Br. J. Pharmacol. (1990), 99, 4SS--460 © Macmillan Press Ltd, 1990

Stereoselective inhibition of muscarinic receptor subtypes by the enantiomers of hexahydro-difenidol and acetylenic analogues

*R. Feifel, *M. Wa~er-Röder, tC. Strohmann, tR. Tacke, §M. Waelbroeck, §J. Christophe, *E. Mutschier & *· G. Lambrecht

*Department of Pharmacology, University of Frankfurt, Tbcodor-Stern-Kai 7, Gebäude 75A, D-6000 Frankfurt/M., Federal Republic of Germany; tinstitute of Inorganic Chemistry, University of Karlsruhe, Engesserstraße, D-7500 Karlsruhe Federal Republic of Germany; §Department of Biochemistry and Nutrition, Medical School, Free University of Brussels, Bo~levard of Waterloo 115, B-1000 Brussels, Belgium

1 Tbc affinities of the (R)- and (S)-enantiomers of hexahydro-difenidol (1) and its acetylenie analogues hexbutinol (2), hexbutinol methiodide (3) and p-fluoro-hexbutinol (4) (stereochemieal purity > 99.8%) for musearlnie receptors in rabbit vas deferens (M 1), guinea-pig atria (M 2) and guinea-pig ileum (M 3) were measured by dose-ratio experiments. 2 The (R)-enantiomers consistently showed higher aßinities than the (S)-isomers. The stereosclectivity ratios [(R)/(S)] wcrc greatest with thc enantiomers of 1 (vas deferens: 550; ilcum: 191; atria: 17) and least with thosc ofthc p-Fluoro-analogue 4 (vas defercns: 34; ileum: 8.5; atria: 1.7). 3 The enantiomerie potency ratios for compounds 1-4 were highest in rabbit vas deferens, intermediate in guinea-pig ileum and much less in guinea-pig atria. Thus, these ratios may serve as a predietor of muscarinic receptor subtype identity.

4 (S)-p-Fluoro-hexbutinol [(S)-4] showed a novel receptor selectivity profile with preference for M 3 receptors: M3 > M 2 ~ M 1• 5 These results do not conform to Pfeiffer's rule that aetivity differences between enantiomers are greater with more potent compounds.

lntroduction Wesset al., 1988) and arecaidine propargyl ester (Mutschler & Hultzseh, 1973; Mutschier & Lambrecht, 1984; Moser et al., A large body of evidence derived from both functional and 1989), it would be of interest to investigate whether the acety­ radioligand binding studies suggests that there are at least lenic analogues of hexahydro-difenidol are also selective for three pharmacological musearlnie receptor subtypes [M., M 2 muscarinic receptor subtypes. In the last few years, data have accumulated that musear­ (M2J and M3 (M21)] (Eglen & Whiting, 1986; Mitehelson, 1988; Giraldo et al., 1988; Waelbroeck et al., 1988a; 1989; inic receptors ean be differentiated on the basis of thcir stereo­ Lambrecht et al., 1989d). This subclassification was recently selectivity to chirat antagonists such as procyelidine confirmed by cloning, sequencing and expression of comple­ (Lambrecht & Mutschler, 1986; Taeke et al., 1986; Wael­ mcntary DNA encoding five musearlnie receptors (m1-m5) broeck et al., 1988b), (Lambrecht et al., 1988b; (Kerlavage et al., 1987; Peralta et al., 1987; Akiba et al., 1988; 1989d), (Lambrecht et al., 1989a), Brann et al., 1988). The antagonist binding properties of (Eltze & Figala, 1988) and (Eveleigh et al., 1989). m1-m3 and their patterns of expression in various tissues Hexahydro-difenidol (J) and its analogues bexbutinol (2), hex­ butinol methiodide (3) and p-fluoro-hexbutinol (4) (Figure 1) eloscly correspond to thosc of the M 1, M 2 and M 3 r,eceptors (Peralta et al., 1987; Akiba et al., 1988; Maeda et al., 1988; possess a centre of chirality and therefore exist in two enantio­ Buekley et al., 1989). Tbc selective muscarinic antagonists mers. We took advantage of this by determining the antago­ pirenzepinc (Hammer et al., 1980; Lambrecht et al., 1988a; nist affinities of the individual enantiomers of these Waelbroeck et al., 1988a; 1989), methoetramine (Melchiorre et compounds at musearlnie receptor subtypes. The results were al., 1987; Wess et al., 1988), AF-DX 116 (Giachetti et al., eompared with those obtained for the selective reference drugs 1986), hexahydro-(sila-)difenidol (Mutschler & Lambrecht, hexahydro-sila-difenidol (M3 ~ M 1 > M 2) and p-tluoro­ 1984; Lambrecht et al., 1988a; 1989d; Waelbroeck et al., hexahydro-sila-difenidol (M3 > M 1 > M 2). The receptors 1988a; 1989; Akiba et al., 1988; Buekley et al., 1989) and p­ studied were presynaptic M1 heteroreceptors in rabbit vas fluoro-hexahydro-sila-difenidol (Lambrecht et al., 1988a; deferens (Eltze, 1988; Eltze et al., 1988; Lambrecbt et al., 1989b,d) have proved to be useful tools in this sub­ 1988a,b), cardiac M 2 receptors present in guinea-pig atria and elassifieation. smooth muscle M3 receptors present in guinea-pig ileum. Among tbese selective musearlnie antagonists, racemie Some of the present results have been briefly presented else­ hexahydr

electrodes. Negative inotropic effects to the agonist were mea­ sured as changes in isometric tension. Responses of ileal longi­ ~./~ tudinal muscle strips (Paton & Zar, 1968) to arecaidine propargyl ester were measured as isotonic contractions. The "-Qi, -D

Hexahydro-difenidol (1) Antagonist affinities

After a 1 h equilibration period, concentration-response curves were constructed by adding doses of the agonists cumula­ tively, according to the method of Van Rossum (1963). When these responses were constant, concentration-response curves were repeated in the presence of antagonists. At least three concentrations of antagonists with log intervals of 0.5 were tested 3 to 5 times (see Table 1) in the three tissues. Each concentration of antagonist was allowed to equilibrate for 15 to 30min (ileum) and 30min (atrium) in guinea-pig prep­ Hexbutinol (2) arations, respectively, and 30 [(S)-isomers] to 60min [(R)­ isomers] in rabbit vas deferens. Preliminary experiments indi­ cated that these intervals were sufficient for equilibration of the antagonist concentrations used. No preparation was exposed to more than three concentrations of antagonists. EC50 values of agonists in the absence and presence of antagonists were determined graphically for calculation of ©l../OHc HJC\ dose-ratios. The slopes of the Arunlakshana-Schild plots (Arunlakshana & Schild, 1959) were determined by linear Cf "c=c-c~ ~NO regression by the method of least squares. pA 2 values were estimated as the intercept on the abscissa scale by fitting to Hexbutinol methiodide (3) the data the best straight line with a slope of unity (Tallarida et al., 1979).

Data analysis

F All data are presented as means ± s.e.mean of 9-17 experi­ ments. Differences between mean values were tested for sta­ ~./~ tistical significance by Student's t test; P < 0.05 was accepted as being significant. Linear regression analyses were carried 0 C "-c ""';C-CHz -N':) out by the method of least squares (Tallarida et al., 1979). Drugs p-Fiuoro-hexbutinol (4) dihydrochloride was obtained from Boehringer Figure 1 Chemical structures of the enantiomers of hexahydro­ difenidol (/), hexbutinol (2), hexbutinol methiodide (3) and p-fluoro­ lngelheim (F.R.G.). 4-(4-Chlorophenylcarbamoyloxy)-2- hexbutinol (4). The asterisk denotes the centre of chirality. butynyltrimethylammonium iodide (4-Cl-McN-A-343) (Nelson et al., 1976), arecaidine propargyl ester (Mutschler & Hultzsch, 1973), racemic hexahydro-sila-difenidol hydro­ Krebs buffer which consisted of (mM): NaCI 118.0, KCI 4.7, chloride (Tacke et al., 1985), (R)- and (S)-hexahydro-difenidol CaCI2 2.5, MgS04 0.6, KH2 P04 1.2, NaHC03 25.0 and ( + )­ hydrochloride [(R)-1· HO and (S)-J· HCl] (Tacke et al., glucose 11.1 ; 1 JLM yohimbine was included to block 1989) as weil as (R)- and (S)-bexbutinol [(R)-2 and (S)-2] oc 2-adrenoceptors. The bathing fluid was maintained at 31°C (Tacke et al., 1989) were synthesized in our laboratories and aerated with 95% 0 2/5% C02 • A basal tension of 750mg according to tbe literature. The enantiomeric excess (ee) of the was applied and after a 30 min period of initial equilibration enantiomers of bexabydro-difenidol and bexbutinol was isometric twitch contractions were elicited by electrical field >99.8%, determined by calorimetric analysis as described by stimulation (0.05 Hz, 0.5 ms, 30 V) with platinum electrodes. Tacke et al. (1987). Racemic p-ftuoro-hexahydro-sila-difenidol The contractions were measured isometrically by a force­ hydrochloride was prepared by analogy to the synthesis of displacement transducer connected to a Heilige amplifier and hexahydro-sila-difenidol (Tacke et al., unpublished results). a Rikadenki polygraph. These effects were concentration­ The enantiomers of p-ftuoro-hexbutinol [(R)-4 and (S)-4; dependently inhibited by the M 1 receptor agonist 4-(4-chlo­ enantiomeric purity: ee > 99.8%; calorimetric analysis] were rophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide synthesized by analogy to (R)- and (S)-hexbutinol (Tacke et (4-Cl-McN-A-343) (Eltze et al., 1988). al., 1989). The other chemieals not described under 'Synthetic chemistry' were of reagent grade and were used as purchased. Guinea-pig isolated left atria and ileallongitudinal muscle Synthetic chemistry Guinea-pigs (3ro-400 g) of either sex were killed by cervical dislocation. The organs required were removed and set up in The enantiomers of hexbutinol methiodide [(R)-3 and (S)-3] 6ml organ baths, under 500mg tension, in oxygenated (95% were prepared as follows: 0 2/5% C02) Tyrode solution (32°C) composed of (mM): NaCl Freshly distilled methyl iodide (18 mmol) was added under 137.0, KCl 2.7, CaC1 2 1.8, MgC1 2 1.05, NaHC03 11.9, an atmosphere of dried nitrogen to a solution of (R)- or (S)­ NaH2P04 0.42 and ( + )-glucose 5.6. Arecaidine propargyl hexbutinol {2) (8mmol) in dry ethanol (50ml). The reaction ester (Mutschler & Hultzsch, 1973; Mutschier & Lambrecht, mixturewas stirred for 3 hat 30°C, dry n-pentane (100ml) was 1984; Moser et al., 1989) was used as an agonist. Left atria added, and the mixture was stirred for 1 h at 20°C. Thereafter, were paced electrically (2 Hz, 3 ms, 5 V) by means of platinum the precipitate was collected by filtration and then recry- STEREOSELECfiVITY OF MUSCARINIC RECEPTORS .ß7

TableI pAz values and slopes of Arunlakshana-Schild Table l Receptor selectivity and stereoselectivity [(R)/(S)] plots (in parentheses) for musearlnie antagonists at M 1, Mz ratios for ehiral musearlnie antagonists and M 3 receptors Receptor selectivity Stereoselectivit y

Rabbit vas Guinea-pig Guinea-pig M 1/Mz MJM1 MJMz Mt Mz M:s Compound deferens (M1) atria (Mz) Ileum (M3) Hexahydro-difenidol (I) HHSiD• 7.92 ± 0.07 6.53 ± 0.05 7.96 ± 0.03 (R)-1 48 0.44 21 p-F-HHSiD• 6.68 ± 0.12 6.01 ± 0.06 7.84 ± 0.03 550 17 191 (S)-1 1.5 1.3 1.9 Hexahydro-difenidol (1) (R)-1 8.71 ± 0.05 7.03 ± 0.06" 8.35 ± 0.04" Hexbutinol (2) [n=-17;6] [n = 9; 8] [n = 14; 11] (R)-2 10 1.0 10 (1.26 ± 0.09)* (0.83 ± 0.30) (0.92 ± 0.07) 107 8.5 44 (S)-1 5.97 ± 0.04 5.80 ± 0.07" 6.07 ± 0.05" (S)-2 0.81 2.5 2.0 [n = 13; 9] [n 9; 8] [n = 9; 6] = Hexbutinol methiodide (3) (0.87 ± 0.10) (0.96 ± 0.19) (0.87 ± 0.17) (R)-3 6.5 0.26 1.7 Hexbutinol (2) 40 17 39 (R)-2 8.78 ± 0.05 7.77 ± 0.04" 8.78 ± 0.04" (S)-3 2.7 0.27 0.72 [n = 13; 6] [n = 10; 10] [n = 13; 10] p-Fluoro-hexbutinol (4) (1.16 ± 0.11) (1.09 ± 0.07) (1.10 ± 0.06) (R)-4 13 2.6 34 (S)-2 6.75 ± 0.07 6.84 ± 0.05" 7.14 0.04" ± 34 1.7 8.5 [n=17;8] [n = 9; 9] [n = 10; 5] (S)-4 0.63 10.5 6.6 (0.84 ± 0.1 5) ( 1.03 ± 0.13) (0.99 ± 0.07) Hexbutinol methiodide (3) The values shown represent the antilogs of the differences (R)-3 9.43 ± 0.06 8.62 ± 0.02 8.85 ± 0.04 between corresponding mean pAz values (Table 1) deter­ [n = 9; 5] [n = 13; 13] [n = 17; 16] mined at M 1 receptors in rabbit vas deferens as weil as at (0.92 ± 0.11) ( 1.07 ± 0.03) (1.14 ± 0.06) atrlal Mz and ileal M 3 receptors of guinea-pigs. (S)-3 7.83 ± 0.05 7.40 ± 0.02 7.26 ± 0.02 [n = 12; 4] [n = 12; 4] [n = 12; 7] (0.92 ± 0.07) (0.91 ± 0.04) (0.96 ± 0.03) stallized from ethanoljdiethylether. After drying the crystals in p-Fluoro-hexbutinol (4) vacuo, analytically pure products were obtained (R)-4 8.08 ± 0.06 6.97 ± 0.04 8.50 ± 0.04 [ characterized by 1 H NMR, 13C NMR, F AB MS measure­ [n = 13; 5] [n = 12; 12] [n = 16; 8] ments and calorimetric analysis (data not given) as weil as by (1.31 0.10)* (0.84 ± 0.06) (0.84 ± 0.06) ± eiemental analyses]. (S)-4 6.55 ± 0.08 6.75 ± 0.05 7.57 ± 0.04 [n = 17; 6] [n = 9; 9] [n = 12; 12] (R)-3: C21H32INO (453.4), yield 92%, m.p. 17~175°C, (0.85 ± 0.18) (1.03 ± 0.09) (0.99 ± 0.06) [oc]~~ 6 ... -3 (c = 0.5, CHC13), ee > 99.8%. Found: C, 58.3; H, 7.1; N, 2.9. Calculated: C, 58.28; H, 7.11; N, 3.09. (S)-3: • Data taken from Lambrccht et al. (1988a). C22H321NO (453.4), yield 94%, m.p. 17~175°C, [oc]~~ 6 = 3 "Data taken from Tacke et al. (1989). (c = 0.5, CH03), ee > 99.8%. Found: C, 58.3; H, 7.1; N, 3.1. The parameters shown represent the mean ± s.e.mean. The Calculated: C, 58.28; H, 7.11; N, 3.09. slopes of Arunlakshana-Schild plots were detennined by linear regression analysis (Tallarida et al., 1979). pAz values were obtaincd after tbe unity constraint bad been imposed. The numbers of total data points (n) and tissues used are Results given in square parentbeses. The slopes shown are not signifi­ cantly dift'erent from unity (P > 0.05), except those for (R)-1 Twitch contractions of rabbit vas deferens elicited by electrical and (R)-4 at M 1 receptors (marked with an asterisk). field Stimulation were inhibited by the M 1 receptor agonist, HHSiD ""'racemic hexahydro-sila-difenidol; p-F-HHSiD­ 4-Cl-McN-A-343 (EC 50 = 250nM). This effect was racemic p-ftuoro-hexahydro-sila-düenidol. concentration-dependently antagonized by the (R)- and (S)­ enantiomers of compounds 1-4. Similarly, all Stereoisomers antagonized the negative inotropic responses in guinea-pig atria (EC50 = 7 nM) and ileal contractions (EC50 = 25 nM) of arecaidine propargyl ester. In the three tissues, parallel shifts of the agonists concentration-response curves without any appreciable changes of basal tension or reduction of 8 maximum responses were obtained and Arunlakshana-Schild plots were linear through tbe concentration range tested for each antagonist, indicating competitive antagonism. Slopes (Table 1) were not significantly different from unity (P > 0.05), except for compounds (R)-1 and (R)-4 at M 1 receptors. Tbe pA 2 values of (R)-J and (R)-4 in rabbit vas deferens (8.71 and 8.08, respectively; Table 1) might therefore be regarded as a purely experimental quantity. However, the binding affinities of (R)-1 and (R)-4 to M1 receptors in NB-OK 1 cells (pK1 = 8.6 and 8.1, respectively; M. Waelbroeck, unpublished results) were very similar to that obtained in rabbit vas deferens (Table 1). In these radioligand binding studies, competition curves with (R)-J and (R)-4 did not deviate significantly from (R)- (S)- (R)- (S)- (R)- (S)- (R)- (S)­ results expected for competitive inhibition of [ 3 H]-N-methyl Hexahydro- Hexbutinol (2} Hexbutinol- p-Fiuoro- ([3H]-NMS) binding. difenidol (1) methiodide (3) hexbutinol (4) The (R)- and (S)-enantiomers of compounds 1-4 showed Fipre l Affinity profiles of the enantiomers of compounds 1-4 at quite wide variations in their affinities for the musearlnie receptor subtypes, their pA 2 values differing by more than musearlnie M 1 receptors in rabbit vas deferens (solid columns), Mz receptors in guinea-pig atrla (diagonally-hatehed eolumns) and M 3 three orders of magnitude (Table 1). lntroduction of a triple receptors in guinea-pig ileum (cross-hatched columns). bond into the basic side chain of the parent compound 458 R. FEIFEL et al. hexahydro-difenidol (1) (-+hexbutinol, 2), quaternization of 2 guinea-pig ileum was not affected (Table 1). Tbus,. N­ ( -+hexbutinol methiodide, 3) and p-ftuoro-substitution of the metbylation of the tertiary amine (R)-2 cbanged the receptor phenyl ring of 2 ( -+p-ftuoro-hexbutinol, 4) changed the affin­ selectivity pattern. The following affinity rank order for (R)­ ities of these compounds for M 1, M 2 and M 3 receptors differ­ hexbutinol methiodide [(R)-3] was observed: Mt> M 3 ~ M 2 ently. Thus compounds with qualitatively and/or (Figure 2). quantitatively different receptor selectivity profiles were A comparison of the antimusearinic potencies of (R)-p­ obtained (Table 2 and Figure 2). ftuoro-hexbutinol [(R)-4] and (R)-bexbutinol [(R)-2] outlines At each of the three musearlnie receptor subtypes, the (R)­ the effect of ftuoro-substitution in the pbenyl ring on anti­ enantiomer of compounds 1--4 was more potent than the (S)­ muscarinic potency. Tbc p-ftuoro substituent reduced the configurated isomer. The difference in potencies between the affinity for the muscarinic receptors up to 6 fold (Table 1). enantiomers of compounds 1--4 was greatest at the M tt less at This decrease in affinity was the least pronounced at the ileal M3 and least at M 2 receptors (Table 2). The degree of stereo­ M3 receptors. Thus, as a result (R)-p-ftuoro-hexbutinol [(R)-4] selectivity (Table 2) was also dependent on the structure of the showed a small preference for M 3 over Mt receptors, but the compounds (M 1 and M 3 : 1 > 2 > 3 > 4; M 2 : 1 = 3 > 2 > 4). selectivity of M3 over M 2 receptors was enhanced (Table 2, Figure 2).

DiKussioa Receptor selectivity of ( S)-enantiomers

Structural variations in the (R)- and (S)-hexahydro-difenidol In general, the (S)-enantiomers of compounds 1--4 were less (1) molecules led to musearlnie antagonists that exhibited a potent than the corresponding (R)-configurated isomers qualitatively or quantitatively different spectrum of receptor (Table 1, Figure 2). However, introduction of a triple bond selectivity to the parent Stereoisomers (R)-1 and (S)-1 (Table 2, into the hexahydro-difenidol molecule (1) as well as quater­ Figure 2). These observed selectivities did not appear to be nization and ftuoro-substitution of hexbutinol (2) bad different associated in generat with high affinity and absolute configu­ effects on affinity for the (S)-isomers in comparison to the (R)­ ration (Figure 2). For example, (S)-p-ftuoro-hexbutinol [(S)-4] enantiomers at the three musearinic receptor subtypes. Thus, was a relatively weak compound but it bad a novel receptor the (S)-enantiomers showed affinity profiles which were quali­ M M ~ • selectivity profile: 3 > 2 M 1 tatively and/or quantitatively different from those obtained for The results of the present study confirm and extend pre­ the (R)-isomers. vious findings that rabbit vas deferens (M 1 receptors) as weil (S)-Hexabydro-difenidol [(S)-1] was a very weak muscarinic as guinea-pig atria (M receptors) and ileum (M receptors) 2 3 antagonist showing no musearinic receptor selectivity (Table possess different musearlnie receptor subtypes (Eltze, 1988; 2, Figure 2). Introduction of a tripte· bond into (S)-hexabydro­ Eltze et al., 1988; Lambrecht et al., 1988a; Waelbroeck et al., difenidol [ -+(S)-hexbutinol; (S)-2] increased the affinity for 1989). Furthermore, our findings provide additional evidence M t• M2 and M3 receptors by factors of 6, 11 and 12, respec­ that these subtypes ean be identified on the basis of their tively (Table 1). Thus, (S)-hexbutinol [(S)-2] showed at most a stereoselectivity. There appears to be a consistent trend 2 fold preference for the ileal M3 receptors (Table 2, Figure 2). showing that the degree of stereoselectivity is always greatest This is different to tbe situation of the (R)-enantiomers. at M 1, intermediate at M 3 and lowest at M 2 receptors N-metbylation of (S)-hexbutinol [(S)-2] increased the aßin• (Lambrecht & Mutschler, 1986; Tacke et al., 1986; Eltze & ity for M and M receptors 12 and 3.6 fold, respectively, Figala, 1988; Lambrecht et al., 1988b; 1989a,c,d). 1 2 whereas the affinity for ileal M3 receptors was nearly uncbanged (Table 1). Thus the receptor selectivity profile of Receptor selectivity of ( R)-enantiomers (S)-hexbutinol methiodide [(S)-3] is slightly different from that obtained for the (R)-enantiomer (Figure 2). (R)-Hexahydro-difenidol [(R)-1] was found to be a potent Tbc inftuence of ftuoro-substitution on potency and selec­ musearlnie antagonist exhibiting high affinity for M 1 recep­ tivity is demonstrated by comparison of (S)-hexbutinol [(S)-2] tors in rabbit vas deferens as well as for M3 receptors in and (S)-p-ftuoro-hexbutinol [(S)-4] (Table 1). Compared to (S)- guinea-pig ileum, whereas its affinity for M2 receptors in 2 the ftuoro derivative (S)-4 exhibited a relatively higher affin­ guinea-pig atria was lower by factors of 48 and 21, respec­ ity (pA = 7.57) for M receptors, whereas its antimuscarinic tively (Table 2, Figure 2). The resulting affinity profile of (R)-1 2 3 potency at M 1 and M2 receptors was lower by factors of 10.5 (M 1 ~ M3 > M2) is qualitatively very similar to that found and 6.6, respectively (Table 1). Thus, ftuoro-substitution in the for the racemic bexahydro-düenidol (Waelbroeck et al., 1989) para-position of the phenyl ring of (S)-hexbutinol [(S)-2] and its racemic silicon analogue bexahydro-sila-difenidol enhanced its M 3-selectivity. The receptor selectivity profile of (Table 1). (S)-p-ftuoro-hexbutinol (M > M ~ M ) is also different from The introduction of a triple bond into the (R)-hexahydro­ 3 2 1 that of the corresponding (R)-enantiomer (M ~ M > M ) difenidol molecule [ -+(R)-hexbutinol; (R)-2] increased the 3 1 2 and of p-ftuoro-hexahydro-sila-difenidol (M > M > M ; affinity for M and M receptors by factors of 2.7 and 3 1 2 3 2 S.S, Table 1). respectively (Table 1), whereas the affinity for Mt receptors was not signifieantly different. Thus, (R)-hexbutinol shows a receptor selectivity profile that is qualitatively similar to that Stereoselectivity of muscarinic receptors of tbe parent compound (R)-bexahydro-difenidol [(R)-1]. It has been suggested (Pfeiffer, 1956) that, with greater However, the selectivity for M 1 and M 3 over M2 receptors is lower than tbat of (R)-1. This might be explained by differ­ potencies of drugs, larger differences in pharmacological ences in the electronic structures and/or the conformational effects will be seen between the enantiomers of chirat com­ behaviour of (R)-bexabydro-difenidol and (R)-hexbutinol. The pounds. The results obtained in this study (Table 1 and 2) do triple bond in (R)-hexbutinol should make it more dißicult for not substantiate the above suggestion and its implications tbis molecule to adopt the active conformation at all subtypes, (Lehmann, 1986). For example, at Mt receptors the affinity thus creating selectivity (Barlow et al., 1988). On the other constants (pA 2 values) of the more potent (R)-enantiomers band, the carbon-carbon triple bond might contribute to affin­ decrease in the order (R)-hexbutinol metbiodide (9.43) > (R)­ ity and thus counteract any selectivity creating effect of rigid­ hexbutinol (8. 78) = (R)-hexahydro-düenidol (8. 71) > (R)-p­ ity. ftuoro-hexbutinol (8.08), whereas the stereoselectivity ratios N-methylation of (R)-hexbutinol [ -+(R)-hexbutinol methio- [(R)/(S)] of these chirat compounds are in the order dide; (R)-3] increased the affinity for Mt receptors in rabbit hexahydro-difenidol (550) > hexbutinol (107) > hexbutinol methiodide (40) > p-ftuoro-hexbutinol (34). Tbus, in this series vas deferens, as weil as for M 2 receptors in guinea-pig atria by factors of 4.5 and 7.1, whereas the affinity for M3 receptors in of compounds the stereoselectivity ratio at, e.g. M 1 receptors, STEREOSELECTIVITY OF MUSCARINIC RECEPTORS 4S9 was higher by more than one order of magnitude for 3 hexahydro-difenidol (1) than for hexbutinol methiodide (3), ..v although compound (R)-3 was 5 fold morepotent than (R)-1. ~ A similar Iack of correlation between potency of the eutomer and stereoselectivity ratios of enantiomers was obtained at M i2 2 ~,__..3 and M 3 receptors (Tables 1 and 2). The findings of the present /.. study confirm and extend previous results obtained with the /./ /.~-~ enantiomers of biperiden (Eltze & Figala, 1988), tri­ / • •4 hexyphenidyl (Lambrecht et al., 1988b; 1989d) and phenglu­ tarlmide (Lambrecht et al., 1989a). In 1982, Robert et al. stated a corollary of Pfeiffer's rule: • "When different receptor subtypes interact with the enantio­ 7 8 9 10 mers of chiral drugs, their stereoselectivity should increase as pA2 eutomer a function of affinity of the more potent enantiomer Fiaure 3 Plot of receptor stereoselectivity [eudismic index (= eutomer; Lehmann, 1986) for the respective subtypes". (EI)= difference of pA 2 valucs of the (R)- and (S)-enantiomers; However, when the magnitude of receptor subtype stereoselec­ Lehmann, 1986] versus pA2 values of the more potent isomer [eutomer (EU)] for compounds 1-4 (sec Figure 1) at musearlnie M tivity (difference in pA2 values of the (R)- and (S)­ 1 receptors in rabbit vas deferens (A.) as weil as at M (e) and M enantiomers- eudismic index; Lehmann, 1986) was plotted 2 3 against the pA value of the more potent isomer for that par­ receptors (•) in guinea-pig atria and ileum. A strong linear corre­ 2 lation was only observed for compound 1: EI .... 0.87 ( ±0.08) pA~u ticular receptor subtype, a strong correlation (correlation coef­ - 4.91 (±0.68), r = 0.995, s.d.::;: 0.182, n = 3. The correlation coeffi­ ficient r = 0.995) was only observed for hexahydro-difenidol cient of 0.995 is significant at the P < 0.05 Ievel. (Figure 3). The stereoselectivity and the affinity of the eutomer of hexahydro-difenidol was greatest at M 1, intermediate at M3 and lowest at M2 receptors. On the other band the enantio­ mers of compounds 2-4 did not fulfill the predictions made by Robert et al. (1982). However, tbe interesting finding of this esting to note that, of tbe enantiomers investigated in this study is that tbe stereoselectivity ratios of all the chirat com­ study, (S)-p-ftuoro-bexbutinol sbows a novel receptor selec­ pounds 1-4 consistently sbow the same order: M 1 > M 3 > tivity profile: M3 > M2 ;;i1: M1 (Figure 2). There was a vari­ M 2 • This implies tbat tbe stereochemical demands made by ation in stereoselectivity ratios on the tbree receptor subtypes the musearlnie receptor subtypes are different for tbe enantio­ (Table 2): M 1 > M 3 > M 2 • These results indicate that stereo­ mers of compounds 1-4 being moststringent at M 1 receptors. selectivity ratios can be successfully used as a parameter to Similar results have been obtained with the enantiomers of characterize musearlnie receptor subtypes providing informa­ telenzepine (Eveleigb et al., 1989), biperiden (Eltze & Figala, tion that racemates cannot give. However, the stereoselectivity 1988), trihexypbenidyl and its methiodide (Lambrecht et al., ratios do not conform to the predictions of the 'classical' Pfeif­ 1988b) and (Lamhrecht & Mutschler, 1986; fer's rule (Pfeiffer, 1956), and that ofits corollary (Robert et al., Waelbroeck et al., 1988b). 1982). In conclusion, the present study shows that tbe anti­ musearlnie potencies and receptor subtype selectivities of the The authors thank Mrs C. Gillessen for skilful technical assistance. (1) enantiomers of hexahydro-difenidol and the acetylenic ana­ R.F., G.L., R.T. and E.M. thank the Fonds der Chemischen Industrie, logues 2-4 (Figure 1) depend on different structural param­ ratiopharm, the Deutsche Forschungsgemeinschaft and Dochringer eters including absolute configuration. Tbe M 1, M 2 and M 3 Ingelheim for financial support. R.T. thanks the Bayer AG for support receptors make qualitatively and quantitatively different with chemicals. C.S. thanks the Land Baden-Württemberg for a post­ stereocbemical demands for the (R)- and (S)-enantiomers, graduale seholarship. All authors thank Dr Lay Khoon Choo for resulting in different receptor selectivity profiles. It is inter- helpful Suggestions. This paper contains parts of the Ph.D. thesis of R. Feifel.

References

AKIDA, I., KUDO, T., MAEDA, A., DUJO, H., NAKAI, J., MISHINA, M. & inic receptors mediating inhibition of neuragenie contractions in NUMA, S. (1988). Primary structure of porcine musearlnie acetyl­ rabbit vas deferens are of the ganglionic Mt-type. Eur. J. Phar­ receptor 111 and antagonist binding studies. FEBS Lett., macol., 1!8, 233-242. 135, 257-261. EVELEIGH, P., HULME, E.C., SCHUDT, C. & DIRDSALL, NJ.M. (1989). ARUNLAKSHANA, 0. & SCHILD, H.O. (1959). Some quantitative uses The existence of stable enantiomers of telenzepine and their stereo­ of drug antagonists. Br. J. Pharmacol., 14, 48-58. selective interaction with musearlnie receptor subtypes. Mol. Phar­ DARLOW, R.D., SHEPHERD, M.K., TYDEMAN, H. & VEALE, M.A. (1988). maco/., 35, 477-483. The aßinity of some acetylenic analogues of 4-DAMP methobro­ FEIFEL, R., AASEN, A., STROHMANN, C., TACKE, R., WAELDROECK, mide for musearlnie receptors in guinea-pig ileum and atria. Br. J. M., CHRISTOPHE, J., LAMBRECHT, G. & MUTSCHLER, E. (1988). Pharmacol., 94,947-951. Stereoselectivity of antagonists of the trihexyphenidyl- and DRANN, M.R., DUCKLEY, NJ. & DONNER, T.l. (1988). The striatum and hexahydro-difenidol-type as a criterion of muscarinic receptor sub­ cerebral cortex express different musearlnie receptor mRNAs. classification. Naunyn-Schmiedebergs Arch. Pharmacol., 338, Suppl. FEBS Lett.,l30, 9()-94. R61. DUCKLEY, NJ., DONNER, T.l., DUCKLEY, C.M. & DRANN, M.R. (1989). GIACHETTI, A., MICHELETTI, R. & MONTAGNA, E. (1986). Cardio­ Antagonist binding properlies of five cloned musearlnie receptors selective profile of AF-DX 116, a musearlnie M2 receptor anlago­ opressed in CHO-Kl cells. Mol. Pharmacol., 35, 469-476. nist Life Sei., 38, 1663-1672. EGLEN, R.M. & WHITING, R.L. (1986). Musearlnie receptor subtypes: GIRALDO, E., VIGANO, M.A., HAMMER, R. & LADINSKY, H. (1988). a critique of the current classification and a proposal for a Characterization of musearlnie receptors in guinea pig ileum lon­ working nomenclature. J. Auton. Pharmacol., 5, 323-346. gitudinal smooth muscle. Mol. Pharmacol., 33,617-625. ELTZE. M. (1988). Musearlnie M1- and M2-receptors mediating HAMMER, R., DERRIE, C.P., BIRDSALL, N.J.M., BURGEN, A.S.V. & opposite effects on neuromuscular transmission in rabbit vas HULME. E.C. (1980). Pirenzepine distinguishes between different deferens. Eur. J. Pharmacol., 151, 205-221. subclasses of musearlnie receptors. Nature, 283, 90-92. ELTZE, M. & FIGALA, V. (1988). Aßinity and selectivity of biperiden KERLAVAGE, A.R., FRASER, C.M. & VENTER, J.C. (1987). Musearlnie enantiomers for musearlnie receptor subtypes. Eur. J. Pharmacol., receptor structure: molecular biological support for 158, 11-19. subtypes. Trends Pharmacol. Sei., 8, 426-431. ELTZE, M., GMELIN, G., WESS, J., STROHMANN, C., TACKE, R., LAMDRECHT, G., FEIFEL, R., FORTH, D., STROHMANN, C., TACKE, R. MUTSCHLER, E. & LAMDRECHT, G. (1988). Presynaptic muscar- & MUTSCHLER, E. (1988a). p-Fiuoro-hexahydro-sila-difenidol: 460 R. FEIFEL et al.

Tbcfirst M2P-selcctive musearlnie antagonist. Eur. J. Pharmacol., PATON, W.D.M. & ZAR, M.A. (1968). The origin of release lSl, 193-194. from guinea-pig intestine and longitudinal musele stripes. J. LAMBRECHT, G., FEIFEL, R., MOSER, U., AASEN, A.J., WAELBROECK, Physiol., 194, 13-33. M., CHRISTOPHE, J. & MUTSCHLER, E. (l988b). Stereoselectivity PERALTA, E.G., ASHKENAZI, A., WINSLOW, J.W., SMITH, D.H., RAMA· of the enantiomers of trihexyphenidyl and its methiodide at CHANDRAN, J. & CAPON, DJ. (1987). Distinct primary structures, musearlnie rcceptor subtypes. Eur. J. Pharmacol., ISS, 167-170. Iigand-binding properlies and tissue-spccific expression of four LAMBRECHT, G., FEIFEL, R. & MUTSCHLER, E. (1989a). Stereoselccti­ human musearlnie acetylcholine receptors. EMBO J., 6, 3923- vity at musearlnie receptor subtypes: Observations with the enan­ 3929. tiomers ofphenglutarimide. Chirality, 1, 170-173. PFEIFFER, C.C. (1956). Optical isomerism and pharmaeological LAMBRECHT, 0., FEIFEL, R., WAONER-RÖDER, M., STROHMANN, C., aetion, a generalization. Science, 124, 29-31. ZILCH, H., TACKE, R., WAELBROECK, M., CHRISTOPHE, J., ROBERT, T.A., HAGAR.DORN, A. N. & DAIGNEAULT, E.A. (1982). Dif­ BODDEKE, H. & MUTSCHLER, E. (1989b). Affinity profiles of ferential stereoselcctivity of methotrimeprazine enantiomers for hexahydro-sila-d.ifenidol analogues at musearlnie rcceptor sub­ selected central nervous system rcceptor types. Mol. Pharmacol., types. Eur. J. PharmDCol., 168, 71-80. ll, 315-319. LAMBRECHT, 0., FEIFEL, R., STROHMANN, C., TACKE, R., MOSER, U. R.OSZKOWSKI, A.P. (1961). An unusual type of sympathetic ganglionic & MUTSCHLER, E. (1989c). Stereoseleetivity at musearlnie receptor stimulant. J. Pharmacol. Exp. Ther., 132, 156-170. subtypes: Observations with the enantiomers of p-ftuoro-hexbu­ TACKE, R., LINOH, H., ZILCH, H., WESS, J., MOSER, U., MUTSCHLER, tinol. Naunyn-Schmiedebergs Arch. PharmDCol., 339, Suppl. R81. E. & LAMBRECHT, 0. (1985). Synthesis and properties of the selcc­ LAMBRECHT, 0., GMELIN, 0., MOSER, U. & WAELBR.OECK, M. tive antimuscarinie agent cyelohexylphenyl(3-piperidinopropyl) (1988c). Selcctivity profiles of antagonists at musearlnie receptor silanol. Liebig's Ann. Chem., 2223-2228. subtypes. Naunyn-Schmiedebergs Arch. Pharmacol., 337, Suppl. TACKE, R., LINOH, H., SCHOMBUR.G, D., ERNST, L., MOSER, U., R92. MUTSCHLER, E. & LAMBRECHT, G. (1986). On the absolute con­ LAMBRECHT, 0., MOSER, U., MUTSCHLER, E., WALTHER, G. & WESS, figuration of the enantiomers of the antimuscarinic agents pro­ J. (1986). Musearlnie ganglionic stimulants: Conformationally cyelidine and tricyclamol iodide: X-ray structural analysis of(R)-1- restrained analogues related to 4-[[N-(3-chlorophenyl)carbamoyl] [3-cyclohexyl-3-hydroxy-3-phenylpropyl]-1-methylpyrrolidinium - oxy]-2-butynyl]trimethylammonium chloride. J. Med. Chem., 19, iodide. Liebig's Ann. Chem., 242-250. 1309-1311. TACKE, R., LINOH, H., ERNST, L., MOSER, U., MUTSCHLER, E., LAMBRECHT, 0. & MUTSCHLER, E. (1986). Chirality as a tool for sub­ SARGE, S., CAMMENGA, H.K. & LAMBRECHT, G. (1987). Prep­ classification of receptors. In Innovative Approaches in Drug aration and properlies of the enantiomers of the antimuscarinic Research, ed. Harms, A.F. pp. 353-370. Amsterdam: Elsevier agents sila-procyclidine and sila-tricyclamol iodide: optically Science Publishers B.V. active silano1s with silicon as the centre of chirality. Chem. Ber., LAMBRECHT, 0., WESS, J., TACKE, R. & MUTSCHLER., E. (1989d). Het­ llO, 1229-1237. erogencity of musearlnie receptors: Evidence from structure­ TACKE, R., STROHMANN, C., SARGE, S., CAMMENGA, H.K., SCHOM· activity relationships of antimuscarinie agents related to BURG, D., MUTSCHLER, E. & LAMBRECHT, G. (1989). Preparation and sila-pridinol. In Trends in Medicinal Chemistry "88", ed. Van and properlies of the enantiomers of the selective antimuscarinic der Goot, H., Domany, G., Pallos, L. & Timmerman, H. pp. 265- agent 1-cyclohexyl-1-phenyl-4-piperidino-1-butanol (hexahydro­ 282. Amsterdam: Elsevier Science Publishers B.V. difenidol). Liebig's Ann. Chem.,137-143. LEHMANN, F.P.A. (1986). Stereoisomerism and drug action. Trends TALLARIDA, RJ., COWAN, A. & ADLER, M.W. (1979). pA2 and recep­ PharmDCol. Sei., 7, 281-285. tor differentiation: a statistical analysis of competitive antagonism. MAEDA, A., KUBO, T., MISHINA, M. & NUMA, S. (1988). Tissue dis­ Life Sei., lS, 637-654. tribution of mRNAs encoding musearlnie acetyleholine receptor VAN ROSSUM, J.M. (1963). Cumulative dose-response curves. II. Tech­ subtypes. FEBS Lett.,l39, 339-342. nique for the making of dose-response eurves in isolated organs MELCHIORRE, C., ANGELI, P., LAMBRECHT, 0., MUTSCHLER., E., and the evaluation of drug parameters. Arch. /nt. Pharmacodyn. PICCHIO, M.T. & WESS, J. (1987). Antimusearinie action ofmethoc­ Ther., 143,299-330. tramine, a new cardioselcctive M-2 musearlnie receptor antago­ WAELBROECK, M., CAMUS, J., TASTENOY, M. & CHRISTOPHE, J. nist, alone and in combination with and gallamine. Eur. (1988a). 80% of musearlnie receptors expressed by the NB-OK 1 J. Pharmacol., 144, 117-124. human neuroblastoma cell line show high affinity for pirenzepine MITCHELSON, F. (1988). Musearlnie receptor differentiation. Phar­ and are comparable to rat hippocampus Mt receptors. FEBS macol. Ther., 37,357-423. Lett., ll6, 287-290. MOSER., U., LAMBRECHT, G., WAGNER, M., WESS, J. & MUTSCHLER, WAELBROECK, M., TASTENOY, M., CAMUS, J., LAMBRECHT, G., E. (1989). Structure-activity relationships of new analogues of are­ MUTSCHLER, E., TACKE, R. & CHRISTOPHE, J. (1988b). Stereosel· caidine propargyl ester at musearlnie M 1 and M 2 receptor sub­ cctivity of three musearlnie receptor subtypes in binding studies. types. Br. J. Pharmacol., 96, 319-324. Naunyn-Schmiedebergs Arch. Pharmacol., 338, Suppl. R61. MUTSCHLER, E. & HULTZSCH, K. (1973). Über Struktur-Wirkungs­ WAELBROECK, M., TASTENOY, M., CAMUS, J., CHRISTOPHE, J., Beziehungen von ungesättigten Estern des Arccaidins und Dihy­ STROHMANN, C., LINOH, H., ZILCH, H., TACKE, R., MUTSCHLER, droarccaidins. Arzneim. Forsch./Drug Res., 23, 732-737. E. & LAMBRECHT, G. (1989). Binding and functional properlies of MUTSCHLER., E. & LAMBRECHT, 0. (1984). Selective musearlnie agon· antimusearinics of the /sila-hexocyclium and ists and antagonists in funetional tests. Trends Pharmacol. Sei., S, hexahydro-difenidol/hexahydro-sila-difenidol type to musearlnie Suppt. 39-44. receptor subtypes. Br. J. Pharmacol., 98, 197-205. NELSON, W.L., FREEMAN, D.S. & VINCENZI, F.F. (1976). Stereochemi­ WESS, J., ANGELI, P., MELCHIORRE, C., MOSER, U., MUTSCHLER, E. cal analogs of a muscarinie, ganglionic stimulant. 2. Cis and trans & LAMBRECHT, G. (1988). selectively blocks olefinic, epoxide, and cyclopropane analogs related to 4-[N-(3- cardiae musearlnie M2 rcceptors in vivo. Naunyn-Schmiedebergs ehlorophenyl)carbamoyloxy]-2-butyny1trimethylammonium chlo­ Arch. Pharmacol., 338, 246-249. ride (McN-A-343). J. Med. Chem., 19, 153-158. (Received June 26, 1989 Revised September I I, 1989 Accepted November 7, 1989)