Enantiomeric Separation, Absolute Configuration by X-ray Crystallographic Analysis, and Functional Evaluation of Enantiomers of the Dual Ligand, SYA0340 at 5-HT_1A and 5-HT_7A Receptors

We have previously identified 5-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (SYA0340) as a dual 5-HT1A and5-HT7 receptor ligand, and we posited such ligands mightfind utility in the treatment of various CNS related illnesses includingcognitive and anxiolytic impairments. However, SYA0340 has a chiralcenter and its enantiomers may confound the readouts for their functionalcharacteristics. Thus, in this study, we resynthesized SYA0340, separatedthe enantiomers, identified the absolute configurations, and evaluatedtheir binding affinities and functional characteristics at both the5-HT1A and 5-HT7A receptors. The results ofthis study show that the (+)-SYA0340-P1 [specific rotation [alpha]= +18.4 (deg.mL)/(g.dm)] has a binding affinity constant, K-i = 1.73 +/- 0.55 nM at 5-HT1AR and K-i = 2.20 +/- 0.33 nM at 5-HT7AR and (-)-SYA0340-P2 [specific rotation [alpha]= -18.2 (deg.mL)/(g.dm)] has K-i = 1.06 +/- 0.32 nM (5-HT1AR) and 4.7 +/- 1.1 nM(5-HT7AR). Using X-ray crystallographic techniques, theabsolute configuration of the P2 isomer was identified as the S-enantiomerand, therefore, the P1 isomer as the R-enantiomer. Functionally, bothSYA0340-P1 (EC50 = 1.12 +/- 0.41 nM; E (max) = 94.6 +/- 3.1%) and SYA0340-P2 (EC50 = 2.21 +/- 0.59 nM; E (max) = 96.8 +/- 5.1%) display similar agonist properties at the 5-HT(1A)Rwhile both enantiomers display antagonist properties at the 5-HT7AR with P1 (IC50 = 32.1 +/- 9.2 nM) displayingover 8 times greater potency as P2 (IC50 = 277 +/- 46nM). Thus, based on the functional evaluation results, SYA0340-P1is considered as the eutomer of the pair of enantiomers of SYA0340.It is expected that these enantiomers will serve as new pharmacologicalprobes for the 5-HT1A and 5-HT7A receptors.