Abstract

Successful development of 5-HT_2C agonists requires selectivity vs. the highly homologous 5-HT_2A receptor as 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, which 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 S242 however in the rat 5-HT_2A receptor it is A242, 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 utilizing site directed mutagenesis both through the mutation of the human receptor S242→A as well as the rat receptor A242→S followed by radioligand binding and second messenger studies. Additionally we attempted to define the space allowed by the alanine by evaluating compounds with larger substitutions at the 9-position. The data indicate 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.