Synthesis and pharmacological characterization of enantiomerically pure muscarinic agonists: Difluoromuscarines

The four homochiral 4-deoxy-4,4-difluoromuscarine stereoisomers (difluoromuscarines) were prepared in very high enantiomeric excess. A convenient sequence based on the use of natural as well. as `'unnatural'' ethyl lactate allowed the synthesis of target compounds, whose absolute configuration is dictated by that of the starting synthon. Quaternary ammonium salts (+)-5, (-)-5, (-)-6, and (+)-6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M(2) (heart) and M(3) (ileum and bladder) muscarinic receptor subtypes. The eutomer (+)-5 and distomer (-)-5 were also tested in vivo on pithed rat, and their muscarinic activity at the M(1) receptor subtype was compared with those of racemic muscarine {[(+/-)-1] and (2S,4R,5S)-4-deoxy-4-fluoromuscarine [(+)-4]. Further pharmacological parameters such as affinity, relative efficacy, and enantioselectivity have been determined for compounds (+)-5 and (-)-5 at M(2) (heart force and rate) and M(3) (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The four homochiral difluoromuscarines behave as muscarinic agonists in all the tests with a potency trend which is different from that previously observed with the 4-deoxy-4-fluoromuscarines and (+/-)-1, thus indicating the intervention of the second fluorine atom on the receptor-ligand interaction. Moreover, the second fluorine atom produces. significant differences in the affinity and relative efficacy values of compounds (+)-5 and (-)-5 at M(2) and M(3) subtypes, which could be attributed to a heterogeneity between the muscarinic receptors mediating heart rate and heart force and those involved in the contraction of ileum and bladder.}