Europeon Journal of Pharmacology - Molecu/ar Pltarmacofogy Section, 189 (1990) 135-142 135 Elsevier

FJPMOL 90098

Stereoselectivity of procyclidine binding to muscarinic receptor

subtypes M 1, M 2 and M 4

1 1 1 2 Magall Waelbroeck , Jean Camus , Micheie Tastenoy • Günter Lambrecht , 2 3 1 Ernst Mutschier , Reinhold Tacke and Jean Christophe 1 Department of Biochemistry and Nutrition, Medical Schoo/, Universtile Libre de Bruxelles. Boulevard of Wmerloo 115. 8-J()()() Brussel'i, Be/gium, ~ Department of Pharmacology, Universi~v of Frtmk{urt IM, Theodor-Stem-Ka1~ 7, D-6000 Frankfurt 1M. F.R.G .• and J lnstirute of Jnorganic Chentistry, Univer.rity of Karlsruhe, Engcsserstrasse, Geh. 30.45, D-7500 Karlsruhe. F.R.G.

Re<:eived 24 January 1990. reviscd MS received 18 April 1990. accepted 7 May 1990

The goals of the present study were: (1) to investigate thc binding properlies oi (R)- and (S)-procyclidine and two

aehiral derivatives of muscarinie M1• M2 and M4 reeeptor subtypes and (2) to identify the interaetions which allow these receptors to diseriminate between the two stereoisomers. (R)-Procyclidine showed a higher affinity for human neuroblastoma NB-OK 1 muscarinie M 1 and rat striatum musearinie M 4 receptors. a~ compared to rat cardiac M 2 receptors. (S)-Procyclidine had a 130-iold lower affinity than (R)-procyclidine for M 1 and M 4 receptors. and a 40-fold lower affinity for M 2 receptors. Pyrrinol. the aehiral diphenyl derivative with the eyclohexyl g.roup of (S}-procyclidine replaeed by a phenyl group, has an eight-fold lower affinity for M1 and M4 receptors. as eompared to (R)-procycli­ dine, and a three-fold lower affinity for M 2 receptors. Hexahydro-procyclidine. the eorresponding achiral dicyclohexyl compound, had a 10- to 20-fold lower affinity than (R)-procyclidine for the three reeeptors. The inerease in binding free energy, which is observed when the phenyl and eyclohexyl groups of procyelidine are separately replaeed by cyclohexyJ and phenyl groups, respectively. was additive in the ease of M 1• M 2 and M 4 receptcrs. This indicates that the musearinic reeeptor s!ereoseleetivity was based on the eoexistence of two binding sites, one preferring a phenylrather than eyclohexyl group and the seeond preferring a cyclohexyl rather than a phenyl group. In addition. there were aiso binding sites for the hydroxy moiety and the protonated amino group of the ligands. The greater affinity and stereoselectivity of M1 and M4 musearinic receptors for (R)-procyelidine reflected the better fit of the eyclohexyl group of (R)-procyclidine to the subsite of M 1 and M 4 as compared to M 2 receptors.

Musearlnie M, reeeptors; Muscarinie M2 reeeptors; Musearinic M 4 receptor~~ (S}-Procyelidine; (R)-Procyclidine; Pyrrinol; Hexahydro-procyclidine; Musearlnie receptors (stereose1ecti\ity)

1. Introduction tors, with a high affinity for , are typi­ cally found in neuronal tissues (Hammer et al., At least four pharmacoiogically and biochem­ 1980). These receptors also have a high affinity for ically distinct muscarinic receptors coexist in 4-DAMP ( 4-diphenylacetoxy-N-mcthylpiperidi••e mammalian tissues (for review: see Mitchelson, methiodide) and HHSiD (hexahydro-sila-difen·· 1988; Levine and Birdsall, 1989): (a) M1 recep- idol) but a low affinity for AF-DX 116 ([11-({(2- [( diethylamino )methyl ]-1-pi peridinyl} acetyl)-5.11- dihydro-6 H·pyrido-(2,3-b) ( 1,4 )-benzodiazepin-6- Correspondence to: J. Christophe. Department of Biochem­ one) (Waelbroeck et al.. 1987b; 1988; 1989); (b) istry and Nutrition, MedicaJ School. Universite Libre de Bruxelles. Boulevard of Waterloo 115. 8-1000 Brussels, Bel· M 2 receptors, wilh a high affinity for AF-DX 116 gium. and a low affinity for pirenzepine are especially

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present in cardiac tissue (Hammer et al.7 1986). They also show a low affmity for 4-DAMP and 0 0 HHSiD (Waelbroeck et al., 1987b; 1988; 1989); HO~~ - CH2 - CH2 - 0 HO~~ - CH2 - CH2 - 0 (c) M3 receptors have high affinities for 4-DAMP (Barlow et al., 1976) and HHSiD (Mutschler and ~ !Sl-PROCYCLIO!NE 6 lll) -PI;('CVCL!OINE Lambrecht, 1984) and low affinities for pirenze­ pine and AF-DX 116. They are typically detected in secretory glands and smooth muscle (Wael­ broeck et al., 1987a; Kore et al., 1987); (d} M 4 Q r~eptors are typically found in NG 108-15 cells 9 HO - C - CH2 - CH2 - HO - C - CH2 - CH2 - 0 0 (Michel et al., 1989) and rat striatum (Waelbroeck e~ al., 1990). They have low affinities for pirenze­ HEXAHYOAO­ PVRR!NOL PROCYCllOlNE pipe and AF!'DX 116 but high affinities for 6 6 m~~hoctramin~ and HHSiD. Fig. 1. Chemical structure of (S}-procy.;lidine, (R)-procycli­ We previously demonstrated that receptors dine, pyrrinol and hexahydro-procyclidine. ln the case of (S)­ Iabeted by [3H]-N-methylscopolamine ([ 3H]NMS) and (R)-procyclidine, the carbinol carbon atorn is a center of in NB-OK 1 cells (a human neuroblastoma cell chirality. line), rat heart, and rat striatum (those receptors 3 showing slow [ H]NMS dissociation) display M1, The first aim of the present study was to com­ M 1 and M 4 selectivities, respectively (Waelbroeck pare the binding properties of (R)- and (S)-pro­ et al., 1986; 1987a,b; 1988; 1989; 1990). We de­ cyclidine to the three reasonably pure musearlnie cided to compare these three systems to an~yze receptor systems at band. The affinity and stereo­ the structure-affinityjselectivity relationships of selectivity of 1\'j ,, M 2 and M4 receptors for pro­ musearlnie antagonists related to procyclidine. cyclidine enantiomers proved to be different in A majority of previous studies comparing the our binding experiments. In order to identify the binding or functional properlies of chiral interactions responsible for musearlnie receptor musearlnie antagonists and agonists used the drugs stereoselectivity, we extended the binding analysis as racemates. While this is sometimes unavoidable to two achiral compounds structurally related to ( fot example if the drug racemizes quickly in (R)- and (S)·procyclidine: pyrrinol (the diphenyl solution), there are important drawbacks in utiliz­ derivative) and hexahydro-procyclidine (the di­ ing a racemate rather than the individual enanti­ cyclohexyl derivative). The structures of these omers (see for example: Lambrecht and Mutschler, compounds are shown in fig. 1. 1986; Lambrecht et al., 1988; Tacke et al., 1986; 1987; 1989; and the Series on Chirality (published in Trends Pharmacol. Sei. 7, 1986, 20-24, 60-65, 2. Materials and methods 112-115, 155-158, 200-205, 227-230, 281-301). Re­ ceptors are indeed asymmetrical macromolecules. 2.1. Human NB-OK 1 neurob/astoma cel/s When studying the binding or functional proper­ lies of a racemic mixture of compounds, the infor­ The NB-OK 1 cells were cultured as previously mation bears at best on the eutomer (high-affinity described (Waelbroeck et al., 1988) in RPMI-1640 enantiomer) but the properties are in some cases medium enriched with 10% fetal calf serum~ 100 affected by the presence of the distomer (low-af­ unitsjrnl p~nicillin and 100 p.gjml Streptomycin. finity enantiomer). If the absolute configuration of F or 1-[N-rr:ethyl- 3H]scopolomine methyl chloride 3 the eutomer is not known, it is, for example, ([ H]NMS) binding experiments, the cells were impossible to map the relative positions of recep­ rinsed, detached and centrifuged in 20 mM sodium tor 'subsites' recognizing the protonated amino phosphate buffer (pH 7 .4) containing 150 mM group and the hydroxyl group of antimuscarinics NaCl and 1 mM EDTA, resuspended and ho­ of the procyclidine type family. mogenized in 20 mM TrisjHCl buffer (pH 7.5) 137

enriched with 5 mM MgCl2 and stored in liquid The eHJNMS concentration used was 1.0 nM, i.e. nitrogen until use. two-fold the tracers' K0 value to M2 bi.nding sites. In rat striatum homogenates, ( 3 H]NMS Iabels 2.2. Rat tissue homogenate prepar~uions M1 and M4 sites but dissociates faster from M 1 receptors (Waelbroeck et al., 1986. 1987b, 1988). We preincubated 80 f.d of the homogenate (equiv­ Male Wistar albino rats (200-250 g) were de­ alent to about 30 p.g protein) in a total volume of capited and the heart and striatum immediately t.2 ml, in the presence of [3H]NMS and unlabeled removed. All following operations were performed at 4°C. drugs. A 2 h preincubation period allowed equi­ librium binding. We then added 1 p.M The heart was rinsed in isotonic N aCI, then and allowed tracer dissociation !or ~5 m.in before homogenized in 2.5 ml of 20 m~.f TrisjHCl buffer filtration. This procedure allowed us to investigate (pH 7.5), enriched with 250 mM sucrose, with an tracer binding to striatum M receptors only Ultraturrax homogenizer (maximal speed for 5 s) 4 (Waelbroeck et al., 1987b; 1988; 1990}. The tracer followed by addition of 12.5 ml of the same buffer, concentration used in these experiments (0.25 nM} seven up and down strokes with a glass-Teflon was equivalent to five·fold the tracers9 K value to hornogenizer and filtration on two layers of medi­ 0 striatum M receptors (Waelbroeck et al., 1988). cal gauze. The resulting homogenate was used 4 All incubations were terminated by addition of inunediately or stored in liquid nitrogen until use. 2 ml of ice-cold filtration buffer (50 mM sodium The striatum was homogenized in 2 ml of 20 phosphate buffer pH 7.4}. Bound and free tracer mM TrisjHCI buffer (pH 7.5) enriched with 250 were immediately separated by fihration on GF;c mM sucrose~ using a glass-Teflon homogenizer glass-fiber filters presoaked ovem.ight in 0.05% (seven up and down strokes). The resulting homo­ polyethyleneimine. The samples were rinsed three genate was stored in liquid nitrogen until use and times wit~ filtration buffer. The filters were then diluted 20-fold with the same buffer immediately dried and the bound radioactivity counted by before the experiment. liquid scintillation. Nonspecific [3 H]NMS binding The protein concentrations were determ.ined was defined as tracer bound in the presence of 1 according to Lowry· et al. (1951) using bovine ~tM atropine. serum albunlin as Standard.

2. 3. Binding studies 2.4. Analysis of binding data

All binding studies were performed at 25 ° C, at All competition curves were repeated in dupli­ equilibrium, in a 50 ml'vf sodium phosphate buffer catet on at least three different preparations. ICso 3 (pH 7.4) enriched with 2 mM MgCl2, [ H]NMS, values were determined by a computer-aided pro­ and the indicated unlabeled dtug concentrations, cedure described by Richardson and Humrich in a total volume of 1.2 ml. (1984), assuming the existence of only one recep­ To measure [3H]NMS binding to human NB­ tor subtype. Indeed, experimental data points were OK 1 cell homogenates, we used 80 111 of homc-­ within 3% of expected values~ assuming that the genate, corresponding to about 200 1-'8 protein per molecules investigated competed with eHJNMS assay. The incubation period was 2 h in the pres· for binding to a single site. 3 ence of 0.25 nM [ H}NMS (this concentration was Ki values were calculated from IC50 va1ues using equivalent to two-fold the tracers' K 0 value to M1 the Cheng and Prusoff equation (Cheng and receptors). Pntsi:>ff. 1973) which assumes competitive inhibi­ For incubation with rat heart homogenates, we tion of tracer binding to a single receptor subtype~ 3 used 80 1.d of the homogenate, corresponding to The [ H]NMS K 0 value for the three systems 400-500 pg protein per assay. The 2 h incubation investigated was determined in separate experi· period was sufficient to allow equilibrium binding. ments, as described by Waelbroeck et al. (1987a.b; 138

1988). The pKi values, mentioned in table 1, cor­ published (Tacke et al., 1986), pyrrinol was responded to -log Ki values. syntbesized according to the Iiterature (Adamson, The standard deviation of piCso (-log IC5o) 1949) and hexahydro-procyclidine was obtained determinations was always equal to or below 0.1 by catalytic hydrogenation of pyrrinol. log unit. Repeated determinations of [3H]NMS

K 0 values were within 10% of each other. 'fhis 3. Results error should be added to errors in IC50 determina­ tions, since [3H]NMS K values were used to 0 As shown in fig. 2, the four compounds in­ calculate pKi values. We therefore estimated the vestigated in this study inhibited [3H]NMS bind­ standard deviation of pK i values as being of ap­ ing to the three musearlnie receptors in a manner proximately 0.15 log unit (40% of Ki value). consistent with competition for a si" · · · binding The binding free energy (.:1G) for the formation site (Hili coefficients were not signif. mtly differ­ of a Iigand-receptor complex is related to i~s affin~ ent from 1). ity constn.nt Ka by equation (1): The affinity of the procyclidine eutomer, (R)­ ßG= -RTln Ka (1) procyclidine, for M1 and M 4 receptors was greater .dG values were therefore calculated according than its affinity for M2 receptors (table 1 and fig . to equation (2), using experimentally determined 2). The procyclidine distomer, (S)-procyclidine, 1 bad similar affinity for the three subtypes (table Ki values (Ka = Kj ): a 1 and fig. 2). As a result, the eudismic index (pKi .:1G = - RT In 1/Ki (2) (eutomer) - pKi (distomer)) at M 1 and M 4 re· ceptors was greater than that at M2 receptors 2.5. Materials (table 1). Pyrrinol and hexahydro-procyclidine bad lower 3 [ H]NMS (80 to 85 Cijrnmol) was obtained affinities than (R)-procyclidine, and higher affini­ from Amersham International (Bucks, England). ties than (S)-procyclidine, at the three subtypes Atropine sulfate and polyethyleneimine were from (table 1 and fig. 2). Sigma Chemical Co. (St Louis, MO, U .S.A.), and Hexahydro-procyclidine bad the same receptor GF;c glass-fiber fdters from Whatman (Maid­ selectivity pattem as (R)-procyclidine. In contrast, stone, England). All the others reagents were of pyrrinol was almost nonselective (table 1 and fig. the highest grade available. All antagonists tested 2), as observed for (S)-procyclidine. were synthesized in our laboratories: the pro­ The binding fr::~ ~nergies of the compounds cyclidine enantiomers were prepared as previously studied in tiüs work and lileir differences are

TADLE 1 1 Comparison of pKi values a and free energies of binding (110) a (in kJ·mol- ) of (R)-procyclidine. (S)-procyclidine. pyrrinol and hexahydro.procyclidine for musearlnie receptor subtypes M1, M2 and M 4•

Muscarinic antagonist Mt (NB·OK 1) M2 (heart) M 4 (striatum) pKi; liG pKi; liG pKi; AG 1) (R}-Procyclidine 8.4 47.95 7.3 41.68 8.1 46.24 2) Pyrrinol 7.5 42.81 6.9 39.37 7.2 41.09 3) Hexahydro.procyclidine 7.1 40.55 6.1 34.81 7.0 39.96 4) (S)-Procyclidine 6.3 35.94 5.8 33.10 6.0 34.27 1-4 ~ 2.1 12.01 1.5 8.58 2.1 11.97 8 The pKi and AG values were calcu1ated as explained in Materials and methods (2.4, analysis of binding data). The standard deviation of pKi values was estimated at ±0.15 log units. b Eudisrnic index (difference of the pKi values) and differences between the free energies of binding of (R)- and (S)-procyclidine at each receptor subtype. 139

100 Heart ~ Hexahydro­ i procyclichne :J 0 lXI 50 \, (SJ -Procyclidine Ul ::E \/ Fig. 3. Interaction pbarmacophores of (ll)-proc}'didine z ' •.. ~ (eutomer) binding to four subsitcs of musearlnie receptors. ~ 0 very shnilar for the three receptors. In contrast, 1001 the difference between the binding free energies. of -I ~--·-··-..~- ..-...... STRIAH:a:yd:I T~S 0 (R)-procyclidine and pyrrinol was smaller at '\ ~prDt1C1idlne cardiac M 2 Ülan at M1 or M4 receptors. The 50 lAI -ProtyCiidtne X ~ "-.. \.ISJ-Procychdine difference observed between the free binding en­ ergies of (R)- and (S) procyclidine corresponded Pyrrtnol •.• ·. "··. ... J •· .. ,_ ... to the sum of the differences between free binding 0 ,-~~_..... g ___ ....e-.--_..-7-_",;":",j-,_s_---"'~_'""'s ..--..:'4 energies of (R)-procyclidine and pyrrinol, and be~ [ORUG] (log M) tween (R)-procyclidine and hexahydro-procycli­ dine, at M , M and M receptors. Fig. 2. [3H)NMS competition curves in membrane.; from hu­ 1 2 4 man neurobhastoma Nß.OK 1 cells (upper panel). rat beart (middle panel) and rat striatum (lower panel). eH]NMS bind· 4. Discussion äng was measured in the absence or presence of (R}procycli­ dine (0). (S)-procyclidine (e), pyrrinol (.&) and hexahydropro. The fact that procyclidine binding was highly cyclidine <•>· as described in Materialsand methods. Average stereoselective indicates that at least three groups of three experiments perfonned in duplicate. surrounding the asymmetrically substituted carl.,on atom contributed to overall drug binding affinity quoted in tables 1 and 2. The difference between {fig. 3). The free energy of (R)-procyclidine bind... the binding free energies of (R)-procyclidine and ing c~., therefore be described by equation (3): 1 hexahydro-procyclidine ( about 7 .kJ • mol- ) was .AG= ~G! + ßllGi. + y.1G3 + &lG4 (3}

TABLE2 Differenc:es in free energies (kJ · mr' -t) for binding of (R}procyclidine. (S)-procyclidine. pyrrinoJ and hcxahydro-prOC)'.,;l.idii.~ i

Musearlnie antagonist M1 (NB-OK 1) M2 (heart) M4 (striatum) (R).Procyclidine/pyninol + 5.14 + 2.31 + 5.15 (R)-Procyelidinc/hexahydro-procyclidine + 7.40 + 6.87 + 6.28 (R)-Procyclidine/(S)-procyclidine obsen·ed • + 12.01 + 8.58 + 11.97 expected b + 12.54 +9.18 + 11.43 • Differcnce betwt:en the free energies or the binding of (R)-procyelidine and (S).procyclidine at each receptor subtype. b Sum or the differences or the free energies of binding of (R)-procyclidinc and pyninot as v.·eß as of (R)-procyclidine al'ld hexahydro-procyclidine. 140

where .401, .402, J1G3 and .4G4 represent the free rieb acceptor atom also affects the hydrogen bond energy achievable by an optimal interaction of, energy. Parameters y and 8 in equation 3 are respectively, the hydrophobic phenyl ring of the therefore strongly dependent on the relative posi­ Iigand with receptor site 1, the cyclohexyl group tions of the nitrogen, oxygen and OH-hydrogen with receptor site 2, the bydroxy group with recep­ atoms of the drug considered, relative to subsites 3 tor site 3 and the prot"nated amino group with and 4 of the receptor. receptor site 4. The value of ~G should be as The phenyl and cyclohexyl groups probably negative as possible to obtain high-affmity bind­ contribute to the binding energy by two other ing. Factors a, ß, y and 8 in equation 3 take into types of interactions: ( a) hydrophobic interac­ account the fact that al1 four groups are not tionst when a nonpolar surface is r"!moved from necessarily simultaneously in optimal position to water and (b) van der Waals interactions ( dipole­ interact with reaptor sites 1 to 4 { at ß: y and 8 dipole, dipole-induced dipole and induced dipole­ values probably vary between 0 and 1, prc,vided induced dipole interactions9 brought about by the that the corresponding group does not obstruct close contact between nonbonded atoms or mole· binding by steric hindrance). cules). The hydrophobic interactions of the phenyl The protonated amino group of procyclidine and cyclohexyl groups with receptor sites 1 and 2, (fig. 3} might conceivably contribute different in­ respectively, are somewhat more independent than teractions with the fourth receptor subsite: an van der Waals interactions on the exact position ion-ion interaction (Iigand + • ... ---- - receptor), of the two ring systems, relative to sites 1 and 2. an ion-dipole interaction (Iigand + - - .. • • • recep.. Thereforet substituting the cyclohexyl and phenyl tor) and a hydrogen bond (N--H------X­ groups . of the musearlnie antagonist in hydro­ receptor). The aveiage intrinsic binding energy of phobic receptor shes 1 and 2 might be less un­ protonated nitrogenswas estimated at 11.5 kcal· favorable than sappressing the interaction of the 1 1 mol- (i.e. 48.1 kJ · mol- ; Andrews, 1986). This hydroxy or ammonium groups of the antagonist very important contrlbution to drug binding is with their respective receptor subsites 3 and 4. To Colnpatible with the Observation tbat all muscarinic test this hypothesis, we investigated the binding antagonists possess a cationic group. lonic interac­ properties of two achiral molecules, in which the tions per se probably made an important contri· phenyl or cyclohexyl groups of (R)-procyclidine bution to binding, since the two enantiomers of were replaced by a cyclohexyl or phenyl group. the quatetnary ammonium derivative tricyclamol We assumed that increases in binding free energy9 (with a pennanent charge and no N-H group), due to the loss of van der W aals interactions with show higher affinities than procyclidine for the receptor sites 1 and 2, should be additive provided three receptors (unpublished results). that the ammonium and hydroxy groups <'f the 4 The hydroxy group of (R)-procyclidine (fig. 3) ligands retain their normal binding position (fig. probably forms a hydrogen bond with the third 3). 1bis was indeed observed experimentally: the receptor subsite: desoxyprocyclidine (without an differences of binding free energies of (R)-pro­ hydroxy group) showed the same low potency as cyclidine .,... pyrrinol and (R)-procyclidine .,... (S)-procyciidine (ched by Lambrecht and hexahydro-procyclidine were small, suggesting that Mutschler, 1986). Misplacing the hydroxy group steric hindrance did not prevent the interaction of of, for example, (S)-procyclidine might be even the (larger) cyclohexyl group with the phenyl-pre­ more unfavorable for binding than replacing it ferring subsite (site 1). They were additive at M1, with a hydrogen atom, if the hydrogen bonds M 2 and M4 receptors (table 2). The stereoselectiv­ · formed with the solvent (water) must be broken to ity of these three receptors for procyclidine bind· allow the drug-receptor interaction. ing apparent!y reflected poor interactions of the The binding energy of ionic and hydrogen phenyl group at the cyclobexyl binding site and bonds depends strongly on the distance between vice versa. the two atoms considered; furthermore, the orien­ Our results also gave valuable information con­ tation of the 0-H bond respective to the electron· cerning the preferential binding of (R)-procycli- 141 dine to M1 and ~{4 receptors: the lower affmity of Andrews, P.. 1986, FunctionaJ groups, drug-receptor interac­ lions and drug design, Trends Pbarmacol. Sei. 7, 148. (R)-procyclidine for M2 sites was apparently due to a poorer fit of the cyclohexyl group in receptor Barlow. R.B .• KJ. Berry, P.A.M~ GJenton. N.M. Nikolaou and K.S. Soh~ 1976. A cornparison ~r affmity constants for subsite 2. This would indeed explain thc following -sensitivc a~tylcholine receprors in guinea-pig Observations: atri.al pacemaJter ceUs at 29°C and in ileum at 29°C and (1) (R)-Procyclidine and the dicyclohexyl de­ 37 ° C. Br. J. Pb(.Jßl3col. 5&. 613. Cheng. Y. and W.H. Ptusoff, 1973, Relationship between the rivative hexahydro-procyclidine were M1, M4 > M selective as a cyclohexyl group was in contact inhibi1ion constant (Ki) and the concentration or inhibitor 2 which causes a SO percent inhibition (lso) of an enzymatic with the 'cyclohexyl receptor site 2'. reaction, Biochern. Phanr.acot 22. 3099. {2) (S)-Procyclidine and pyrrinol, the diphenyl Hammer. R., C.P. Berrie. N.J.M. Birdsall, A.S.V. Bargen and derivative, were not selective as the cyclohexyl E.C. Hulrne, 1980, Pirenzepin~ distinguisbes betwC(:D dif~ receptor site 2 was occupied by a phenyl group. ferenr subc!asses of musearlnie reccptors. Nature 283,90. Hammer, R., E. Giraldo. G. B. Schiavi. E. Monferini üiid H. (3) The affinity loss when replacing the Ladinsky, 1986, Bindingprofile of a no\·el cardiose!e(:tive cyclohexyl group of (R)-pr~Jfclidine by a phenyl muscarine reccptor antagonist. AF-DX 116, to membranes 1 group was much smaller at M 2 (2.31 kJ · mol- ) of perlpberal tissues and brain in the rat. Life Sci. 38, 1653. 1 Kore, M., M.S. Ackerman and W.R. Roeske. 1987, A tban at M1 and M 4 receptors (5.15 kJ · mol- ). cboü.nergic a.ntagonist identifies a subdass of muscarinie In conclusion, musearlnie M 1, M 2 and M4 re­ receptors in isolatcd rat pancreatic acini. J. Phannacol. ceptors clearly discriminated between the two pro­ Exp. 11ter. 240, 118. cyclidine enantiomers, and preferred (R)-pro­ Lunbrecllt. G.. R. Feifcl. U. Moser. AJ. Aasen, M. cyclidine. This is in line with functional studies on Waelbrocck. J. Christopb.: and E. Mutschler. 1988. Stereo­ guinea-pig ileum. (Tacke et al., 1986). The enanti­ sctectivity of the enantiomers of and its methiodide at muscuinic receptor subtype:S, European J. oselectivity of cardiac M 2 receptors was lower Phanna«·l. ISS. J.67. neuroblastoma than that of M1 and striatum M4 Lambrecht. G. and E. Mutschler. 1986. Chirality as a tool Cor receptors. A systematic comparison of the binding subclass!fication of rcceptors, in: Innovative Approaches in properlies of the two procyclidine enantiomers Dr-..:g Research. :d. A.F. Harms (Elsevier Science Pub-­ and of the related achiral compounds pyrrinol and lisbers. Amsterdam) p. 353. hexahydro-procyclidine suggested that the recep­ Levine, R.R. and NJ.M Binfsall (eds.). 1989, Subtypes of musc.arinic receptors. IV. Trends Phatma(:OL Sei. 10 tors' stereoselectivity reflected the loss of van der (Suppl.). W aals interactions of the hydrophobic receptor Lowry. O.H.. NJ. Rosebrough. A.L. Farr and RJ. ~ subsites recognizing the phenyl and cyclohexyl 1951, Protein measurement witb the Folin phenol reagent. groups of the Iigand. The lower affinity and J. Biol Chem. 193. 265. eudismic index of musearlnie M receptors were Michel, A.D.• R. Delmendo, E. Stefanich and R.L. Whiting. 2 1989, Binding cbaractemtics of the mu.scarinic rcceptor due to the poorer interaction of their subsites with subtype of the NG108-15 ceU line. Naunyn-Sch.miedeb. the cyclohexyl group (as compared to the Arcb. Pbarmacol. 340, 62. cyclohexyl subsite of M1 or M 4 receptors). Mitchelson. F., 1988., Musearlnie receptor differentiation, Phannacol. Ther. 37, 357. Mutschler, E. and G. Lambrecht, 1984, Sel«tr.•e musearlnie Acknowledgements agonists and antagonis'ts in functional tests, Trends Pharmacol. Sei. s. Suppl. 39. Richardson. A. and A. Humrich. 1984, A mierocomputer pro­ M.W., J.C., M.T. and J.C. thank the Fund for Medical gram for tbe analysis of radioligand binding curves and Scientific Research (Belgium) for grant 3.4S71.85. R. T. thank.s otber dosc-response data, Trends Pb.armacot Sei. 5. -+7. the Deutsche Forschungsgemt=..nscbaft, and G.L, E.M. and Tack:e. lt. H. Lino~ L. Ernst, U. Moser, E. Mutschler, S.. R. T. thank the Fonds der Chemischen Industrie !or financial Sarge, H.K. Cammenga and G. Lambrecht. 1987. Prepara· SUpport. tion and properlies of tbe enantiomers of the anti­ mUY.~inic agents sila·procyclidine and sila·tricyclamol ieJide: optica11y active silanols with silicon as the c:enter oJ References clürality. Chem. Berat. 120, 1229. Tackc, R., H. Linoh. D. Schombarg. L. Ernst. U. Moser~ E. Adamson, D.W~ 1949, Aminoalkyl tertiaty carbinols and de­ Mutsch1er and G. Lamb:rccbt. 1986, On tbe absolute con­ rived products. Part 1. .3-Amino--1: l--c!ipbenylpropan-1-ols, figuration of the enantiomus of the antimuscarinic agents J. Chem. Soc. Suppl, s:44. procyclidine and tricydamol iodide: X·ray st:r'UCtiU'al anaJy- 142

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