β-Lactam TRPM8 Antagonists Derived from Phe-Phenylalaninol Conjugates: Structure-Activity Relationships and Antiallodynic Activity

The protein transient receptor potential melastatin type 8 (TRPM8), a non-selective, calcium (Ca²⁺)-permeable ion channel is implicated in several pathological conditions, including neuropathic pain states. In our previous research endeavors, we have identified β-lactam derivatives with high hydrophobic character that exhibit potent and selective TRPM8 antagonist activity. This work describes the synthesis of novel derivatives featuring C-terminal amides and diversely substituted N'-terminal monobenzyl groups in an attempt to increase the total polar surface area (TPSA) in this family of compounds. The primary goal was to assess the influence of these substituents on the inhibition of menthol-induced cellular Ca²⁺ entry, thereby establishing critical structure-activity relationships. While the substitution of the tert-butyl ester by isobutyl amide moieties improved the antagonist activity, none of the N'-monobencyl derivatives, regardless of the substituent on the phenyl ring, achieved the activity of the model dibenzyl compound. The antagonist potency of the most effective compounds was subsequently verified using Patch-Clamp electrophysiology experiments. Furthermore, we evaluated the selectivity of one of these compounds against other members of the transient receptor potential (TRP) ion channel family and some receptors connected to peripheral pain pathways. This compound demonstrated specificity for TRPM8 channels. To better comprehend the potential mode of interaction, we conducted docking experiments to uncover plausible binding sites on the functionally active tetrameric protein. While the four main populated poses are located by the pore zone, a similar location to that described for the N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist cannot be discarded. Finally, in vivo experiments, involving a couple of selected compounds, revealed significant antinociceptive activity within a mice model of cold allodynia induced by oxaliplatin (OXA).