteric agonists. Molecular docking study displayed that both lactone in A5 and lactam in A10 formed H-bonds with the key amino acid residue Thr131, which are critical for TGR5 allosteric effect. Unexpectedly, none of target compounds A1–10 could activate farnesoid X receptor (FXR), indicating that final compounds are potent and selective TGR5 allosteric agonists. More importantly, both A1 and A10 exhibited significant glucose-lowering effects in the oral glucose tolerance test (OGTT) in mice. Taken together, we discovered two potent and selective TGR5 allosteric agonists A1 and A10 , which could be employed as lead compounds to further study TGR5 allostery with potential antidiabetic effects.
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