Abstract: Using the preclinical drug CVL-231 as a lead compound and introducing the 2-trifluoromethylpyridine moiety from the high-activity compound VU6000918, 27 novel 2-trifluoromethylpyridine cyclobutylamide derivatives were designed and synthesized by modifying the heterocyclic moiety. The allosteric modulatory activity of the compounds on M4 receptor cells was assessed using the FLIPR fluorescence detection technique. The results revealed that several compounds exhibited significant cellular activity, especially for compounds Ⅳ1, Ⅳ12, Ⅳ20, Ⅳ23, Ⅳ25, Ⅳ26, and Ⅳ27. The results of the EC50 determination indicated that compound Ⅳ23 had an EC50 value as low as 979 nmol/L, which is slightly higher than that of the positive control VU0467154. Structure–activity relationship (SAR) analysis revealed that the introduction of a quinoline ring, particularly the 4-methylquinoline moiety, significantly enhanced the activity of the compounds. Molecular-docking simulations revealed that compound Ⅳ23 forms both hydrogen-bond and π–π stacking interactions with the M4 receptor protein (7TRP), providing a plausible mechanistic for its recognition by the target protein. In summary, compound IV23, as a promising lead compound for M4 positive allosteric modulators, provides an important research basis for subsequent structural optimization and drug development.

Key words: M4 positive allosteric modulator, Trifluoromethylpyridine, Cyclobutylamine; Synthesis, Molecular docking