Table 2.

Screening of the Second Set Compounds 20–28 (Receptor Binding Affinities to M₁, M₂, and M₃ (K_i in μM), M₂ Receptor Activation and Docking Data)

compd	R1	R2	X	Ki [μM]a			IP accumulation assayb		docking
				M1R	M2R	M3R	EC50 [μM]c	Emax [%]d	docking score active state	H-bond to Asn6.52	predicted agonist?
20				0.74 ± 0.13	1.0 ± 0.25	0.78 ± 0.10	8.2 ± 5.5	−12 ± 1.5	−40.99	Y	+
21	OH	H	H,H	20 ± 11	14 ± 4.7	13 ± 4.8	10 ± 4.1	44 ± 10	−36.29	Y	+
22	OMe	F	H,H	7.1 ± 2.5	0.98 ± 0.61	14 ± 5.9	23 ± 10	44 ± 4.7	−29.73	Y	+
23	OMe	H	H,OH	13 ± 5.5	29 ± 8.7	12 ± 4.2		<10e	−32.23/−30.97g	Y/N	+/−
24	OMe	H	O	0.17 ± 0.014f	2.4 ± 0.42f	0.21 ± 0.028f		<10e	−34.56	Y	+
25	OEt	H	H,H	42 ± 14	27 ± 9.9	43 ± 10		40e	−23.00	Y	+
26	Cl	H	H,H	4.5 ± 1.1	4.8 ± 1.4	5.8 ± 1.8		<10e	−39.07	N	−
27	OCF3	H	H,H	7.8 ± 1.6	9.2 ± 2.1	11 ± 6.6		<10e	−28.6	Y	+
28				6.4 ± 0.96	13 ± 3.3	6.3 ± 1.7	21 ± 7.6	62 ± 6.6	−23.47	Y	+

^aK_i values ± SEM derived from three to eight individual competition binding experiments using the radioligand [³H]N-methyl-scopolamine bromide.

^bSecond, less sensitive IP accumulation assay with COS cells coexpressing M₂R and Gα_qi5HA.

^cEC₅₀ values ± SEM from three individual experiments each done in triplicate.

^dE_max values ± SEM relative to the full effect of carbachol.

^eMaximum effect at 100 μM; no complete dose–response curve could be determined.

^fK_i values ± SD derived from 2 individual competition binding experiments.

^gRacemic mixture.