Muscarinic activity in the isolated guinea pig ileum of some carboxamides related to oxotremorine

European Journal of Pharmacology, 85 (1982) 79-83

79

Elsevier Biomedical Press

M U S C A R I N I C ACTIVITY IN THE ISOLATED GUINEA PIG I L E U M OF S O M E C A R B O X A M I D E S RELATED TO O X O T R E M O R I N E BJ()RN R I N G D A H L * ' * * * , B A H R A M RESUL **, D O N A L D J. J E N D E N * and R I C H A R D D A H L B O M **

• Department of PharmacoloyA', School of Medicine, University of California, Los Angeles, Cahfornia 90024, U.S.A. and * * Department of Organic Pharmaceutical Chemistry, Biomedical Center, Universi O, of Uppsala, Box 574, S-751 23 Uppsala. Sweden Received 15 June 1982, revised MS received 10 August 1982, accepted 30 August 1982

B. RINGDAHL, B. RESUL, D.J. JENDEN and R. DAHLBOM, Muscarinic aetivi(v in the isolated guinea pig ileum of some carboxamides related to oxotremorine, European J. Pharmacol. 85 (1982) 79-83. A series of structural analogues of the potent oxotremorine-like agent N-(4-pyrrolidino-2-butynyl)-N-methylacetamide (1) was investigated for muscarinic activity in the isolated guinea pig ileum. Substitution of larger alkyl groups for the acetyl methyl group of 1 results in an attenuation of muscarinic potency. The observation that the agonist N-(4-dimethylamino-2-butynyl)-N-methylpropionamide (6) has a dissociation c o n s t a n t ( K A --5.1 X 10-5 M), estimated after elimination of spare receptors with dibenamine, similar to that of the antagonist N-(4-dimethylamino2-butynyl)-N-methyl-2,2-dimethylpropionamide (11) suggests that the decrease in muscarinic agonist activity with increasing substitution is due mainly to a loss of efficacy. The N*methyl group of 1 is essential for muscarinic activity since its replacement by a hydrogen atom or an ethyl group yields antagonists. Muscarinic action

Oxotremorine analogues

Structure-activity relationships

1. Introduction

Bebbington et al. (1966) reported that N-(4-pyrrolidino-2-butynyl)-N-methylacetamide (1), which may be regarded as a close structural analogue of oxotremorine, is a parasympathomimetic agent almost as potent as oxotremorine in vitro. In vivo, 1 is a powerful central and peripheral muscarinic agonist (Bebbington et al., 1966; Svensson et al., 1978) surpassed in potency only by oxotremorine and its azetidine analogue (Resul et al., 1980; 1982). The dimethylamino analogue of 1 elicits effects similar to those seen after the administration of 1, although at higher doses (Bebbington et al., 1966). We prepared a number of analogues of 1 and tested them for tremorogenic (central muscarinic) and tremorolytic activity in intact mice (Svensson et al., 1978; Resul et al., 1979). Only the propionic acid amide related to 1 and its dimethyl-

*** To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 r,~ 1982 Elsevier Biomedical Press

Guinea pig ileum

Spare receptors

amino analogue were found to be tremorogenic. Distributional factors may play a major role in determining the central potency of tertiary amines (Brodie et al., 1960; Karl6n and Jenden, 1970). Results obtained from in vivo experiments with such drugs, although informative in many other respects, therefore provide little information regarding drug-receptor interactions. In order to obtain a more sophisticated analysis of the relationship between chemical structure and muscarinic activity in compounds related to 1, we have examined a number of analogues of 1 for muscarinic and antimuscarinic activity in the isolated guinea pig ileum. //O

CH3- C \N/ CH2-C---C-C H2-N~ CHs 1

~0

N~/~ -CH2-C---C-CH 2OXOTREMORINE

Fig. 1. Structural formulas for NI(4-pyrrolidino-2ibutynyl)-N_ methylacetamide (1) and oxotremorine.

80 The pharmacological potency of muscarinic agonists, as measured by the contractile responses in the guinea pig ileum, is determined by both affinity and efficacy (Stephenson, 1956). The question therefore arises whether differences in potency observed among members in a series of agonists is to be attributed to differences in the affinity for the receptor or to differences in the efficacy of the compounds in producing agonist activity after they have combined with the receptor. If there are spare receptors differences in potency may be due to either of these causes. Accordingly, we have evaluated the receptor reserve with respect to the contractile response in the guinea pig ileum of a representative agonist, N-(4-dimethylamino-2butynyl)-N-methylpropionamide (6), having intermediate potency. For this purpose we used the irreversible antagonist dibenamine. The dissociation constant (KA) of 6 at muscarinic receptors in the guinea pig ileum was determined according to the method of Furchgott (1966).

2. Materials and methods

2.1. Drugs Compounds 1--15 (Svensson et al., 1978; Resul et al., 1979) and oxotremorine sesquioxalate (Bebbington and Shakeshaft, 1965) were prepared as previously described. Other drugs and their sources were the following: carbamylcholine chloride (Aldrich Chemical Co., Milwaukee, WI, U.S.A.), hexamethonium chloride ( K & K Laboratories, Plainview, NY, U.S.A.), atropine sulphate (Mallinckrodt Inc., Paris, KY, U.S.A.), dibenamine (N,N-dibenzyl-2-chloroethylamine) hydrochloride (gift of Dr. P.T. Ridley, Smith Kline & French Laboratories, Philadelphia, PA, U.S.A.). The Tyrode solution had the following composition (mM): NaC1 137; N a H C O 3 12; glucose 5.0; KC1 2.7; MgSO 4 1.0; N a H 2 P O 4 0.4; CaC12 1.8.

moved and suspended in a 10 ml organ bath containing Tyrode solution at 37°C and aerated with 02 containing 5% CO 2. Contractions were recorded isotonically at l g of tension, using an electromechanical displacement transducer and a potentiometric recorder. Agonists were compared on the same preparation with carbachol using the cumulative dose-response technique. Potencies were expressed as pD 2 values (negative logarithm of the EDs0 values). The preparation was allowed to equilibrate with each concentration of antagonist for 15 rain before dose-response curves to carbachol were obtained. pA 2 values were calculated according to the method of Arunlakshana and Schild (1959). 2.3. Dissociation constant of the agonist 6 The dissociation constant of compound 6 at muscarinic receptors of guinea pig isolated ileum was determined according to the method of Furchgott (1966) and Furchgott and Bursztyn (1967) using cumulative additions of the agonist. After the determination of the control dose-response curve, the preparation was treated with an adequate amount (successive 20 min incubations with 5 × 10 6, 10 5 and 5 × 10 -6 M) of dibenamine to occlude a fraction of receptors. The tissue was then washed several times, allowed to rest for 20 min and challenged with a submaximal dose of 6 until constant responses were obtained. The complete dose-response curve of 6 was then obtained in the dibenamine-treated tissue. Several equipotent doses of 6 before (A) and after (A') dibenamine treatment were determined graphically. 1/(A) was plotted vs. 1/(A') and the points were fitted on a straight line by linear regression analysis. The dissociation constant (K A) was calculated from the slope of the regression line and the intercept on the ordinate.

3. Results

2.2. Isolated guinea pig ileum Guinea pigs (male, English short hair, 350400 g) were killed by a blow to the head and bled. Segments of the ileum (2-3 cm long) were re-

The results of the pharmacological tests are shown in table 1 which also includes carbachol and oxotremorine as reference compounds. As previously reported (Bebbington et al., 1966), the

81 TABLE 1 Muscarinic and antimuscarinic activity of some N-(4-tert-amino-2-butynyl) carboxamides in the isolated guinea pig ileum. R-CO- N -CH2-C~C-CH2-Am W Compound

R

Rl

Am

pD 2 a

pA 2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Carbachol Oxotremorine

CH 3 CH 3 H H CH3CH 2 CH3CH 2 CH3CH2CH 2 CH3CH2CH 2 (CH3)2CH (CH3)3C (CH 3 )3C C6H 5 CH 3 CH3CH 2 CH 3

CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH3CH 2 CH3CH 2 H

NC4H 8 N(CH3) 2 NC4H 8 N(CH3) 2 NCnH s N(CH3) 2 NC4H 8 N(CH3) 2 NC4H s NC4H 8 N(CH 3)2 NC4H 8 NC4H 8 NC4H 8 NC4H 8

7.15-+0.02 6.37+0.03 5.40+0.05 4.77-+0.04 6.38+0.04 5.86+0.05 5.18+0.04 4.68 -+0.05

(4) (4) (3) (3) (4) (4) (3) (3) 4.36-+0.03 4.45 -+0.04 4.29-+0.05 4.39+0.02 5.44 + 0.04 5.20+0.04 4.50+0.03

(4) (4) (4) (4) (4) (4) (4)

7.20-+0.04 (8) 7.58 -+0.05 (5)

a pD 2 and pA 2 values given-+ S.E. Number of estimates given in parentheses.

100 25

80 ~.

20

60

I

[A]

40

x I0 5

15

o~ 20 I0 -7

-6

-5

-4

-3

LOGIo CONCENTRATION (mol/litre) Fig. 2. Dose-response curves for compound 6 in the isolated guinea pig ileum. The curve to the left (©) is the dose-response curve of 6 before dibenamine treatment (control curve). The intermediate curves are the dose-response curves of 6 after two successive 20 min incubations with dibenamine at 5 × 10 6 (@) and 10 -5 M (/',) illustrating the existence of a receptor reserve. To the right ( A ) is depicted the dose-response curve of 6 after an additional incubation with dibenamine at 5 × 10 -6 M showing a decrease in the maximum response. Data for one typical experiment.

I

I

I

I

1

2

4

6

8

I0

I

Ix]

x I04

Fig, 3. Double reciprocal plot of A vs. A' for compound 6. Values for A and A' were obtained from the control dose-response curve and the plotted points ( A ) after the third dibenamine incubation, respectively (fig. 1).

82 acetamides 1 and 2 are potent muscarinic agonists in vitro. The formamides 3 and 4 are about 50 times less potent than their corresponding acetamides (table l). Replacement of the acetyl methyl group in 1 and 2 by progressively larger alkyl groups results in an attenuation of muscarinic agonist activity. Thus the propionic acid amides 5 and 6 retain considerable muscarinic agonist potency whereas the butyric acid amides 7 and 8 are rather weak muscarinic agonists. It is interesting to note that the branched isomer of the 'full' agonist 7, i.e. the isobutyric acid amide 9, is a pure antagonist. The more heavily substituted analogues 10, 11 and 12 also behave like competitive antagonists. The dimethylamino derivatives are consistently less potent than their corresponding pyrrolidino analogues. Substitution of an ethyl group for the N-methyl group in the potent agonists 1 and 5 yields competitive antagonists (13 and 14). Removal of the N-methyl group of 1 also gives an antagonist (15), although it is weaker than 13 and 14. The muscarinic nature of the response produced by compound 6 is confirmed by the observation that hexamethonium (3 × 10 4 M) has no appreciable effect on the dose-response curve of 6 whereas atropine (10 ~ M) causes a parallel shift of the curve to the right. Dibenamine (two successive 20 rain incubations with 5 × 10 6 and 10 5 M, respectively) produces a shift to the right of the dose-response curve to 6 without any noticeable decline in the maximum response obtainable (fig. 2). After another incubation with dibenamine (5 X 10 6 M for 20 min) a decrease in the maximum response to 6 is observed. The double reciprocal plot of A vs. A' gives a straight line (fig. 3) from which a dissociation constant for the agonist-receptor complex of (5.07 ± 1.6) × 10 5 M (n = 4) was calculated.

4. D i s c u s s i o n

The K A estimated for compound 6 exceeds the concentration required to produce a half-maximal contraction (EDs0) prior to dibenamine treatment by about 37 times. These results indicate that there is a substantial spare receptor capacity for corn-

pound 6 with respect to contractile responses in the guinea pig ileum. Using the estimated K A it may be calculated (Furchgott and Bursztyn, 1967) that prior to receptor inactivation a maximum response is obtained when about 10% of the total active receptors are occupied by compound 6. The dissociation constant of oxotremorine at muscarinic receptors in the isolated guinea pig ileum, estimated under similar experimental conditions as the K A of compound 6, was recently shown to be 1.09 X 10 ~ M (Ringdahl and Jenden, unpublished observations). Compound 6, therefore, has about 50 times lower affinity than oxotremorine for the receptors studied. Since the EDs0 for contraction of compound 6 is about 53 times greater than the EDs0 of oxotremorine (table 1), it appears that the efficacy of 6 is similar to that of oxotremorine (Furchgott and Bursztyn, 1967). The existence of a receptor reserve in the guinea pig ileum for agonists of the type studied implies that the observed decrease in potency on increasing substitution (table 1) is due to either a decrease of affinity or efficacy or both. However, there is evidence that the effect of replacing the ethyl group in the agonists 5 and 6 by larger alkyl groups is mainly to reduce efficacy. Firstly, the higher homologues, being antagonists, retain measurable affinity, but are devoid of efficacy. Secondly, replacement of the ethyl group of 6 by the bulkier t-butyl group yields an antagonist (11), having about the same affinity as 6. An almost universal feature of potent muscarinic agonists is the presence of a methyl group, corresponding to the acetyl methyl group of acetylcholine. It has been proposed (Baker et al., 1971; Triggle 1979) that the steric relations between this methyl group and the trimethylammonium group are the most important determinants of muscarinic activity. The tertiary amino group of 1, like that of oxotremorine (Hanin et al., 1966), is mostly protonated at physiological pH. It seems likely that it has a similar function in the drug-receptor interaction as the trimethylammonium group of conventional muscarinic agonists. The observation (Bebbington et al., 1966) that the trimethylammonium analogue of 1 is about equipotent with 1 lends support to this view. How-

83

ever, the agonist 1 has two methyl groups. It is known that in the acyl cholines (Wurzel, 1959) and the tertiary acyl 3-quinuclidinols (Mashkovsky and Yakhontov, 1969) there is a rapid attenuation of muscarinic potency as the carbon chain of the carboxyl group is lengthened. Thus propionylcholine has about 1/19 and butyrylcholine 1/510 of the potency of acetylcholine on the guinea pig intestine (Wurzel, 1959). Moreover, replacement of the acetyl methyl group in acetylcholine, or its equivalent in other muscarinic agonists, with a hydrogen atom drastically reduces muscarinic activity (see Triggle and Triggle, 1976 for a review). The effects of structural variations in the acyl group of ! or its dimethylamino analogue 2, although qualitatively similar to those observed for acetylcholine, are less dramatic. These results would suggest a somewhat less specific role in the drug-receptor interaction for the acetyl methyl group of 1 than for the acetyl methyl group of acetylcholine. In contrast, the N-methyl group of 1 seems to be essential for muscarinic activity since its replacement by either a hydrogen atom or an ethyl group completely abolishes efficacy (table 1). Obviously, the N-methyl group of 1 is capable of a very specific and highly effective interaction with the receptor.

Acknowledgements This work was supported by USPHS grant MH-17691. We wish to acknowledge the excellent editorial assistance of Ms. D. Matushek.

References Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Baker, R.W., C.H. Chothia, F. Pauling anzl T.J. Petcher, 1971, Structure and activity of muscarini¢ stimulants, Nature 230, 439. Bebbington, A., R.W. Brimblecombe and D. Shakeshaft, 1966, The central and peripheral activity of acetylenic amines related to oxotremorine, Br. J. Pharmacol. 26, 56. Bebbington, A. and D. Shakeshaft, 1965, An improved synthesis of oxotremorine, J. med. Chem. 8, 274.

Brodie, B.B., H. Kurz and L.S. Shanker, 1960, The importance of dissociation constant and lipid solubility in influencing the passage of drugs into the cerebrospinal fluid, J. Pharmacol. Exp. Ther. 130, 20. Furchgott, R.F., 1966, The use of fl-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes, Adv. Drug Res. 3, 21. Furchgott, R.F. and P. Bursztyn, 1967, Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathetic receptors, Ann. N.Y. Acad. Sci. 144, 882. Hanin, I., D.L Jenden and A.K. Cho, 1966, The influence of pH on the muscarinic action of oxotremorine, arecoline, pilocarpine and their quaternary ammonium analogs, Mol. Pharmacol. 2, 352. Karl6n, B. and D.J. Jenden, 1970, The role of distribution as a determinant of central anticholinergic specificity in a series of oxotremorine analogs, Res. Commun. Chem. Pathol. Pharmacol. 1,471. Mashkovsky, M.D. and LN. Yakhontov, 1969, Relationships between the chemical structure and pharmacological activity in a series of synthetic quinuclidine derivatives, Prog. Drug Res. 13, 293. Resul, B., T. Lewander, B. Ringdahl, T. Zetterstr6m and R. Dahlbom, 1982, The pharmacological assessment of a new, potent oxotremorine analogue in mice and rats, European J. Pharmacol, 80, 209. Resul, B., T. Lewander, T. Zetterstr/3m, B. Ringdahl, Z. MuhiEldeen and R. Dahlbom, 1980, Synthesis and pharmacological properties of N-[4-(l-azetidinyl)-2-butynyl]-2-pyrrolidone, a highly potent oxotremorine-like agent, J. Pharm. Pharmacol. 32, 439. Resul, B., B. Ringdahl, U. Hacksell, U. Svensson and R. Dahlbom, 1979, Acetylene compounds of potential pharmacological value. XXXI. Studies on N-(4-tert-amino2-butynyl)-carboxamides as muscarinic agonists, Acta Pharm. Suecica 16, 225. Stephenson, R.P., 1956, A modification of receptor theory, Br. J. Pharmacol. 11, 379. Svensson, U., U. Hacksell and R. Dahlbom, 1978, Acetylene compounds of potential pharmacological value. XXV. N(4-pyrrolidino-2-butynyl)-N-alkylcarboxamides, Acta Pharm. Suecica 15, 67. Triggle, D.J., 1979, The muscarinic receptor: Structural, ionic and biochemical implications, in: Recent Advances in Receptor Chemistry, eds. F. Gualtieri, M. Giannella and C. Melchiorre (Elsevier/North-Holland Biomedical Press) p. 127. Triggle, D.J. and C.R. Triggle, 1976, Chemical Pharmacology of the Synapse (Academic Press, New York) p. 233. Wurzel, M., 1959, A suggested mechanism for the action of choline esters on animal organs, inferred from a study of the effect of choline, /3-methylcholine and thiocholine esters, Experientia 15, 430.