Hexahydro-Diphenidol/Hexahydro-Sila-Diphenidol Type to Muscarinic Receptor Subtypes M

Br. J. Pharmacol. (1989), 98, 197-205 Binding and functional properties of antimuscarinics of the hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-sila-diphenidol type to muscarinic receptor subtypes M. Waelbroeck, M. Tastenoy, J. Camus, 1J. Christophe, *C. Strohmann, *H. Linoh, *H. Zilch, *R. Tacke, tE. Mutschler & tG. Lambrecht Department of Biochemistry and Nutrition, Medical School, Universite Libre de Bruxelles, Boulevard of Waterloo 115, B-1000 Brussels, Belgium, *Institute of Inorganic Chemistry, University of Karlsruhe, Engesserstrasse, D-7500 Karlsruhe, F.R.G. and tDepartment of Pharmacology, University of Frankfurt, Theodor-Stern Kai 7, D-6000 Frankfurt am Main, F.R.G. 1 In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2 The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3 Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4 A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5 The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro- diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle > cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 > glandular/smooth muscle, cardiac). Introduction Three muscarinic receptors with different selectivities nists such as 4-diphenylacetoxy-N-methyl-piperidine for antagonists have been described in binding methiodide (4-DAMP) (Barlow et al., 1976; Wael- studies as well as in in vitro and in vivo pharmaco- broeck et al., 1987a; Doods et al., 1987), hexahydro- logical assays. M1 receptors are above all found in diphenidol (hexahydro-difenidol) and the nervous system and are characterized by a high hexahydro-sila-diphenidol (hexahydro-sila-difenidol) affinity for pirenzepine (Hammer et al., 1980; Bird- (Mutschler & Lambrecht, 1984; Waelbroeck et al., sall et al., 1980; Brown et al., 1980; Hammer & Gia- 1987b; Lambrecht et al., 1988a, c), AF-DX 116 chetti, 1982; Lambrecht et al., 1988b). M2 receptors, (Hammer et al., 1986; Giachetti et al., 1986; Doods which show low affinity for pirenzepine, have been et al., 1987) and methoctramine (Melchiorre et al., further subdivided into cardiac type and glandular/ 1987; Michel & Whiting, 1988). smooth muscle subtypes by use of selective antago- Among these selective muscarinic antagonists, hexahydro-diphenidol (3a) and hexahydro-sila-di- 1 Author for correspondence. phenidol (3b) have been shown to have high affinity C) The Macmillan Press Ltd 1989 198 M. WAELBROECK et al. II:3, / OH [1 wOH El - f El f--\ X,0H3 \CH N N-CH3 iiIIIii\CH2-N NQ( \ kJ ~~~\-- CH3 la/lb 2a/2b (~</ OH /OH CH3 El ( El \CH2 - CH2 -CH2 -ND \CH2-CH2 CH2- N 3al3b 4al4b >xO OH OH El El/,, OHC3 r1Ii/ \CH2-CH2- NO rtII/uii \CH2 -CH2- N2 5al5b 6a/6b a: El = C, b: b: El = Si Figure 1 Structural formula of antagonists studied: demethyl-hexocyclium (la), demethyl-sila-hexocyclium (lb), hexocyclium (2a), sila-hexocyclium (2b), hexahydro-diphenidol (3a), hexahydro-sila-diphenidol (3b), N-methyl- hexahydro-diphenidol (4a), N-methyl-hexahydro-sila-diphenidol (4b), procyclidine (5a), sila-procyclidine (5b), tricyclamol (6a), and sila-tricyclamol (6b). for M1 receptors in neuronal tissues as well as for The experiments were carried out using the glandular/smooth muscle receptors in exocrine racemates of compounds la/lb to 6a/6b but some glands and smooth muscle, but a much lower affinity data of functional experiments with the individual for cardiac receptors (Mutschler & Lambrecht, 1984; enantiomers of Sa/5b and 6a/6b have been published Waelbroeck et al., 1987b; Lambrecht et al., 1988a,c). elsewhere (Tacke et al., 1986; 1987a) and binding In order to assess the structural requirements for the studies are in progress. observed selectivity of 3a/3b, the binding character- istics of a series of C/Si pairs structurally related to 3a/3b were determined (compounds la/lb, 2a/2b, 4a/ 4b-6a/6b; Figure 1). These experiments were carried Methods out using a NB-OK 1 cell line (M1 receptors type) and rat heart (cardiac receptors type) and pancreas Radioligand binding studies (glandular/smooth muscle receptors type) homogenates (Waelbroeck et al., 1987a,b,c). Furthermore Tissue preparations Male Wistar rats (200-250g) the binding affinities of compounds la/lb-6a/6b to were killed by decapitation and the pancreas and glandular/smooth muscle and cardiac receptor types heart were immediately removed. were compared with the functional antimuscarinic The pancreas was minced with scissors and properties in guinea-pig isolated ileum and atria. homogenized in 8 ml per pancreas of 300 mm sucrose Atropine, pirenzepine and methoctramine were used enriched with 0.2mgmlP1 bacitracin and 500 kalli- as reference drugs. krein inhibitor unitsml-1 of aprotinin, with a glass- SELECTIVE ANTAGONISTS AT MUSCARINIC RECEPTORS 199 Teflon homogenizer (7 up and down strokes). The homogenates). An incubation period of 2 h at 25°C resulting homogenate was filtered on two layers of was sufficient to allow equilibrium binding of [3H]- medical gauze, and immediately diluted 11 fold with NMS in this tissue (Waelbroeck et al., 1988). the incubation buffer (66 mm sodium phosphate Non-specific [3H]-NMS binding, using pancreas, buffer (pH 7.4) enriched with 2.6mm MgCl2 and heart and NB-OK 1 homogenates was defined as with 1.32% bovine serum albumin, 0.24mgml-' [3H]-NMS binding in the presence of 1 pM atropine. bacitracin and 600 kallikrein inhibitor units ml' In our binding conditions, non-specific binding re- aprotinin). presented less than 5% of total tracer binding, and The hearts were rinsed in isotonic NaCI, then was fully accounted for by binding to GF/C glass- homogenized in 2.5ml of Tris-HCI buffer (pH 7.5) fibre filters (see below). enriched with 250 mm sucrose, in an Ultraturrax To separate bound and free [3H]-NMS, in pan- homogenizer (5 s at maximum setting). The resulting creas, heart or NB-OK 1 homogenates, 2ml of ice- homogenate was diluted 8 fold with the same buffer cold 50mM sodium phosphate buffer (pH 7.4) was and filtered on two layers of medical gauze. added to each sample, followed by immediate fil- NB-OK 1 cells were cultured in RPMI 1640 tration on GF/C glass-fibre filters (Whatman, Maid- medium, enriched with 10% foetal calf serum, 100 stone, Kent, England) presoaked overnight in 0.05% unitsml-1 penicillin and 100pgmlPl streptomycin, polyethyleneimine. The filters were rinsed three times as described previously (Waelbroeck et al., 1988). with the same phosphate buffer, dried, and the radio- For [3H]-N-methylscopolamine ([3H]-NMS) bind- activity counted by liquid scintillation. ing experiments, the cells were rinsed, detached and centrifuged in 20 mm sodium phosphate buffer Analysis of binding data All competition curves (pH 7.4) containing 156 mm NaCl and 1 mm EDTA, were repeated at least three times, in duplicate. IC50 resuspended and homogenized in 20mm Tris-HCI values were determined by a computer-aided pro- buffer (pH 7.5) enriched with 5 mM MgCI2 and stored cedure described by Richardson & Humrich (1984), in liquid nitrogen. assuming the existence of only one receptor subtype. Experimental data points were within 3% of the Binding experiments: On rat pancreas homogenates, expected values, assuming that the molecules investi- 1 ml of diluted homogenate (800 to 1000 ug protein gated competed with [3H]-NMS for binding to a per assay) was added to 200 M1 of [3H]-NMS and single site. unlabelled drugs (in water) to obtain final concentra- Ki values were determined from IC50 values, by tions of 50 mm sodium phosphate buffer (pH 7.4), the Cheng & Prusoff (1973) equation which assumes 2 mM MgCl2, 1% bovine serum albumin, competitive inhibition of tracer binding to a single 0.2mg ml1 bacitracin and 500 kallikrein inhibitor receptor subtype. The [3H]-NMS KD value for the units ml-' of aprotinin. The [3H]-NMS concentra- three systems investigated was determined in tion was 240pM (with a KD value of 120PM in separate experiments, as described by Waelbroeck et pancreas). This incubation was prolonged for 4 h at al. (1987a,b; 1988). The pKi values, indicated in 25°C to allow equilibration of tracer binding to Table 1, corresponded to -log Ki values. pancreas receptors (Waelbroeck et al., 1987b). The standard deviation of p'C50 (-log IC50) On rat cardiac homogenates, 80 p1 of homogenate determination was equal to or below 0.10 log unit. (400 to 500jug of protein) was used per assay, in a Repeated determinations of [3H]-NMS KD values total volume of 1.2 ml containing (final were within 10% of each other. This error should be concentrations): 50 mM sodium phosphate buffer added to errors in IC0Sodeterminations, since [3H]- (pH 7.4), 2 mm MgCl2, 1% bovine serum albumin, NMS KD values were used to calculate pKi values.

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