The original hit (1) and selected analogs (16, 20, and 39) were screened for binding against a panel of common neurological off-targets conducted by the National Institute of Mental Health Psychoactive Drug Screening Program (PDSP). In total the panel includes 44 targets including various GPCRs and ion channels. The molecules were assessed for the ability to displace radioligands from each target at a single concentration of 10 µM. In general, all molecules revealed limited significant binding with many neurological targets including the chronic pain relevant MOR, DOR, and KOR (Table ). Molecule 1 was selected for secondary follow-up binding assays in dose-response to validate the primary assay binding against four targets with elevated mean % binding values: serotonin 2B (5-HT2B), benzodiazepine rat brain site, muscarinic acetylcholine-2 receptor, and sigma-2 receptor. Of the four targets selected for secondary binding molecule 1 only displayed binding to 5-HT2B with a Ki value of 1.3 µM while the other three did not exhibit displacement of the radioligand at concentrations up to 10 µM in the secondary validation. The same was observed for analog 16 upon secondary dose-response against 5-HT2B with a Ki value of 0.9 µM. Both analogs 20 and 39 also displayed primary displacement of the radioligand from 5-HT2B at 49 and 32%, respectively. While binding to 5-HT2B may indicate a potential undesired off-target liability the binding assays do not assess functional efficacy at the receptor. Thus, all four analogs were further evaluated for functional efficacy against 5-HT2B transfected into HEK cells in both agonist and antagonist modes. Non-transfected HEK cells served as controls. The compounds displayed no agonist activity against the 5-HT2B receptor for Gq stimulation or -arrestin recruitment in the Tango assay that monitors -arrestin translocation (Table ). Likewise, no antagonist activity was observed in the Gq/Ca 2+ assay using a calcium sensitive reporter; however, there was consistent inhibitory activity observed in the antagonist Tango assay with IC50 values ranging from 0.42 -0.60 µM. The somewhat contrasting results in the Gq/Ca 2+ and -arrestin antagonist assays are perplexing and may reflect a complex pharmacology requiring further study or may be due to compound interference with the Tango assay. The latter is supported by data revealing that compounds 20 and 39 that weakly bind the 5-HT2B receptor (<50% displacement at 10 μM; Table ), yet inhibit in the Tango assay with submicromolar potency.