Side-Chain Homologs of Δ9-THC, Δ8-THC, and HHC Reveal Pathway Bias at CB1R and CB2R Cannabinoid Receptors.

Minor cannabinoids and semisynthetic cannabis constituents, such as derivatives of tetrahydrocannabinol (THC) and hexahydrocannabinol (HHC), are increasingly encountered in consumer products, yet their signaling profiles at cannabinoid receptors remain incompletely characterized. Here, we describe how structural modifications of THC and HHC, including variations in alkyl side-chain length (C3-C8), double-bond position (Δ9 vs Δ8), and HHC stereochemistry (9R vs 9S), shape signaling at CB1R and CB2R cannabinoid receptors. Using a BRET-based platform in HEK293 cells, we determined pathway-specific potency, efficacy, receptor selectivity, and signaling bias in isoform-specific inhibitory G protein activation (Gi1, Gi2, Gi3, GoA, GoB, Gz) and β-arrestin recruitment (β-arr1, β-arr2) of a panel of Δ9-THC, Δ8-THC, (9R)-HHC, and (9S)-HHC side-chain homologs. C3 side-chain ligands were additionally evaluated in antagonist mode. Across both receptors, C4-C8 homologs behaved as high-efficacy agonists in Gi/o pathways while remaining partial agonists in β-arrestin recruitment. Side-chain elongation was the dominant determinant of signaling efficiency, producing progressive potency gains that peaked for C7-C8 homologs. Δ9/Δ8 isomerism and hydrogenation effects were context-dependent, while HHC epimerism imposed a consistent separation, with 9R-HHC homologs exhibiting higher potency than matched 9S-HHC homologs. In contrast, C3 homologs exhibited minimal agonism and instead displayed functional CB1R antagonism, with partial inhibition of G protein pathways and near-maximal inhibition of β-arrestin recruitment. These findings provide quantitative, pathway-resolved benchmarks for how closely related cannabinoid scaffolds signal through receptor- and transducer-specific pathways and indicate structural features that may be leveraged to tune CB1R/CB2R selectivity and G protein versus β-arrestin engagement.