Optimization of Chemical Functionalities of Indole-2-carboxamides To Improve Allosteric Parameters for the Cannabinoid Receptor 1 (CB1)

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (1; ORG27569) is a prototypical allosteric modulator for the cannabinoid type I receptor (CBI). Here, we reveal key structural requirements of indole-2-carboxamides for allosteric modulation of CBI: a critical chain length at the C3-position, an electron withdrawing group at the CS-position, the length of the linker between the amide bond and the phenyl ring B, and the amino substituent on the phenyl ring B. These significantly impact the binding affinity (K-B) and the binding cooperativity (alpha). A potent CB1 allosteric modulator 5-chloro-N-(4(dimethylamino)phenethyl)-3-propyl-1H-indole-2-carboxamide (12d) was identified. It exhibited a K-B of 259.3 nM with a strikingly high binding alpha of 24.5. We also identified 5-chloro-N-(4-(dimethylamino)phenethyl)-3-hexyl-1H-indole-2-carboxamide (120 with a K-B of 89.1 nM, which is among the lowest K-B values obtained for any allosteric modulator of CB1. These positive allosteric modulators of orthosteric agonist binding nonetheless antagonized the agonist-induced G-protein coupling to the CB1 receptor, yet induced beta-arrestin mediated ERK/2 phosphorylation.