Exploration of synthetic approaches and pharmacological evaluation of PNU-69176E and its stereoisomer as 5-HT_2C receptor allosteric modulators

Allosteric modulators of the serotonin (5-HT) 5-HT_2C receptor (5-HT_2CR) present a unique drug design strategy to augment the response to endogenous 5-HT in a site- and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT_2CR. For the first time, an optimized synthetic route to readily access PNU-69176E (1) and its diastereomer 2 has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid. This synthetic approach not only enables a feasible preparation of a sufficient amount of 1 for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT_2CR allosteric modulators. Compound 1 and its diastereomer 2 were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT _2CR using an intracellular calcium (Ca_i²⁺) release assay. Compound 1 demonstrated efficacy and potency as an allosteric modulator for the 5-HT_2CR with no intrinsic agonist activity. Compound 1 did not alter 5-HT-evoked Ca_i²⁺ in CHO cells stably transfected with the highly homologous 5-HT_2AR. In contrast, the diastereomer 2 did not alter 5-HT-evoked Ca_i²⁺ release in 5-HT_2AR-CHO or 5-HT_2CR-CHO cells or exhibit intrinsic agonist activity.