Design, synthesis and biological activity of novel pyrimidine piperazine ureas as μ-opioid and TRPV1 dual-target ligands for pain management

The pathophysiology of pain involves multiple signaling pathways, making its management a persistent clinical challenge. Transient receptor potential vanilloid 1 (TRPV1) acts as a molecular integrator of nociceptive stimuli in primary C-fiber sensory neurons and plays a crucial role in nociception, as well as in neuropathic and inflammatory pain. Numerous TRPV1 antagonists have been evaluated in clinical trials for various pathologies, including pain. However, their clinical development has been hindered by side effects such as hyperthermia and impaired noxious heat sensation. Additionally, these antagonists have limited efficacy when used as standalone therapies. Furthermore, studies have demonstrated a complex interplay between TRPV1 and μ-opioid receptor (MOR). In this study, dual-acting compounds targeting both TRPV1 and MOR were designed and synthesized using a pharmacophore fusion strategy, aimed at enhancing pain treatment, overcoming drug resistance, and minimizing the adverse effects typically associated with single-target drugs. Among these, compound 2ac demonstrated the highest in vitro potency, with an IC₅₀ of 29.3 nM for TRPV1 antagonism and a K_i of 60.3 nM for MOR binding affinity. In vivo analgesic experiments conducted using a formalin-induced pain model in mice showed that compound 2ac exhibited a potent, dose-dependent anti-nociceptive effect. Target engagement studies confirmed that the analgesic effect of compound 2ac was attributed to both TRPV1 antagonism and MOR activation. Notably, further testing indicated that compound 2ac did not induce hyperthermia (a common side effect of TRPV1 antagonists) or lead to analgesic tolerance (a typical opioid-related adverse effect). Additionally, molecular docking studies showed strong compatibility of compound 2ac with the active sites of hMOR and hTRPV1, supporting its potential as a promising lead compound for pain management.