Abstract There is a significant need for new treatments for opioid use disorder (OUD) to manage patients abusing heroin, fentanyl, oxycodone and related drugs. Buprenorphine and methadone are both effective medications for OUD but are agonists with potential for abuse and diversion, and with methadone the risk of respiratory depression and overdose death. Moreover, these compounds are not effective in relapse situations. The antagonist naloxone is an alternative that does prevent relapse but has poor patient compliance due to its complete blockade of the mu-opioid system. We have discovered compounds that act as non-competitive antagonists of the mu-opioid receptor (MOR). These compounds interact with a novel site on the receptor to function as allosteric antagonists, also called NAMs or Negative Allosteric Modulators. They reduce the activity of MOR agonists in a fashion that is non-surmountable and that does not completely shut down the receptor, thereby providing the ability to reduce opioid actions in individuals with OUD, potentially without the negative side-effects of current agonist or antagonist treatments. Our hypothesis is that NAMs of MOR (mu-NAMs) could be developed as effective non-agonist treatments for OUD by targeting MOR in a novel way, leading to our goal of identifying proprietary mu-NAMs as an alternative, non-agonist treatment for OUD. In a previous U18 “Step up for Substance Abuse” grant (DA DA052371) we identified a lead mu-NAM that is active in vitro, shows good brain penetration after intraperitoneal injection, and is effective in vivo in blocking MOR-mediated agonist actions in the mouse. However, this compound is not patentable (i.e., it is not a new chemical entity) and lacks drug-like characteristics. The objective of the current application is to develop our original lead molecule to obtain a mu-NAM that has high affinity for the allosteric site on MOR, is selective for MOR, has appropriate solubility and pharmacokinetic properties, is functional in vivo, and around which we can develop intellectual property. Towards this aim in this Phase 1 application, we will 1) Synthesize and evaluate in vitro derivations of our initial mu-NAM lead to identify structurally novel mu-NAMs, and 2) De-risk the project by confirming our lead mu-NAM has activity in both the mouse and the rat and in both males and females.