TRPM8 antagonist 4 is a compound with high functional selectivity and favorable physicochemical properties, serving as a CB2R partial agonist (EC50=54.2 μM, Ki=3.2 μM) and a TRPM8 (IC50=42.3 nM) antagonist. It possesses notable anti-inflammatory and analgesic effects, demonstrating good safety by reducing the mRNA expression of TNF-α, IL-6, and IL-1β.

TRPM8:42.3 nM

TRPM8 antagonist 4 (Compound 6b) exhibits neither antagonistic nor agonistic activity on CB1R in HTRF cAMP assays. It inhibits the hTRPM8 current induced by 30.0 μM menthol in HEK293 cells, with an IC50 of 42.3 nM at concentrations ranging from 1 nM to 1000 nM. Additionally, TRPM8 antagonist 4 binds to CB2R as shown by radioligand binding assays at concentrations of 1 μM to 1 M. At 0.3 μM for 8 hours, it reduces the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 macrophages.

Compound 6b, a TRPM8 antagonist, exhibits anti-inflammatory effects at 2.5 mg/kg through intraperitoneal injection in lipopolysaccharide-induced C57BL/6J mice. Additionally, at a dosage of 5 mg/kg, administered in the same manner, it demonstrates analgesic properties in acetic acid-induced C57BL/6J mice.