Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N,N'-dibenzyl tryptophan were prepared and characterized in vitro by Ca-imaging and patch-clamp electrophysiology assays. Molecular modeling studies led to identification of a broad and well-defined interaction network of these derivatives inside the TRPM8-agonist binding site, underlying their antagonist activity. The (5R,11aS)-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)-dione () emerged as a potent (IC50 4.10 ± 1.2 nM), selective, and metabolically stable TRPM8 antagonist. In vivo, showed significant target coverage in an icilin-induced WDS (at 11.5 mg/kg i.p.), an oxaliplatin-induced cold allodynia (at 10-30 μg s.c.), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg i.p.) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.