ANNULATED HETEROCYCLIC BIOISOSTERES OF NORARECOLINE - SYNTHESIS AND MOLECULAR PHARMACOLOGY AT 5 RECOMBINANT HUMAN MUSCARINIC ACETYLCHOLINE-RECEPTORS

A series of O-alkylated analogs of 5,6,7,8-tetrahydro-4H-isoxazolo[4,5 -c]azepine-3-ol (THAO) were synthesized and characterized as ligands f or muscarinic acetylcholine receptors (mAChRs). O-Methyl-THAO (4a), O- ethyl-THAO (4b), O-isopropyl-THAO (4c), and O-propargyl-THAO (4d) were shown to be potent inhibitors of the binding of tritiated quinuclidin yl benzilate (QNB), pirenzepine (PZ), and oxotremorine-M (Oxo-M) to ti ssue membrane preparations. In the [H-3]Oxo-M binding assay, receptor affinities in the low nanomolar range were measured for 4a (IC50 = 0.0 10 mu M), 4b (IC50 = 0.003 mu M), 4c (IC50 = 0.011 mu M), and 4d (IC50 = 0.0008 mu M). Pharmacological effects (EC(50) or K-i values) and in trinsic activities (per cent of maximal carbachol responses) were dete rmined using five recombinant human mAChRs (m1-m5) and the functional assay, receptor selection and amplification technology (R-SAT). Compou nd 4c antagonized carbachol-induced responses at m1, m3, and m5. With the exception of 4b, which was an antagonist at m5, 4a,b,d showed part ial agonism at m1-m5 with very similar subtype selectivity (m2 > m4 > m1 > m3 > m5). Agonist index values for 4a-d, which were calculated fr om [H-3]QNB (brain) and [H-3]Oxo-M (brain) binding data, were shown to be predictive of pharmacologically determined intrinsic activities at m1-m5, the same rank order of intrinsic activity being observed at al l five mAChRs (4a > 4d > 4b > 4c). It is concluded that within this cl ass of high-affinity mAChR (m1-m5) ligands, containing secondary amino groups, minor changes of the bioisosteric ester alkyl groups have mar ked effects on potency and, in particular, intrinsic activity.