Synthesis and biological evaluation of new dual APN/NEP inhibitors as potent analgesics

An alternative approach for the management of acute and chronic pains involves prolonging the half-life of endogenous opiates, such as enkephalins that are released in response to nociceptive stimuli. This can be achieved through the inhibition of enzymatic pathways responsible for the hydrolysis of these peptides, particularly targeting Aminopeptidase N (APN) and Neutral Endopeptidase (NEP). In this study, we designed and synthesized a series of dual enkephalinase inhibitors (DENKIs) targeting both APN and NEP as novel analgesic treatments. Notably, SDUY812, SDUY816 and SDUY817 exhibited potent inhibition of APN activity with IC₅₀ values of 0.38 µM, 0.68 µM and 0.29 µM, respectively, whereas their IC₅₀ values against NEP were 6.9 µM, 6.9 µM and 7.4 µM, separately. In in-vivo antinociceptive assays, SDUY816 and SDUY817 demonstrated superior analgesic efficacy compared to Thiorphan and Bestatin in mice models of acute, inflammatory and neuropathic pains with jumping latencies exceeding 100 s and withdrawal thresholds more than 0.13 g. Moreover, the analgesic activity of these inhibitors was significantly diminished by a potent opioid antagonist, naloxone, indicating the contribution of opioid receptors to the robust analgesic properties of these newly developed DENKIs. In addition, SDUY816 and SDUY817 exerted the analgesic activity in a concentration- and time-dependent manner with SDUY816 possessing acceptable pharmacokinetic properties (t_1/2 = 4.02 h and F = 27 %) and low toxicity. These findings provide alternative analgesic therapeutics that are potentially devoid of opioid-associated side effects.