Synthesis and pharmacological evaluation of dimeric muscarinic acetylcholine receptor agonists

Two dimeric analogs of the muscarinic acetylcholine receptor (mAChR) agonis t phenylpropargyloxy-1,2,5-thiadiazole-quinuclidine (NNC 11-1314) were synt hesized and pharmacologically evaluated. In radioligand binding assays on C hinese hamster ovary (CHO) cell membranes expressing the individual human M -1 to M-5 mAChR subtypes, both dimers [(3S)-1,4-bis(3-[(3-azabicyclo[2.2.2] octanyl)-1,2,5-thiadiazol-4-yloxy]-1- propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1607) and (3S)1,3-bis-(3-[(3-azabicyclo[2.2.2]octanyl)-1,2,5-thiadi azol-4-yloxy]-1-propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1585)] exhibit ed higher binding affinities than the monomeric NNC 11-1314. Only NNC 11-15 85, however, displayed significant selectivity for the M-1 and M-2 mAChRs r elative to the other subtypes. Although binding studies in rat brain homoge nates supported the selectivity profile of NNC 11-1585 observed in the CHO membranes, rat heart membrane experiments revealed complex binding behavior for all three agonists that most likely reflected differences in species a nd host cell environment between the heart and CHO cells. Subsequent functi onal assays with phosphatidylinositol hydrolysis revealed that all three no vel ligands were partial agonists relative to the full agonist oxotremorine -M at the CHO M-1, M-3, and M-5 mAChRs, with NNC 11-1607 displaying the hig hest functional selectivity. In the CHO M-2 and M-4 mAChR cells, agonist-me diated effects on forskolin-stimulated cAMP accumulation were characterized by bell-shaped concentration-response curves, with the exceptions of NNC 1 1-1607, which had no discernible effects at the M-2 mAChR, and NNC 11-1585, which could only inhibit cAMP accumulation at the M-4 mAChR. Thus, we iden tified NNC 11-1607 as a novel functionally selective M-1/M-4 mAChR agonist. Our data suggest that dimerization of mAChR agonists is a viable approach in designing more potent and functionally selective agonists, as well as in providing novel tools with which to probe the nature of agonism at these r eceptors.