The design and pharmacology of novel selective muscarinic agonists and antagonists

Life Sciences, Vol. 56, No.s 11/l& pp. 815422, 1995 Copyright Q1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/95 $9.50 + .OU

Pergamon 0024-3205(95)00015-l

THE DESIGN AND PHARMA COLOGY OF NOVEL SELECTIVE AGONISTS AND ANTAGONISTS

MUSCARINIC

G. Lambrecht*l, J. Gross’, U. Hacksellz, U. Hermannil, C. Hildebrandtl, X. Hou3, U. Moser’, B. M. Nilsson2, 0. Pfaffl, M. Waelbroeck3, J. Wehrlel and E. Mutschlerl lDept. of Pharmacology, University of Fra&i.nt, D-60439 Frankl?ut/M., Germany, 2Dept. of Organic Pharmaceutical Chemistry, University of Uppsala, S-75 123 Uppsala, Sweden and 3Dept. of Biochemistry and Nutrition, Free University of Brussels, B-1070 Brussels, Belgium.

The muscarinic pharmacology of C 1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent Ml-selective agonist known today (pECs0: Ml/rabbit vas deferens = 7.83; Mzlguinea-pig atria = 6.35; M&uinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)4-F-MePyMcN was a potent partial agonist (pECs0 = 7.22; apparent efficacy = 0.83). These results indicate that (S)4-F-MePyMcN might be a useful tool to study Ml receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7,86/atria; pKi = 7.8/rat heart) with lower affinities for the Ml (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6,92/guinea-pig ileum; pKi = 6.7/rat pancreas) and Mb receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal HI receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging. Key Words: McN-A-343

analogues,

(S)-dimethindene,

tripitramine,

anococcygeus

muscle

Each of the four muscarinic receptor subtypes, Ml - M4, has been demonstrated to exist in the CNS (1,2). Ml receptors are particularly enriched in the cortex and the hippocampus, two brain areas known to play an important role in learning and memory processes. Activation of these postsynaptic Ml receptors causes cellular excitation. M2 receptors appear to be most often expressed as presynaptic autoreceptors on, e.g. cortical and hippocampal, cholinergic nerve terminals, the stimulation of which results in an inhibition of acetylcholine release (3). Based on this knowledge, selective Ml receptor agonists capable of penetrating into the CNS may be con*To whom correspondence

should be addressed.

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Ligands

Hfi) 7’ N-C-0-C-C=C-CH,-Am ;r

R’ = H, CH,, X

C,H,

Am (R* = H, CHJ

3-Cl, -F

+NR*(CH,),

4-Br, -Cl, -F

+NR*CH&H,

3,4-dichloro

+NR2G2W2

R’\ /N' R’\ /N'

3 3

Fig. 1 Chemical structure of tertiary and quaternary analogues of McN-A-343 [X = 3Cl, Am = +N(CH,),, R1 = H]. BN 228: X = 4-C], Am = +N(CH,),, RI = CH3; BN 225: X = 4-Cl, Am = +NH(CH&, R1 = CH3 ; 4-F-MePyMcN+: X = 4-F, Am = N-methylpyrrolidinium, RI = CH,; 4-F-MePyMcN: X = 4-F, Am = pyrrolidino, R1 = CH3; 4-Cl-McN-A-343: X = 4-Cl, Am = +N(CH,),, R1 = H; 4-F-PyMcN: X = 4-F, Am = pyrrolidino, RI = H; 4-F-PyMcN? X = 4-F, Am = N-methylpyrrolidinium, RI = H.

sidered to be candidates for the treatment of the cholinergic deficit involved in Alzheimer’s disease (4). Furthermore, lipophilic M2 antagonists, which enhance the release of acetylcholine by blockade of M, autoreceptors, may also be of use in the treatment of cholinergic deficits (3). An ideal compound would act both as a selective postsynaptic Mr agonist and as a selective presynaptic M2 antagonist, Lipophilic M, antagonists could also be useful as diagnostic tools for quantifying the loss of central M2 receptors in Alzheimer’s disease with PET imaging (5). The tixrctionally Ml-selective agonist McN-A-343 (Fig. 1) has been used in our laboratory as a starting point for the design of CNS active muscarinic ligands, the primary objective being to replace the quaternary ammonium group of McN-A-343 by a tertiary bioequivalent. Structure-activity relationship studies culminated in the first tertiary amino analogue of McN-A-343, 4-F-PyMcN (Fig. l), that acts as a functionally Ml-selective agonist (6). In order to increase the potency of 4-F-PyMcN at Ml receptors and to alter its functional selectivity in favour of the Ml subtype, we chose to incorporate an element of asymmetry into the butynyl chain of this series of compounds, The new chiral Cl-methyl-substituted analogues of McN-A-343 (Fig. 1) were assayed for muscarinic activity on the rabbit vas deferens (putative Ml receptors) as well as on the guinea-pig atria (M2 receptors) and ileum (M3 receptors) (Moser et al., this volume). In this paper we describe the fimctional pharmacological profile of the pure enantiomers of BN 225, BN 228, 4-F-MePyMcN and 4-F-MePyMcN+ (Fig. 1). As a result of our studies of the stereoselective interaction of chiral antagonists with muscarinic receptor antagonist, receptor subtypes (7) the (S)-enantiomer of the histamine HI

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Fig. 2 Chemical structure of dimethindene denotes the centre of chirality.

(left) and tripitramine

(right). The asterisk

dimethindene, was found to be an Mz-selective antagonist. In this paper we also describe the antimuscarinic properties of both enantiomers of dimethindene (Fig. 2) using the M2 antagonist tripitramine (Fig. 2) (8, 9) as the reference drug.

Methods Functional activity at muscarinic receptor subtypes has been studied in one cardiac and in various smooth muscle preparations (see TABLE I and II). These methods have been described in detail earlier (6, 10-13; Gross et al., this volume and Pfaff et al., this volume). Muscarinic binding selectivity was assessed using [3H]N-methylscopolamine ([3H]NMS) binding to homogenates of NB-OK 1 cells (MI) and rat heart (Mz), pancreas (M-J and striatum @Id) (14). Data analvsis The potencies and intrinsic activities of the agonists were expressed by their pEC5o and apparent efficacy (a.e.) values, Antagonism was evaluated by Schild analysis (15). [3H]NMS competition binding data were analyzed by a computer-assisted curve-fitting procedure, and unlabelled drug apparent pKi values were calculated (14). Results are given as arithmetic means (n =4 - 18). The following drugs were used: pirenzepine and AQ-RA 741 (Thomae, Tacke, Dr. R. p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; BiberacWGermany), Karlsruhe/Germany), tripitramine (Dr. C. Melchiorre, Bologna/Italy), (R)- and (S)-dimethindene (Zyma, Munich/Germany). Arecaidine propargyl ester as well as McN-A-343 and analogues were synthesized in our laboratories. All other chemicals were of the highest grade available.

McN-A-343 and analogues All agonist responses were determined to be muscarinic in nature in that their activities were blocked by: pirenzepine (O.luM; pA2 = 8.14 - 8.51) in rabbit vas deferens (MI), AQ-RA 741 (100 - 300 nM; pA2 = 8.31 - 8.49) in guinea-pig atria (I$) and p-F-HHSiD (0.5 uM; pA2 = 7.5 1 - 7.91) in guinea-pig ileum (M3). Tetrodotoxin (0.1 uM) and hexamethonium (100 @I) did

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TABLE I In Vitro Functional Activity of McN-A-343 Analogues at Ml Receptors in Rabbit Vas Deferens (RVD), M2 Receptors in Guinea-Pig Atria (GPA) and M3 Receptors in Guinea-Pig Ileum (GPI). Abbreviations for the Muscarinic Agents are Explained in Legend of Fig. 1 GPAM2

RVD/M,

McN-A-3434 4-Cl-McN-A-343d (R)-BN 228 (S)-BN 228 (R)-BN 225 (S)-BN 225 (R)+F-MePyMcN+ (S)-4-F-MePyMcN+ (R)-4-F-MePyMcN (S)4-F-MePyMcN

GPI/M,

pECsc

a.e.3

pA2

pEC&

a.e.h

pA2

6.77s 7.13 6.92 7.83 b 6.51 6.37 7.25

1.00 1.00 1.00 1.00

4.87f 5.26 5.52 6.35

0.49 0.77 1.00 1.00

1.00 0.27 0.25

7.22

0.83

h 5.651 7.26’ 5.41’ 7.11’

5.10 -

0.51 -

4.48k 5.901 6.33’ 5.6Oi 7.39i

pEC=,n a.e.E 5.511 5.71 5.99 6.29 5.40 -

0.83 1.00 1.00 1.00 0.79 -

pA2 4.54k 5.76i 6.421 5.29i 6.821

aJ,c Apparent efficacy: the maximum response to McN-A-343a and to arecaidine propargyl esterQ = 1.OO. d Data taken from Lambrecht et al. (6). c pKA (= 5.17) was estimated using irreversible antagonism (17). f,g pKA values determined by the technique of Waud (18) and using arecaidine propargyl ester as full agonist, were: M, = 4.79, M, = 5.17. h No effect (up to 30 PM). i Slopes of Schild plots were not significantly different from unity (P>O.O5). k pA2 values (30pM) were calculated using individual dose ratios (15).

not block agonist activities in guinea-pig atria and ileum. The potency (pEC&, apparent efficacy (a.e.) and affinity (PAZ) for McN-A-343 and analogues are shown in TABLE I. As shown in TABLE I, the effects of McN-A-343 and of most of its analogues differed clearly in the three fUnctiona assays. McN-A-343 and its 4-chloro derivative had comparable potency in rabbit vas deferens, whereas in both the atria and ileum they behaved as partial or ml1 agonists, being more potent and more efficacious in the M, assay. The incorporation of chirality into this series of acetylenic derivatives related to McN-A-343 has had a beneficial effect upon the potency (affinity) and muscarinic receptor subtype-selectivity. In general, the introduction of a methyl group at the Cl position in 4-Cl-McN-A-343, 4-F-PyMcN and 4-F-PyMcN+ (6) led to an increased potency and affinity at the three receptor subtypes, the (S)-enantiomers consistently yielding compounds with higher activity than the corresponding (R)-isomers. The two enantiomers of the quaternary compound BN 228 were full agonists in all three tissue preparations, the weaker (R)-isomer displaying about the same selectivity profile as the parent compound 4-C% McN-A-343. (S)-BN 228 was found to be the most potent Ml-selective agonist known today (potency ratios: Ml/M, = 30; MI/M3 = 35). It was also more potent than McN-A-343 (56-fold) at ganglionic Ml receptors in the pithed rat, and it exhibited higher affinity than the parent compound for Ml receptors in rabbit sympathetic ganglia (pKi/MCN-A-343 = 5.06; pKi/(S)-BN 228 = 6.74) (16). The more potent (S)-enantiomer of the tertiary compound BN 225 exhibited a

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selectivity profile similar to that of McN-A-343, whereas the corresponding very weak antagonist in atria and ileum and inactive in the vas deferens.

(R)-isomer

was a

Among the amino terminally modified analogues of McN-A-343, the tertiary amine, (S)4-F-MePyMcN, was a potent partial agonist at Ml receptors in rabbit vas deferens. In contrast, at M2 receptors in atria and at ileal M, receptors (S)-4-F-MePyMcN lacked et%cacy and was shown to act as a competitive antagonist with pA2 values of 7.4 and 6.8, respectively. Interestingly, the corresponding quaternary analogue, (S)-4-F-MePyMcN+, was found to be a relatively potent Ml-selective antagonist. The (R)-enantiomers of 4-F-MePyMcN and 4-FMePyMcN+ were weak non-selective muscarinic antagonists (pAz values = 5.3 - 5.9). The stereoselectivity ratios (= antilogs of the difference of respective pA2 values) for 4F-MePyMcN at Ml, M2 and M, receptors were very similar [(S)/(R) = 50, 63 and 32, respectively]. In contrast , these ratios of the quaternary analogue, 4-F-MePyMcN+, differed in the three functional pharmacological assays. They were very low at M, and M, [(S)/(R) = 2.5 and 4, respectively], but high at Ml receptors [(S)/(R) = 401. This implies that the stereochemical requirements of the muscarinic receptor subtypes are different for the enantiomers of 4-FMePyMcN+, being most stringent at MI receptors. Dimethindene and tripitramine Tripitramine and the enantiomers of dimethindene did not elicit an agonist response itself but surmountably antagonized responses to the agonists in all preparations studied. They behaved as simple competitive muscarinic antagonists in the fUnctiona pharmacological assays (TABLE II). In all the tissues tested (TABLE III) (R)- and (S)-dimethindene inhibited [3H]NMS binding to muscarinic receptors. The competition curves did not deviate significantly from results expected for competitive inhibition of tracer binding to a single receptor subtype. Functional (pA2 values) and binding (pKi values) affinities for the three antagonists are shown in TABLE II and III. Binding data for tripitramine were taken from work previously published (8). In general, (S)-dimethindene was more potent than the (R)-enantiomer in all muscarinic assays. However, the stereoselectivity ratios were found to be different at the four muscarinic receptor subtypes, being greatest at M, receptors (32- to 41-fold). In contrast, the stereoselectivity was inverse at histamine HI receptors, (R)-dimethindene being the eutomer (TABLE II). The pA2 values for (S)-dimethindene at Ml receptors in rat duodenum and rabbit vas deferens were not significantly different (P>O.O5) either from each other or from pA2 values determined at M3 receptors mediating contractions of the guinea-pig ileum and trachea or from the pKi value determined at M4 receptors in rat striatum. However, the pA2 (pKi) values for (S)dimethindene at muscarinic M2 receptors in guinea-pig atria, rabbit vas deferens, rabbit anococcygeus muscle* and rat heart were significantly (PcO.05) greater (up to 32-fold) than those obtained at Ml, M3 and Ma receptors. The fUnctional affinity estimates (TABLE II) of (R)- and (S)-dimethindene at Ml, M, and M3 receptors were highly correlated (r = 0.98) with their binding affinities (TABLE III) in NB-OK 1 cells and rat heart and pancreas. This correlation supports the suggestion that the receptor subtypes studied in the fUnctional preparations are equivalent to the muscarinic binding sites in the tissues investigated. As far as muscarinic receptor specificity is concerned, no specific binding (up to a concentration of 10 @I) was found for (S)-dimethindene by Nicholson et al. (19) at a2- and P-adrenoceptors, 5HT1 receptors as well as at benzodiazepine receptors. With respect to al-adrenoceptors, 5-I-IT, receptors and dopamine D2 receptors, interactions of (S)-dimethindene occur only at concentrations in the micromolar range (pKi: ccl = 6.3; 5-HT2 = 5.7; D2 = 6.5). *The rabbit anococcygeus muscle is a novel tinctional M2 and, respectively, Ma receptor model, recently developed in our laboratory (Gross et al., this volume).

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TABLE II Affinity Estimates (pA2 Values) for (R)-Dimethindene, Various Functional Muscarinic Receptor Subtypes

(S)-Dimethindene

and Tripitramine

at

pA2 valuesa Subtype Ml Ml M2 M2 M2 M3 M3 M3 M4 Hl

Preparation Rabbit vas deferen& Rat duodenums Guinea-pig atriaf Rabbit vas deferensg Rabbit anococcygeusf Guinea-pig ileumf Guinea-pig tracheaf Rat duodenumf Rabbit anococcygeu& Guinea-pig ileumi

(R)-Dimethindene 5.81

5.49 6.25 6.22 n.d.h 5.61 5.59 n.d.h n.d.b9.42

(S)-Dimethindene 6.83 6.36 7.86 7.74 7.69 6.92 6.96 n.d.b n.d.h 7.48

Tripitramine 8.55 8.33 9.69s 9.14c 9.10s 7.29 n.d.h 7.60 7.69 n.d.h

a Slopes of Schild plots were not significantly different from unity (P>O.O5). Thus, pA2 values were calculated from regression lines whose slopes were constrained to 1.OO. b n.d. = not determined. E There was hardly any antagonism below 30 nM. Thus, the concentrations tested were 30, 100, 300 and 1000 nM. &e,f&h,j Agonists used: 4-Cl-McN-A-343d (Fig. l), 4-F-PyMcN+c (Fig. l), arecaidine propargyl esterf, carbacholg, (+)-muscarin&, histamine!

TABLE III Apparent AfIinities (pKi Values) for (R)-Dimethindene, (S)-Dimethindene Muscarinic Binding Sites in Various Tissues and Cell Lines

and Tripitramine

at

pKi values Subtype

Tissue/Cell Line

MIb ml M2h m2 M3h m3 M4h m4

NB-OK 1 cells CHO-K 1 cells Rat heart CHO-K 1 cells Rat pancreas CHO-Kl cells Rat striatum CHO-K 1 cells

(R)-Dimethindene

(S)-Dimethindene

5.6

7.1

6.3

7.8

5.6

6.7

6.0

7.0

Tripitraminti

8.8 9.6 7.4 8.2

a Data taken from Maggio et al. (8). b Muscarinic binding sites were labelled using [3H]N-methylscopolamine. Hill coefficients of the competition curves were not significantly different from unity (P>O.O5).

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The order of apparent affinities for tripitramine in the various tissues (TABLE II) were atria (Mz) 1 vas deferens (M2) = anococcygeus* (Mz) > vas deferens (MI) = duodenum (MI) > anococcygeus* @Id) 2 duodenum (I$) and ileum (M3). Tripitramine was, therefore, about 8 fold selective for M2 receptors versus MI receptors but 40- and 80-fold, respectively, selective for M2 versus M4 and M, receptors. The fUnctiona affinities of tripitramine for native muscarinit receptor subtypes MI - M4 (TABLE II) and the binding affinities for cloned ml - m4 receptors (TABLE III) were very similar, both individually and in rank order. Thus, tripitramine appears to be a potent M2 antagonist. However, it is worth noting that tripitramine displayed competitive antagonism at M2 receptors (atria, vas deferens and anococcygeus muscle*) only at high concentrations (2 30 nM). In addition, the binding affinities of tripitramine at cloned ml (pKi = 8.8) and m3 receptors (pKi = 7.4) (TABLE III) were found to be much higher than the corresponding afftnities at native Ml (rat cortex; pKi = 7.6) and M3 receptors (rat submaxillary gland; pKi = 6.19) (9). The reasons for these discrepancies are unclear. Further experiments are needed to clarifjl these issues.

Conclusions The present results describe novel muscarinic agonists related to McN-A-343. The high potency and functional MI selectivity of (S)-BN 228 and (S)-4-F-MePyMcN make these compounds suitable for the study of muscarinic receptor mechanisms. Due to its MI-agonistic and M2-antagonistic properties, the non-quatemary compound, (S)-4-F-MePyMcN, may be considered as an important tool for future drug research in the field of cognitive disorders. Its partial agonist nature may be advantageous in therapeutic applications, as chronic administration of the drug would be less likely to result in receptor desensitization. The experiments also demonstrate that (S)-dimethindene is a novel MTselective muscarinic antagonist. Since studies in man showed that (S)-dimethindene penetrates readily into the brain (19 - 21) this compound could be a valuable tool to test the hypothesis that lipophilic M2-selective antagonists show beneficial effects in the treatment of Alzheimer’s disease. (S)dimethindene might also become the starting point for the development of appropriate PET ligands useful as antemortem diagnostic tools for quantifying the loss of M2 receptors in the brain of Alzheimer’s patients. One interesting aspect of using chiral antagonists, such as dimethindene, in PET studies is the availability of the less potent optical isomer, which can be used to measure nonspecific binding under the imaging condition. Based on its selectivity profile, tripitramine might also be a candidate to study M2 receptor mechanisms in the CNS. However, this type of compound is not expected to penetrate into the brain readily (3, 22).

The authors thank the Deutsche Forschungsgemeinschatt and the Fonds der Chemischen Industrie (Germany) for financial support and gratefully acknowledge the donation of drugs.

*The rabbit anococcygeus muscle is a novel functional M2 and, respectively, M4 receptor model, recently developed in our laboratory (Gross et al., this volume).

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