European Journal of Pharmacology, 168 (1989) 71-80 71 Elsevier EJP 50940 Affinity profiles of hexahydro-sila-difenidol analogues at muscarinic receptor subtypes Giinter Lambrecht *, Roland Feifel, Monika Wagner-Rôder, Carsten Strohmann \ Harald Zilch Reinhold Tacke \ Magali Waelbroeck Jean Christophe ^, Hendrikus Boddeke ^ and Ernst Mutschler Department of Pharmacology, University of Frankfurt, D-6000 Frankfurt / M, F.R.G., ' Institute of Inorganic Chemistry, University of Kartsruhe, D-7500 Karlsruhe, F.R.G., ' Department of Biochemistry and Nutrition, Médical School, Free University of Brussels, B-lOOO Brussels, Belgium, and Preclinical Research, Sandoz Ltd., CH-4002 Basel. Switzerland Received 20 April 1989, accepted 13 June 1989 In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a séries of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M,­ (rabbit vas deferens), Mj­ (guinea­pig atria) and M 3­ (guinea­pig ileum) receptors. Ail compounds were compétitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p­fluoro­hexahydro­difenidol displayed the same affinity profile as the parent compound, hexahydro­sila­difen­ idol: M, = M3 > M2. A différent selectivity pattem was observed for p­fluoro­hexahydro­sila­difenidol: M3 > M, > Mj. This compound exhibited its highest affinity for Mj­receptors in guinea­pig ileum (pA2 = 7.84), intermediate affinity for M,­receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the Mj­receptors in guinea­pig atria (pAj = 6.01). This receptor selectivity profile of p­fluoro­hexahydro­sila­difenidol was confirmed in ganglia (M,), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M,) or hexahydro­ sila­difenidol (M2 and M3) and the p­fluoro analogue used in combination suggested that the antagonism was additive, implying mutual compétition with a single population of muscarinic receptor subtypes. Thèse results indicate that p­fluoro­hexahydro­sila­difenidol represents a valuable tool for characterization of muscarinic receptor subtypes. Muscarinic receptor subtypes; Muscarinic Mj­selective antagonists; Hexahydro­sila­difenidol analogues; p­Fluoro­hexahydro­sila­difenidol; Pirenzepine; Methoctramine: Vas deferens (rabbit); Ganglia (rat); (Structure­activity relationships) 1. Introduction tors: M, (neuronal type), Mj (cardiac type; M2„, Mutschler et al., 1988) and Mj (smooth muscle/ Radioligand binding and functional data, have glandular type; M2/3, Mutschler et al., 1988) (for allowed the clear démonstration that there are at récent reviews, see Birdsall and Hulme, 1983; least three major subtypes of muscarinic recep­ Eglen and Whiting, 1986; Mitchelson, 1988; Mutschler et al., 1988). M,­receptors are found in high density in neuronal tissues such as autonomie ganglia, cérébral cortex and hippocampus, whereas * To whom al! correspondence should be addressed: Depart­ ment of Pharmacology, University of Frankfurt, Theodor­ M 2" and Mj­receptors are mainly présent in lower Stern­Kai 7. Gebâude 75A, D­6000 Frankfurt/M, F.R.G. brain areas and in peripheral effector organs such 0014­2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomédical Division) 72 as heart (Mj), smooth muscle (M3) and glands (M3). This pharmacological subclassification of muscarinic receptors is based mainly on the différ• ent affinities of sélective antagonists such as HO-Si -CH2-CH2-CH2-NRJ pirenzepine (M]>M2 = M,) (Hammer et al., o 1980; Hammer and Giachetti, 1982; Doods et al., 1987; Lambrecht et al., 1988c; Waelbroeck et al., "Hexahydro-sila-difenidol 1986; 1988), methoctramine and AF-DX 116 (M^ > M, > M3) (Melchiorre et al., 1987; Micheletti No. El R et al., 1987; Melchiorre, 1988; Waelbroeck et al., 4 SI 0-OCH3 1988) , and hexahydro-sila-difenidol (M, = M3 > M2) (Fuder et al., 1985; Giraldo et al., 1988; 5 Si P-OCH3 S Si p-CI Lambrecht et al., 1988a; Waelbroeck et al., 1988). H0-El-CH2-CH2-CH2-N^^ More recently, différences in the amino acid JS C p-F séquences of muscarinic receptor subtypes have Ib Si p-F been demonstrated by cloning, sequencing and expression of complementary DNA encoding thèse Fig. 2. Chemical structure of hexahydro-sila-difenidol (1) and receptors (Kerlavage et al., 1987; Peralta et al., its analogues 2-6. 7a and 7b. 1987; Akiba et al., 1988; Brann et al., 1988). The antagonist binding properties of the individual cloned receptors (ml, m2 and m3) and their pat- difenidol analogues are controlled by the nature of terns of expression in various tissues correspond the central atom (El = C, Si) and the structure of closely to those of the pharmacologically defined the ring System 'R' bound to this atom, the ab- M,-, M,- and Mj-receptors, respectively (Peralta solute configuration at the central atom and the et al., 1987; Akiba et al., 1988; Buckley et al., length of the alkylene chain (Tacke et al., 1986; 1989) . 1987; 1989; Eltze et al., 1988; Lambrecht et al., Hexahydro-sila-difenidol (1, fig. 2) was dis- 1988b; 1989). The main goal of the présent study covered in the course of structure-activity (selec- was to characterize the structural demands for tivity) relationship studies of a séries of difenidol potency and selectivity among hexahydro-sila-di• and sila-difenidol analogues (fig. 1) (for récent fenidol analogues modified in the cyclic amino reviews, see Tacke and Becker, 1987; Mutschler et group and in the phenyl ring System. Conse- al., 1988). Structurally, thèse molécules consist of quently, the antagonist affinities of compounds a central carbinol (silanol) carbon (silicon) atom 2-6, 7a and 7b (fig. 2) were determined and com- carrying an OH group, two ring Systems and an pared with those obtained for the parent com- aminoalkyl group [(CH2)„-NR2] (figs. 1 and 2). It pound hexahydro-sila-difenidol (1) and the référ• was found in preceding investigations that the ence drugs pirenzepine and methoctramine. The potency and selectivity of the difenidol and sila- receptors studied were M,-receptors in rabbit vas deferens (Eltze, 1988; Eltze et al., 1988), cardiac M 2-receptors in guinea-pig atria and smooth muscle M3-receptors in guinea-pig ileum. Further- more, we report on in vitro experiments with hexahydro-sila-difenidol and its p-fluoro deriva- HO {CH2),- tive 7b in ganglionic (M,), atrial (M2) and ileal (M3) préparations of the rat. The agonist independence of the antagonism Fig. 1. General formula of antimuscarinic agents of the difen• idol (El = C, R = phenyl, n = 3) and sila-difenidol (El = Si, was investigated to verify the mechanism of action R = phenyl, n = 3) type. El = C, Si; R = aryl, cycloalkyl; n = of p-fluoro-hexahydro-sila-difenidol (7b) (Lam• 1-4. brecht et al., 1988a). In addition, antagonist com- 73 bination experiments were carried out and sion. A force-displacement transducer connected analyzed according to the dose-ratio method of to a Hellige amplifier and a multichannel recorder Paton and Rang (1965). The results are discussed were used for thèse measurements. Strips of ileal in terms of their implications for muscarinic re- longitudinal muscle (1.5 cm length) were prepared ceptor classification. according to Paton and Zar (1968). The tissue responses to the cumulative addition of arecaidine propargyl ester were measured as isotonic contrac• 2. Materials and methods tions and recorded as with the atria. 2.1. Rabbit isolated vas deferens 2.3. Isolated superior cervical ganglion of the rat Experiments on rabbit isolated vas deferens Experiments on ganglia were performed as de• were performed as described by Eltze (1988). scribed by Brown et al. (1980). Superior cervical Maie New Zealand white rabbits were killed by ganglia were excised from maie Sprague-Dawley i.v. injection of pentobarbital sodium (120 mg/kg). rats (200-300 g) that had been anaesthetized with Vasa deferentia were removed and segments of 1.5 urethane (1.2 g/kg i.p.). Each ganglion was de- cm length were suspended in 7 ml organ baths sheathed, suspended vertically in a separate heated containing modified Krebs buffer (composition in chamber (36 °C) and superfused with oxygenated mM: NaCl 118.0, KCl 4.7, CaClj 2.5, MgS04 0.6, (95% 0,-5% CO2) Krebs solution (1 ml/min) KH2PO4 1.2, NaHCOj 25.0, ( + )-glucose 11.1; which consisted of (mM): NaCl 124.0, KCl 3.0, 10"^ M yohimbine was included to block aj- NaHCO, 26.0, NaH2P04 1.25, CaCl2 2.0, MgCl2 adrenoceptors) gassed with 95% Oj-S^o CO, at 2.0 and (-I-)-glucose 10.0. The muscarine-induced 31° C. A resting tension of 0.75 g was applied and depolarization (EC50 ^ ^0 recorded dif- isometric twitch contractions were elicited by elec- ferentially, via two calomel électrodes, between trical field stimulation (0.05 Hz, 0.5 ms, 30 V). ihe ganglion and its postganglionic trunk. The DC Thèse effects were concentration dependently in- potentials were amplified by microvoltmeters hibited by the Mj-selective agonist, 4-(4-chloro- (Keithley 177) and were monitored on a chart phenylcarbamoyloxy)-2-butynyltrimethylammoni- recorder. Concentration-response curves were um iodide (4-Cl-McN-A-343) (Eltze et al., 1988) made with single doses of muscarine applied at and recorded as with the atria. 20-45 min intervais, followed by a washout phase until the baseline was reached. 2.2. Isolated atria and ileum of the guinea-pig and rat 2.4. Antagonist affinities Adult guinea-pigs and Wistar rats of either sex The tissues were allowed to equilibrate for 30-60 were killed by a blow to the head. Left atria and min.