Synthesis and Evaluation of Potent and Selective μOR and KOR Ligands Derived from the Akuamma Alkaloids

The use of structure-activity relationships (SAR) of opioid receptor ligand natural products such as morphine, salvinorin A, and mitragynine has been a fundamental tool in understanding the intricacies of the opioid receptors leading to the discovery of signaling mechanisms, crystal structures of activated receptors, and the design of safer ligands. Identification of structurally distinct opioid receptor probe molecules may identify novel opioid receptor interactions which will ultimately further the understanding of opioid receptor signaling. This study delves into the semi-synthetic diversification of underexplored indole alkaloids as molecular tools to probe mu-opioid receptor (μOR) and kappa-opioid receptor (κOR) activation. Alkaloids from akuamma seeds were isolated to examine their SAR at the μOR and κOR. The alkaloids akuammine and pseudo-akuammigine selectively bind and activate the μOR, but are inactive in animal models of antinociception. Through strategic chemoselective modifications targeting the C10, C11, C16, and N1 positions, a suite of 22 analogues was synthesized, culminating in the discovery of N-1-phenethyl pseudo-akuammigine (PhEtPAK). PhEtPAK demonstrated significantly increased in vitro potency and in vivo efficacy in animal models of antinociception. The akuamma alkaloid akuammicine potently and selectivity activates the κOR. Selectively modifying functional handles of akuammicine yielded 26 semi-synthetic analogues. Halogenation of the C10 position of the aromatic ring dramatically increased the κOR potency while other modifications were not tolerated. Synthesis of dually substituted analogues containing both C10 halogens and modifications to the C19,20 olefin, C2,16 olefin, and N4 nitrogen rescued κOR activity corroborating the key role of the halogens in κOR stabilization and activation. Furthermore, akuamma alkaloid scaffolds served as a basis for the synthesis of structurally similar akuammiline alkaloids and analogues, cabuamine and vincorine, facilitating efforts to identify their biological targets. High-throughput screening of these natural products and their analogues revealed that cabuamine and vincorine do not have CNS activity; however, the analogue 10-demethoxy vincorine selectivity binds to the κOR. This comprehensive investigation elucidates the SAR of three new classes of opioid receptor-selective indole alkaloids. The development of these unique opioid receptor ligand classes has provided valuable insights for understanding opioid receptor signaling and future drug development.