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 (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; : 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 -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. Concentration-response curves to the agonists ileum were set up under 0.5 g tension in 6 ml were obtained before and after adding an antago• organ baths containing oxygenated (95% 02-5% nist. ECso values were determined for the control CO2) Tyrode solution (32° C; composition in mM: and the antagonist-shifted concentration-response NaCl 137.0, KCl 2.7, CaClî 1.8, MgCl, 1.05, curves. Unless indicated otherwise (table 1), at NaHCOj 11.9, NaH2P04 0.42, ( + )-glucose 5.6). least three concentrations (log interval = 0.48) of The atria were paced at 2 Hz, with bipolar antagonists were tested in the three tissues, allow- platinum électrodes, square-wave puises of thresh- ing 15-60 min équilibration time. Each concentra• old ( + 50%) voltage and 3 ms puise width. Néga• tion of antagonist was tested 3-5 times and the tive inotropic responses to the cumulative addition ratios of agonist molar EC50 values obtained in the of the , arecaidine propargyl absence and présence of antagonists were calcu- ester (Mutschler and Hultzsch, 1973; Moser et al., lated. Schild plots were made, using linear régres• 1989) were measured as changes in isometric ten• sion, by the method of least squares. The slopes of 74 thèse plots were not significantly (P > 0.05) différ• chloride) (Serva); arecaidine propargyl ester ent from unity. The pAj values were thus esti- (Mutschler and Hultzsch. 1973), 4-Cl-McN-A-343 mated by fitting the best straight line with a slope (4-(4-chlorophenylcarbamoyloxy)-2-butynyltri- equal to unity (Tallarida et al., 1979). methylammonium iodide) (Nelson et al., 1976) as well as compounds 1-6, 7a and 7b were synthe- 2.5. Antagonist combination experiments sized in our laboratories (Tacke et al., 1985: 1; silicon compounds 2-6 and 7b were prepared by The effects of p-fluoro-hexahydro-sila-difenidol analogy to the synthesis of the parent compound 1 (7b) in combination with pirenzepine in rabbit vas and the carbon compound 7a was prepared by deferens or hexahydro-sila-difenidol (1) in guinea- analogy to the synthesis of hexahydro-difenidol; pig atria and ileum were investigated on the basis unpublished results). The other chemicals were of of dose ratio analysis (Paton and Rang, 1965). The reagent grade and were used as purchased. concentration-response curves to the agonists in Compounds 1-6, 7a and 7b possess a centre of thèse experiments were made under control condi• chirality and therefore exist in two enantiomers. tions then in the présence of pirenzepine (50 nM), They were used as racemates because indications hexahydro-sila-difenidol (atria; 3 juM; ileum: 100 were found that the enantiomers of silanols of this nM) or p-fluoro-hexahydro-sila-difenidol (vas de• type may racemize in aqueous solution (Tacke et ferens: 1 juM; atria: 3 juM; ileum: 100 nM) alone al., 1987). or with the respective two antagonists combined. The contact time for antagonists was always 30 3. Results min and the agonists used were 4-Cl-McN-A-343 (vas deferens) and arecaidine propargyl ester (atria The inhibition of twitch contraction induced by and ileum), respectively. The expérimental data 4-Cl-McN-A-343 (EC50 = 250 nM) in field-stimu- for individual antagonist applications were used to lated rabbit vas deferens and the muscarine-in- calculate dose ratios (DR) expected for the combi• duced depolarization in rat gangha as well as the nation for two models: expected single-site = DR, arecaidine propargyl ester-mediated responses in -I- DR2 — 1 and expected independent sites = DRj atria and ileum of guinea-pigs and rats were X DR 2. Thèse dose ratios were compared with the antagonized by compounds 1-6, 7a and 7b. The experimentally observed combined mean dose antagonists produced concentration-dependent ratios. parallel shifts of the agonist concentration-re• sponse curves without either basai tension or max• 2.6. Statistics imum response being affected at the concentration The data are presented as means ± S.E.M. of range tested. The Schild plots were linear and the 8-18 experiments with each préparation. The dif• slopes were not significantly différent from unity férences between mean values were tested for stat- (P > 0.05). Thus, ail compounds were apparently istical significance (P < 0.05) by means of Stu- simple compétitive muscarinic antagonists. Thèse dent's t-test. results are summarized in tables 1 and 2.

2.7. Drugs 3.1. Antagonist affinities in rabbit vas deferens and guinea-pig atria and ileum (carbamylcholine chloride), d,l- muscarine chloride and yohimbine hydrochloride Compounds 1-6, 7a and 7b showed quite wide (Sigma); sesquifumarate (Ega-Che- variations in their affinities to the mu.scarinic re• mie); pirenzepine dihydrochloride (Boehringer In- ceptors in vas deferens, atria and ileum, their pA2 gelheim); methoctramine tetrahydrochloride values (table 1) differing by more than two orders (kindly provided by Dr C. Melchiorre, University of magnitude. As with the parent compound, of Bologna, Italy); McN-A-343 (4-(3-chlorophen- hexahydro-sila-difenidol (1), the derivatives 2-6 ylcarbamoyloxy)-2-butynyltrimethylammonium and 7a exhibited similar affinities at receptors 75

TABLE 1 Affinity profiles of pirenzepine. methoctramine and compounds 1-6, 7a and 7b at muscarinic M,-receptors in rabbit vas deferens (RVD) and at Mj-receptors in guinea-pig atria (GPA) as well as at M,-receptors in guinea-pig ileum (GPI). pAj values and slopes of Schild plots (in parenthèses) were calculated as described in Methods (antagonist affinities) and are presented as means + S.E.M. The ratios of affinity constants are given as a measure of receptor selectivity. Thèse values were calculated from the antilogs of the différences between respective pA2 values.

Selectivity ratios

RVD-M, GPA-Mt GPI-M , M,/M, M3/M, M3/M2

Pirenzepine " 8.24 ±0.06 6.82 ±0.03 6.88 ±0.04 26 0 04 1.2 (1.19±0.10) (0.98 ±0.02) (1.07 ±0.04) Methoctramine 6.85 ±0.07 " 7.69 ±0.03 6.17 ±0.05 0.15 0.21 0.03 (1.19±0.10) (1.05 ±0.05) (0.99 + 0.09) 7.92 ±0.07 6.53 ±0.05 7.96 ±0.03 25 1.1 27 {l.yjy ± U.Uj) (U.yo ± o.yjy) yyj.yj ± U.UO} 2 7.78 ±0.09 ^ 6.73 ±0.04 8.13 ±0.05 11 2.2 25 (1.07 ±0.04) (0.96 ±0.04) (0.88 ±0.08) 3 7.13±0.10 5.92 ±0.04' 7.48 ±0.02 16 2.2 36 (1.23 ±0.07) (1.06 ±0.05) 4 6,77 ±0.10 5.79 ±0.06 ' 6.74 ±0.05 9.5 0.93 8.9 (1.21 ±0.06) (0.99±0.11) 5 6.74 + 0.08'' 5.59±0.03 ^ 7.09 ±0.06 14 2.2 32 (1.06 ±0.04) (0.94±0.12) 6 6.82 ±0.07 5.50 ±0.05 7.06 ±0.03 21 1.7 36 (1.08±0.21) (0.93 ±0.07) 7â 7.56 ±0.07 6.58 ±0.03 7.93 ±0.03 9.5 2.3 22 (0.95 ±0.16) (1.05±0.08) (0.89 ±0.04) 7b'' 6.68 ±0.03' 6.01 ±0.06 7.84 + 0.03 ' 4.7 15 68 (1.12±0.21) (1.08 + 0.06)

" Data taken from Eltze and Figala (1988) and Lambrecht et al. (1988a). Hexahydro-sila-difenidol. ' Only one or two concentra• tions of the antagonists were investigated due to the négative inotropic effects of the antagonists themselves at higher concentrations: 3 (1 fiM). 4 (3 nM), 5 (3 and 10 fiM). 6 (1 fiM), 7b (1 and 3 fiM). The pAj values were therefore determined from the individual dose ratios according to Tallarida et al. (1979). pA, values and slopes of Schild plots taken from Eltze et al. (1988). ' A pAj value of 6.95 ±0.04 was obtained using McN-A-343 as agonisl. ' pA, values of 7.83 ±0.08 and 7.89 ±0.07 were obtained using carbachol and oxotremorine, respectively, as agonists.

TABLE 2

The affinities of hexahydro-sila-difenidol (1) and p-fluoro-hexahydro-sila-difenidol (7b) for muscarinic Mpreceptors in rat superior cervical ganglia (RG), Mj-receptors in rat atria (RA) and Mj-receptors in rat ileum (RI). pA, values and slopes of Schild plots (in parenthèses) are presented as means±S.E.M. The ratios of affinity constants are given as a measure of receptor selectivity. Thèse values were calculated from the antilogs of the différences between respective pAj values.

pA, Selectivity ratios

RG-M, " RAM, RI-M, M3/M, Mj/M, 1 7.54 ±0.07 6.22 ±0.05 7.77 ±0.06 21 1.7 36 (0.98 ±0.04) (1.20±0.11) (1.00±0.11) 7b 7.21 ±0.04 5.99 ±0.04 7.88 ±0.02 16 4.7 78 (1.04 ±0.06) (0.91 ±0.10) (0.98+-0.05)

" The pAj value observed in this préparation for pirenzepine was 8. 30 (Lambrecht et al., 1988c). 76

lleum pA2 lleum

8 9 pA2 Atria pA2 Vas deferens

pA2 Atria

pA2 Vas deferens

Fig. 3. Comparison of the affinities (pAj; table 1) of pirenzepine (Pz), methoctramine (Me) and compounds 1-6, 7a and 7b at muscarinic M,-receptors in rabbit vas deferens (panels b and c), Mj-receptors in guinea-pig atria (panels a and c) and Mj-receptors in guinea-pig ileum (panels a and b). If the affinities are not the same for two receptors. the results deviate from the theoretical equality line (y = x), and the distance from this line is then a measure of receptor selectivity. The dotted Unes represent the following receptor selectivity ratios, panel a: M, (ileum)/M2 (atria) = 25; panel b; M, (ileum)/M, (vas deferens) = 2.2; panel c: M, (vas deferens)/M2 (atria) = 15. présent in the vas deferens and ileum (dotted line A différent selectivity pattern was observed for in fig. 3, panel b), but the pAj values obtained in p-fluoro-hexahydro-sila-difenidol (7b). This anti- atria were much lower. This selectivity profile of muscarinic agent exhibited the highest affinity the compounds is demonstrated by the selectivity (pA2 = 7.84) for the receptors in the ileum, an ratios given in table 1 and is illustrated in fig. 3. intermediate affinity for the receptors in vas de- 77

ferens (pA2 = 6.68) and lowest affinity for the the parent compound 1 was investigated in rabbit cardiac receptors (pA2 = 6.01). vas deferens and guinea-pig atria and ileum, re- spectively, on the basis of dose ratio analysis 3.2. Antagonist affinities in rat ganglia, atria and (Paton and Rang, 1965). The results of thèse ex• ileum periments are summarized in table 3. The com- bined dose ratios obtained experimentally in the Hexahydro-sila-difenidol (1) and its p-fluoro three préparations were very close to those calcu- derivative 7b were tested in three isolated prépara• lated for two antagonists acting at a single site. tions of the rat: superior cervical ganglia (M,), The results are not consistent (P < 0.05) with a atria (Mj) and ileal longitudinal muscle (M3). The multiplication of dose ratios which would be ex• pAj values and corresponding Schild slopes are pected if two antagonists interacted at indepen• shown in table 2. Although there were some dent sites. quantitative différences, both compounds showed qualitatively the same receptor selectivity profile as was found in rabbit vas deferens and guinea-pig 4. Discussion atria and ileum (table 1); hexahydro-sila-difen• idol: Mj = M, > M2, p-fluoro-hexahydro-sila-dif- 4.1. General considérations enidol: M3 > M, > Mj. The presynaptic muscarinic receptors in rabbit 3.3. Combination experiments vas deferens and the postsynaptic receptors in guinea-pig atria and ileum have been well estab- The mutual compétition between p-fluoro- lished to belong to différent subclasses: ganghonic hexahydro-sila-difenidol (7b) and pirenzepine or Mj-, cardiac M 2- and smooth muscle/glandular M^-receptors (Eltze, 1988; Eltze and Figala, 1988; TABLE 3 Eltze et al., 1988; Lambrecht et al., 1988a-c; 1989). This heterogeneity is based mainly on the différ• Summary of dose ratio analysis from the p-fluoro-hexahydro- sila-difenidol (7b) combination experiments. Each dose ratio ent affinities of sélective antagonists such as (DR) is the mean + S.E.M. and the number of observations is pirenzepine (M, > = M3), methoctramine (Mj given in parenthèses. > M) > M3) and hexahydro-sila-difenidol (1; M, Mean dose ratio = M3>M2) (table 1).

Rabbit vas deferens (M/) The présent study concerned the effects of Pirenzepine (50 nM) 6.8±0.8 (n = 5) changing the size of the cychc amino group (com• Compound 7b (1 /iM) 15 + 2.3 (n = 5) pounds 2 and 3) and introducing substituents in Expected single site DR " 20.8 the phenyl ring (compounds 4-6, 7b) of hexahy• Expérimental combination DR 27 ±2.5 (n = 5) dro-sila-difenidol (1) as well as of replacing the Expected independent site DR 102 central silicon atom of compound 7b by a carbon Guinea-pig atria (M2) atom (compound 7a) on muscarinic potency. Compound l(3 /iM) 12±1.2 (n = 6) The principal conclusions drawn from thèse Compound 7b (3 jiM) 3.9±0.1 (n = 6) experiments are that: (1) compounds 1-6, 7a and 7b Expected single site DR " 14.9 act as compétitive muscarinic antagonists in rabbit Expérimental combination DR 18 + 4.5 (n = 6) Expected independent site DR 47 vas deferens as well as in guinea-pig atria and ileum. They produced an antagonism that was Guinea-pig ileum (M^) réversible and surmountable and their apparent Compound 1 (100 nM) 16 + 2.2 (n = 8) Compound 7b (100 nM) 8.7 + 0.8 (n = 8) affinity in each tissue appeared to be independent Expected single site DR " 23.7 of the concentrations used. Thus, Schild analysis Expérimental combination DR 20 ±2.7 (n = 8) yielded slopes which were not significantly différ• Expected independent site DR 139 ent from unity (table 1). The affinity constants of " DRi +DR2 -1. DR, xDRj. = Hexahydro-sila-difenidol. p-fluoro-hexahydro-sila-difenidol (7b) in rabbit 78 vas deferens and guinea-pig ileum were indepen- up to more than one order of magnitude (table 1). dent of the agonist used (rabbit vas deferens: Thus, compounds 4-6 were even less potent than McN-A-343 = 6.95; 4-Cl-McN-A-343 = 6.68; the hexamethyleneimino derivative 3. On the other guinea-pig ileum: arecaidine propargyl ester = hand, compounds 4-6 retain the same selectivity 7.84; carbachol = 7.83; oxotremorine = 7.89) (ta• profile as the parent compound 1 and the deriva- ble 1). In addition to the data obtained from the tives 2 and 3: M, = M, > (table 1 and fig. 3). Schild analysis, combination studies (table 3) A novel affinity profile was observed for p- showed compound 7b to act in a manner indis- fluoro-hexahydro-sila-difenidol (7b): M3 > M, > tinguishable from 'classical' compétitive antago- M2. This compound exhibited high affinity for nists. (2) Structural variations in the hexahydro- smooth muscle Mj-receptors in guinea-pig ileum sila-difenidol molécule led to new muscarinic (pA2 = 7.84) while its antimuscarinic potency at antagonists which exhibit a qualitatively and/or M,-receptors in rabbit vas deferens and cardiac quantitatively différent spectrum of receptor selec- M 2-receptors in guinea-pig atria was lower by tivity to the parent compound. The selectivity factors of 15 and 68, respectively (table 1, fig. 3). observed does not appear to be associated with This novel selectivity profile of 7b is based on the high affinity as there are less potent compounds fact that the para-fluoro substituent leads to a with appréciable selectivity (compounds 4-6; table decrease in M,- and Mj-antimuscarinic potency 1 and fig. 3). while only shghtly affecting the affinity towards M 3-receptors. 4.2. Structure-selectivity relationships The pAj values determined for p-fluoro-hexa- hydro-sila-difenidol (7b) in rat atria and ileum The influence of the ring size of the cyclic (5.99 and 7.88, respectively; table 2) were very amino group on potency and selectivity can be close to that found in the corresponding guinea-pig demonstrated by comparison of compounds 1-3. tissues (6.01 and 7.84, respectively; table 1). The As can be seen from the data in table 1 the pA2 value observed for compound 7b at M,-re• affinity to the three muscarinic receptor subtypes ceptors in rat superior cervical ganglia (7.21 using dépends on the nature of the heterocyclic ring muscarine as agonist; table 3) was slightly higher System and varies up to about 6-fold. Compound than that found in rabbit vas deferens (6.68 using 2, containing a pyrrolidino ring, shows nearly the 4-Cl-McN-A-343 and 6.95 using McN-A-343 as same affinity to M,-, Mj- and Mj-receptors as the agonists; table 1). However, the data obtained parent compound, hexahydro-sila-difenidol (1). In with the three rat tissues show that the receptor contrast, ring extension to the hexamethyleneimino selectivity profile of p-fluoro-hexahydro-sila-difen- derivative 3 results in a decrease in affinity for ail idol (M3 > M, > M2) is species-independent. This subtypes. However, the influence of the size of the also holds for the parent compound, hexahydro- cyclic amino group on selectivity is moderate as sila-difenidol (M, = M3 > M2; tables 1 and 2). compounds 2 and 3 showed the same selectivity Replacement of the central silicon atom in pattern as the parent compound I : Mj = M3 > Mj. compound 7b by a carbon atom (7b 7a) in- Therefore compounds 1-3 deviate significantly creased the affinity for M,-receptors in rabbit vas from the line of équivalence when the pAj values deferens as well as for cardiac M j-receptors in in guinea-pig atria are compared to those in the guinea-pig atria by factors of 8 and 4, respectively, ileum and rabbit vas deferens, respectively (fig. 3, whereas the affinity to M 3-receptors in guinea-pig panels a and c). ileum was not changed significantly (P > 0.05) (ta• Comparison of compounds 1, 4-6 and 7b de- ble 1). Thus, the selectivity profile of p-fluoro- fines the effect of substituents in the phenyl ring hexahydro-difenidol (Ta) is similar to that of the of hexahydro-sila-difenidol. Either a methoxy sub• silicon compounds 1-6 (fig. 3), but differs from stituent in the ortho or para position (compounds that of its sila analogue 7b). This is a further 4 and 5) or a para-chloro substituent (compound example indicating that sila substitution (C/Si 6) reduces the affinity to the muscarinic receptors exchange) may be a useful tool to improve recep- 79 tor selectivity (for a récent review on C/Si-bioiso- Buckley, N.J., T.I. Bonner, CM Buckley and M.R. Brann, sterism, see Tacke and Linoh, 1989). 1989, Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-Kl cells, Mol. In conclusion this report describes structure-ac- Pharmacol. 35, 469. tivity relationships of antimuscarinics related to Doods, H.N., M.I. Mathy, D. Davidesko, K.J. Van Charldorp, hexahydro-sila-difenidol (1). The antimuscarinic A. De Jonge and P.A. Van Zwieten, 1987, Selectivity of potency and selectivity in this séries of compounds muscarinic antagonists in radioligand and in vivo experi- (1-6, 7a and 7b; fig. 2) is controlled by the sub• ments for the putative M,, M2 and M3 receptors, J. Pharmacol. Exp. Ther. 242, 257. stitution pattern of the phenyl moiety, the struc• Eglen, R.M. and R.L. Whiting, 1986, Muscarinic receptor ture of the cyclic amino group and the nature of subtypes: A critique of the current classification and a the central atom 'El' (carbon or silicon). Com• proposai for a working nomenclature, J. Auton. Pharmacol. pounds with qualitatively and/or quantitatively 5, 323. différent receptor selectivity profiles were ob- Eltze, M., 1988, Muscarinic M,- and Mj-receptors mediating opposite effects on neuromuscular transmission in rabbit tained. Among the antimuscarinic agents tested in vas deferens, European J. Pharmacol. 151, 205. this study, p-fluoro-hexahydro-sila-difenidol (7b) Eltze, M. and V. Figala, 1988, Affinity and selectivity of is the only compound which showed appréciable enantiomers for muscarinic receptor subtypes, Mj-receptor selectivity (M3 > M, > Mj). Its affin- European J. Pharmacol. 158, 11. ity profile is différent from that of the parent Eltze, M., G. Gmelin. J. Wess, C. Strohmann, R. Tacke, E. Mutschler and G. Lambrecht. 1988, Presynaplic muscarinic compound, hexahydro-sila-difenidol (1) (M, = M3 receptors mediating inhibition of neurogenic contractions > Mj), and its selectivity for M,-receptors is com• in rabbit vas deferens are of the ganglionic M,-type, parable to that of pirenzepine for M,- and European J. Pharmacol. 158, 233. methoctramine for Mj-receptors (fig. 3). p- Fuder, H., H. Kilbinger and H. Millier, 1985, Organ selectivity Fluoro-hexahydro-sila-difenidol (7b) therefore of hexahydrosiladifenidol in blocking pre- and postjunc- tional muscarinic receptors studied in guinea-pig ileum and represents a useful tool for the subclassification of rat heart. European J. Pharmacol. 113, 125. muscarinic receptors. Giraido, E.. M.A. Vigano. R. Hammer and H. Ladinsky, 1988, Characterization of muscarinic receptors in guinea-pig ileum longitudinal smooth muscle. Mol. Pharmacol. 33, 617. Acknowledgements Hammer, R., CP. Berrie, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between dif• férent subclasses of muscarinic receptors. Nature 283, 90. The authors gratefully acknowledge the skillful technical Hammer, R. and A. Giachetti, 1982, Muscarinic receptor sub• assistance of Mrs. C. Gillessen. 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