Abstract

Successful development of 5-HT_2C agonists requires selectivity versus the highly homologous 5-HT_2A receptor, because agonism at this receptor can result in significant adverse events. (R)-9-Ethyl-1,3,4,10b-tetrahydro-7-trifluoromethylpyrazino[2,1-a]isoindol-6(2H)-one (compound 1) is a potent 5-HT_2C agonist exhibiting selectivity over the human 5-HT_2A receptor. Evaluation of the compound at the rat 5-HT_2A receptor, however, revealed potent binding and agonist functional activity. The physiological consequence of this higher potency was the observation of a significant increase in blood pressure in conscious telemeterized rats that could be prevented by ketanserin. Docking of compound 1 in a homology model of the 5-HT_2A receptor indicated a possible binding mode in which the ethyl group at the 9-position of the molecule was oriented toward position 5.46 of the 5-HT_2A receptor. Within the human 5-HT_2A receptor, position 5.46 is Ser242; however, in the rat 5-HT_2A receptor, it is Ala242, suggesting that the potent functional activity in this species resulted from the absence of the steric bulk provided by the -OH moiety of the Ser in the human isoform. We confirmed this hypothesis using site-directed mutagenesis through the mutation of both the human receptor Ser242 to Ala and the rat receptor Ala242 to Ser, followed by radioligand binding and second messenger studies. In addition, we attempted to define the space allowed by the alanine by evaluating compounds with larger substitutions at the 9-position. The data indicate that position 5.46 contributed to the species difference in 5-HT_2A receptor potency observed for a pyrazinoisoindolone compound, resulting in the observation of a significant cardiovascular safety signal.