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Structure-activity requirements for hypotension and α-adrenergic receptor blockade by analogues of atropine
European Journal of Pharmacology, 90 (1983) 75-83
75
Elsevier
STRUCTURE-ACTIVITY R E Q U I R E M E N T S FOR H Y P O T E N S I O N AND et-ADRENERGIC R E C E P T O R BLOCKADE BY ANALOGUES OF A T R O P I N E E L I N O R H. C A N T O R , S H L O M O A B R A H A M *, E U G E N E A. M A R C U M and SYDNEY SPECTOR **
Roche Institute of Molecular Biology, Roche Research Center, Department of Physiological Chemistry and Pharmacology, Nutley, New Jersey 07110, U.S.A. Received 10 November 1982, revised MS received 9 February 1983, accepted 24 February 1983
E.H. CANTOR, S. ABRAHAM, E.A. MARCUM and S. SPECTOR, Structure-activity requirements for hypotension and a-adrenergic receptor blockade by analogues of atropine, European J. Pharmacol. 90 (1983) 75-83. The hypotensive action of various antimuscarinic compounds structurally related to atropine was studied in conscious, unanesthetized rats. The a-adrenolytic activity of these agents was assessed both in vivo (blockade of norepinephrine-induced pressor response) and in vitro (displacement of [3 H]WB-4101 binding). Benztropine, homatropine and hyoscyamine caused hypotension and produced a-adrenergic receptor blockade similar to atropine. Other analogues were either inactive (atroscine, scopolamine, tropic acid and tropine) or evoked nonspecific changes in blood pressure and lacked a-adrenolytic activity (benactyzine, eucatropine, methylatropine, methylhomatropine and methylscopolamine). Based on these data, we propose the following structure-activity relationship for hypotension and c~-adrenolytic activity: (a) the tropine moiety is inactive unless it is attached to another group by an ester linkage, (b) chemical modification of the tropine moiety, including quaternization, decreases potency, (c) the d-stereoisomer appears to be more potent than the corresponding 1-form. Hypotension
a-Adrenergic receptor blockade
Atropine
1. Introduction
Several lines of evidence indicate that atropine may act as an a-adrenergic receptor blocking agent in addition to its commonly recognized action as a muscarinic cholinergic antagonist. Such effects had previously been demonstrated in isolated vascular tissue (Bussell, i 940; Furchgott, 1955; Nedergaard and Schrold, 1977, Sakanashi et al., 1979) and perfused rabbit ear vessels (Burn and Dutta, 1948; Fleckenstein, 1952). We recently reported that this phenomenon is manifested in vivo as a decrease in blood pressure both in normotensive (Abraham et al., 1981a) and hypertensive rats with the hypertensive animals showing a greater sensitivity to * Present address: Department of Pharmacology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, Israel. ** To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
atropine (Abraham et al., 1980; 1981c). In support of these observations, radioligand binding studies have confirmed that atropine interacts with ~ladrenergic receptors, but is virtually ineffective in binding to a2-adrenergic receptors (Abraham et al., 1981a; Cantor et al., 1980). Although the induced hypotension was mediated via a peripheral mechanism, presumably at vascular ~-adrenergic sites, the peripherally active antimuscarinic agent methylatropine, a quaternary analogue of atropine, was devoid of c~-adrenolytic activity in vivo (Abraham et al., 1981a) and failed to displace a-adrenergic receptor ligands in vitro (Cantor et al., 1980). Thus, the a-adrenolytic activity of atropine may be dissociated from its antimuscarinic activity. We have, therefore, undertaken a systematic evaluation of several structurally-related analogues of atropine, the majority of which have established antimuscarinic activity, to elucidate the structural requirements for decreas-
76
ing blood pressure and blocking al-adrenergic receptors. The hypotensive activity of these compounds was characterized in vivo. The interaction of these analogues with a]-adrenergic receptors was evaluated both in vivo (blockade of norepinephrine-induced pressor response) and in vitro (displacement of [3H]WB-4101) and compared to their activity in corresponding cholinergic systems (a preliminary note of these results has already been published (Abraham et al., 1981 b)).
2. Materials and methods 2.1. Animals
Male Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats were purchased from Taconic Farms, Germantown, NY and maintained on Purina Chow CHz--CH--CH 2 I J CH$ C H - - O R
i
I
CH2--CH--CH 2 TROPINE
and tap water ad libitum. At the time of the experiments, all animals were between 12-20 weeks of age.
2.2. Drugs Atropine and the structurally related compounds were obtained from the following sources: Atropine sulfate, atropine methyl nitrate, eucatropine HC1, dl-homatropine HBr, dl-homatropine methyl bromide, 1-hyoscyamine HC1, l-scopolamine HC1, 1-scopolamine methyl nitrate, tropic acid and tropine were purchased from Sigma Chemical Co., St. Louis, MO. Atroscine HBr and benactyzine HC1 were purchased from Adams Chemical Co., Round Lake, IL and Aldrich Chemical Co., Inc., Milwaukee WI, respectively. Benztropine mesylate was generously supplied by Merck Sharp & Dohme Research Laboratories, Rahway,
0
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TROPIC ACID(dL)
GENERAL FORMULA
COMPOUND
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HYOSCYAMINE ATROPINE METHYLATROPINE
STEREOISOMER
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EIENACTYZINE
Fig. 1. Structural formulae of analogues of atropine tested in this study. * Indicates optically active carbon.
C6H5 / OH ~C6H5 /
77 NJ. The structural formulae of these analogues are illustrated in fig. 1. Other compounds used in this study were acetylcholine iodide (Sigma), 1-norepinephrine HC1 (Sigma), pentolinium tartrate (Sigma), phentolamine HC1 (generously supplied by Ciba-Geigy Corp., Summit, NJ), [3H]QNB (12 Ci/mmol, Amersham Corp., Arlington Heights, IL) and [ 3H]WB-4101 (30-50 Ci/mmol, Amersham Corp., Arlington Heights, IL.).
2.3. Measurement of blood pressure and i.v. administration of drugs Blood pressure was measured in conscious, freely-moving WKY rats as previously described (Abraham et al., 1981a). Drugs were dissolved in saline and, with the exception of pentolinium, were administered via a venous catheter in a volume of 0.1 ml/kg body weight. Pentolinium was injected i.m. in a volume of 0.05 ml/kg body weight. Doses of all analogues of atropine are expressed as #mol/kg. Unless otherwise specified, doses of other compounds are expressed on a weight basis in terms of the salt. Blood pressure and heart rate were monitored for 30-60 min after the administration of the various analogues of atropine. In some experiments, animals were pretreated with a ganglionic (pentolinium, 30 mg/kg) or a-adrenergic (phentolamine, 5 mg/kg) blocking agent to differentiate a direct effect on vascular smooth muscle from an effect on sympathetic function (Constantine et al., 1973). In preliminary experiments, it was established that the above doses of blocking agents were sufficient to fully block the pressor responses elicited by l,l-dimethyl-4-phenyl piperazinium iodide (DMPP, 0.1 mg/kg) or norepinephrine (0.001 mg/kg), respectively. Analogues of atropine were administered 15 min after pentolinium or 5 min after phentolamine, at which times blood pressure had stabilized.
2.4. Evaluation of a-adrenergic receptor blockade by analogues of atropine 2.4.1. In vivo: blockade of norepinephrine-induced pressor response The effects of the analogues of atropine on the
norepinephrine-induced pressor response were assessed in WKY rats by generating dose-response curves to norepinephrine (0.05-5 nmol/kg) at the time of the maximal change in mean arterial blood pressure elicited by the analogues. The extent of blockade was expressed as a percent of the pressor response elicited by a given dose of norepinephrine in the absence of the blocker. The blocking dose 50 (BDs0) was defined as the dose of the analogue which blocked the pressor response produced by norepinephrine (0.5 nmol/kg) by 50%. BDs0 values were calculated by the linear regression analyses of log-probability plots of the response curves obtained with each analogue.
2.4.2. In vitro: displacement of ct-adrenergic ligand binding Rat brain was used for in vitro displacement studies as this tissue is rich in both a-adrenergic and muscarinic receptors. The brains from SD rats were removed following decapitation, dissected free of membranes, and the cerebella discarded. Tissues were then homogenized with a Brinkmann Polytron in 20 vol of ice cold Tris-HCl buffer (50 mM, pH 7.7). The homogenates were centrifuged at 40000 × g for 10 min at 4°C and the pellets resuspended in the buffer at a concentration of 50 mg wet weight/ml. Binding studies were performed according to U'Prichard et al. (1977) and as previously described by Cantor et al. (1981). Displacement curves were generated with 0.2 nM [3H]WB-4101 using 1.5-2.5 mg protein per reaction. Protein concentration was measured by the method of Lowry et al. (1951), using bovine serum albumin as the standard. IC50 values were determined by linear regression of the log probability plots of the displacement curves obtained with each analogue. Apparent K~ values were calculated according to the formula K i = I C s 0 / ( C / K D ) , where C is the concentration of the ligand. The K D value for [3H]WB-4101 was previously determined in our laboratory to be 0.5 nM. 2.5. Evaluation of muscarinic receptor blockade by analogues of atropine Although most of the analogues of atropine included in this study have well-defined anti-
78 muscarinic activity we have, for comparative purposes, included an evaluation of the antimuscarinic properties of these compounds using methods equivalent to those used for the assessment of adrenoceptor blocking activity. 2.5.1. In vivo: blockade of acetylcholine-induced hypotension Acetylcholine was administered to WKY rats in a dose (36 nmol/kg) which caused a submaximal reduction in mean arterial blood pressure. Animals were pretreated with different doses of the analogues of atropine and at various time intervals thereafter the animals were challenged with acetylcholine. The ability of the analogues to block the hypotension produced by acetylcholine was expressed as the BDs0 (for definition, see above). 2.5.2. In vitro: displacement of muscarinic ligand binding Rat brain was prepared as previously described using sodium phosphate buffer (50 mM, pH 7.2). Binding studies were performed according to the procedure described by Yamamura and Snyder (1974) as modified by Cantor et al. (1981). The reaction mixture contained 0.06 nM of [3H]QNB and 0.2-0.4 mg protein. The K D value for [3H]QNB was previously determined in our laboratory to be 0.1 nM. Analysis of the results was performed as described above. 2.6. Statistical analysis Data are presented as mean ± S.E.M. Statistical analysis were performed using Student's t-test (two-tailed). Slopes of regression lines were compared by an F-test for analysis of covariance (Snedecor and Cochran, 1967), the parameters of the lines having first been determined by a computer-assisted linear regression analysis using the method of least squares. 3. Results
3.1. Effects on blood pressure and heart rate Analogues of atropine were evaluated at doses ranging from 1 to 150 /xmol/kg except in cases
where the appearance of behavioral responses typical of anticholinergic toxicity (ataxia, convulsions) precluded testing the higher doses. The changes in blood pressure produced by the analogues of atropine could be grouped into three categories. Analogues included in Category I were atroscine, scopolamine, tropic acid and tropine. These agents were inactive at doses as high as 100 /~mol/kg (tropine) or 150 /~mol/kg (atroscine, scopolamine and tropic acid). When tropine was administered at a dose of 150 /~mol/kg, a transient fall in blood pressure of about 20 mmHg was observed, but the blood pressure returned to control levels within the first minute following injection. This decrease in blood pressure was not affected by pentolinium pretreatment. Category II included those analogues which produced mixed responses as follows: (a) benactyzine (3 and 15/~mol/kg) and eucatropine (30 and 75 /~mol/kg) produced slight pressor responses of 10-20 mmHg. (b) Methylatropine, methylhomatropine, and methylscopolomine, at doses up to 15 /~mol/kg, also produced slight pressor responses of 10-20 mmHg. At 30//mol/kg, however, the pressor response was immediately followed by a 10-20 mmHg reduction in blood pressure below control levels. Neither the increase nor the decrease in blood pressure were fully blocked by pretreatment with pentolinium. Analogues in Category III (benztropine, homatropine, hyoscyamine) were those which produced hypotensive responses with characteristics similar to that elicited by the reference compound, atropine. As shown, in fig. 2, the decrease in blood pressure was dose-dependent. The order of potency for these agents for producing hypotension was benztropine > atropine > homatropine > hyoscyamine. As was the case with atropine, the hypotensive response was blocked by pentolinium. Like atropine, the analogues included in this study, except tropic acid and tropine, produced tachycardia within the first minute. The magnitude of the increase in heart rate (40-80 beats/min) was not dose-dependent. At the highest doses tested, benztropine (30 #mol/kg), eucatropine, and hyoscyamine (150 /~mol/kg) lowered heart rate 30-50 beats/min. This bradycardia was of short duration (< 5 min) and then reverted to tachycardia as above.
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Fig. 2. Blood pressure response to i,v. administration of atropine and Category III analogues. Only one dose of any given c o m p o u n d was tested in each rat. Blood pressure was measured one minute after injection, at which time the magnitude of the response was maximum. Each point represents the mean responses of 5 - 6 animals. Control mean arterial blood pressure was 129+ 1 m m H g (n = 98).
3.2. c~-Adrenergic receptor blockade Atropine, benztropine, hyoscyamine, and homatropine caused a shift to the right of the norepinephrine dose-response curve (data not shown). This inhibition was dose-dependent when tested against a single concentration of norepinephrine (0.5 nmol/kg) (fig. 3). The analogues in Categories I and II failed to block the pressor response to norepinephrine over the range of doses tested. BDs0 values were calculated for each active compound (table 1). The effects of the analogues in Category III on the binding of [3H]WB-4101 are shown in fig. 4. The relative potency of these analogues in displacing the ligand paralleled that for the hypotensive action and the blockade of the norepinephrine-induced pressor response. Apparent K i values for all the analogues included in this study are given in table 2. These data indicate that the tertiary analogues are more potent than the corresponding quaternary N-methyl compounds e.g. (methylatropine vs. atropine) and that the dl-isomers were more active than the 1-forms (atropine vs. hyoscyamine and atroscine vs. scopolamine). Furthermore, replacing the tropic acid moiety of atropine with mandelic acid to form homatropine resulted in a decrease in potency. In view of the a-adrenolytic activity exhibited
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Fig. 3. Dose-related blockade of the norepinephrine-induced pressor response by atropine and Category III analogues, Pressor responses were elicited by i.v. administration of a single dose of norepinephrine (0.5 n m o l / k g ) which increased blood pressure to 85% of the m a x i m u m attainable increase. Comp o u n d s were administered 0.5-1 min prior to norepinephrine. Each point is the mean of 3 - 5 separate experiments.
by the analogues of Category III, the hypotensive action of these compounds was evaluated following an a-adrenergic blockade. The analogues were administered to phentolamine-pretreated rats. Un-
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[M] Fig. 4, Displacement of the a-adrenergic receptor ligand [3H]WB-4101, by atropine and Category IIl analogues. Binding was measured in rat brain homogenates in the presence of the indicated concentrations of the various compounds as described in Materials and methods. Control specific binding was 4 4 + 2 f m o l / m g protein ( n = l l ) . Each point is the mean of 3-5 separate experiments performed in duplicate.
80 TABLE 1 Blockade of norepinephrine-induced pressor and acetylcholine-induced depressor responses by atropine and Category III analogues. The BDs0 value was defined as the dose needed to block the indicated responseby 50%. Values were calculated as described in Materials and methods. Acetylcholine (36 nmol/kg) or norepinephrine (0.5 nmol/kg) was administered i.v. at the time of the maximum change in blood pressure elicited by the test compound. Compound
Benztropine Atropine Homatropine Hyoscyamine
BDs0 (~tmol/kg) vs. norepinephrineinduced pressor response
vs. acetylcholineinduced depressor response
9 35 45 85
4.0 0.3 17 0.4
der this condition, the hypotensive action of these analogues was fully blocked (data not shown).
3.3. Muscarinic cholinergic receptor blockade Acetylcholine (36 n m o l / k g ) decreased blood pressure by 61 +__2 m m H g (n = 31). All analogues TABLE2 K i values for displacement of aradrenergic and muscarinic cholinergic ligand binding in rat brain by analogues of atropine. K i values were determined as described in Materials and methods. Each value is the mean :t:S.E.M. of 3-4 separate determinations. Compound
K i value a 1-Adrenergic receptor (~.M)
Benztropine Atropine Homatropine Hyoscyamine Atroscine Benactyzine Scopolamine Eucatropine Methylscopolamine Methylatropine Methylhomatropine Tropic acid Tropine
0.7 + 0. I 2.05:0.3 6.0± 0.9 14 5:2.4 33 ± 6.0 33 5:1.0 75 + 23 130 5:20 160 5:30 190 5:30 720 5:80 > 10-3 > 10-3
Muscarinic cholinergic receptor (nM) 8.6 5:0.4 7.05:0.5 310 +40 3.2+ 0.4 7.25:0.9 65 + 6.0 4.2 5:0.5 780 5:30 4.6 5:0.8 9.3___ 1.0 350 5:40 > 10 -3
> 10-4
except for tropic acid and tropine blocked the acetylcholine-induced hypotension in a dose-dependent manner. None of the compounds caused hypotension at the dose which blocked acetylcholine-induced hypotension. K i values for the displacement of [3H]QNB are shown in table 2. In contrast to the data for anti-adrenergic properties, neither the BDs0 nor the K i values for antimuscarinic activity discriminate among the categories into which these analogues were grouped. For example, dl- and 1-stereoisomers or quaternary and tertiary analogues exhibited similar potency. There was, however, fairly good agreement in both the cholinergic and adrenergic systems between the relative potencies of the compounds in vivo and in vitro.
4. Discussion In the present study, we evaluated the hypotensive and a-adrenolytic activities of several structural analogues of atropine and demonstrated that, like atropir~e, some of these agents possessed both anticho!inergic and a:adrenolytic activity~ The hypotensive activity produced ~ by benztropine, homatropine, and hyoscyamine resembled that reported for atropine (Abrahamet aL, 1980; 1981a,c). That this hypotensive action wasclue to a-adrenergic blockade was confirmed by the' findings that these analogues 'blocked the increase in 'blood pressure caused by norepinephrine and displaced the binding of the a~-adrenergic ligand [3H]WB4101. Furthermore, the hypotension produced by these analogues was itself blocked by phentolamine. The total loss of hypotensive activity after blockade of sympathetic ganglia by pentolinium precluded a papaverine-like action of the analogues directly on the smooth muscle (Constantine et al., 1973). The persistence of the hypotensive effect when hexamethonium was used as the ganglionic blocking agent in the study of prazosin (Oates et al., 1976) or of phentolamine and atropine (Abraham et al., 1981a), could be attributed to incomplete ganglionic blockade by hexamethonium (Oates et al., 1976). The relative potency for a-adrenergic blockade in vivo and in vitro was comparable to that ob-
81 served by Nedergaard and Schrold (1977) who evaluated the ability of four antimuscarinic compounds to block the contractile response of rabbit pulmonary artery to electrical stimulation. They also found, as in this study, that methylatropine and scopolamine were relatively inactive. Greenberg and Snyder (1978) and Rehavi et al. (1980) r e p o r t e d K i values for atropine and benztropine vs ~t-adrenergic receptor ligands which are consistent with our data. In contrast to analogues assigned to Category III, those grouped in Categories I and II were devoid of a-adrenolytic activity in vivo and displayed considerably lower activity in vitro. Although the analogues in categories I and II produced some changes in blood pressure, the changes were not specific in nature as they persisted after ganglionic blockade. Since these compounds did not produce the pattern of response typical of atropine, we did not pursue further the mechanism of their action on blood pressure. With the exception of tropic acid and tropine, the analogues included in this study have established antimuscarinic activity (Weiner, 1980) and the structural and conformational requirements for antimuscarinic activity have been defined (Inch and Brimblecombe, 1974). Nevertheless, in the course of this investigation, we re-evaluated this parameter in order to compare the relative potencies of the antimuscarinic and a-adrenolytic activities of these analogues using similar pharmacological approaches. As was the case for antimuscarinic properties, neither tropic acid nor tropine exhibited hypotensive or ct-adrenolytic activity. When these two components were linked by an ester or ether bond, the compound was active. Replacing the tropic acid moiety of atropine with mandelic acid to form homatropine did not markedly decrease a-adrenolytic activity although antimuscarinic potency was reduced. On the other hand, the tropine portion of the molecule was essential for a-adrenolytic activity. Substituting scopine for tropine to yield atroscine resulted in a loss of activity at the c~-adrenergic receptor with no change in antimuscarinic potency. This was further demonstrated in the case of eucatropine in which one of the carbon-carbon bonds in the tropine moiety is cleaved, rendering the compound inactive on the a-adrenergic system. In addition, benactyzine,
which has no tropine structure, has no ct-adrenolytic activity. It is interesting to note that the tropine structure contained in cocaine probably contributes to the reported a-adrenergic blocking activity of that compound (Bussell, 1940). The quaternary N-methyl analogues of atropine, homatropine and scopolamine were inactive as ct-adrenolytic agents in vivo. This lack of effect is not due to the inability of these agents to enter the central nervous system, but to their failure to interact with ct-adrenergic receptors as was demonstrated in vitro. Furthermore, centrally acting aadrenergic antagonists would be expected to produce a hypertensive response. Thus, it is imperative for an action on the a-adrenergic receptor that the nitrogen atom be tertiary, while tertiary and quaternary analogues were equipotent on the muscarinic receptor both in vivo and in vitro. This preservation of full muscarinic activity in quaternary derivatives has been previously demonstrated (Weiner, 1980). Due to the presence of an asymetric carbon atom in tropic and mandelic acids, the corresponding analogues were examined for stereochemical specificity. In line with our results, it has been reported that the l-isomers (scopolamine and hyoscyamine) are more potent antimuscarinic agents than the respective dl-forms (atroscine and atropine) (Buckett and Haining, 1965; Long et al., 1956; Marshall, 1955). In contrast to the stereospecific requirements for antimuscarinic activity, we found that for a-adrenergic activity, the dlforms were more potent than the corresponding l-forms. A preliminary evaluation of d-atropine (generously supplied by Dr. Russell Miller, Howard University College of Medicine, Washington, D.C.) indicated that, compared to dl-atropine, datropine was 10 times less active as an antimuscarinic agent whereas its a-adrenolytic effects were fully preserved both in vivo and in vitro. Thus, the d-stereoisomers appear to be more potent as hypotensive agents than the corresponding 1forms. There are other instances in which the d-stereoisomer has a greater effect than the 1-form on the t~-adrenergic receptor. For example, Cheng et al. (1980) reported that the d-enantiomers of medroxalol were 10 times more potent than the corresponding 1-enantiomers as c~-adrenergic blockers in vitro.
82
The structural requirements outlined above for the a-adrenolytic and hypotensive activities of the analogues of atropine are entirely different from those for their antimuscarinic activity. The total loss of activity of scopolamine is therefore attributed to both replacing tropine with scopine and to the fact that scopolamine is the l-stereoisomer. One can look at racemic mixtures as having two components, one with predominant ct-adrenolytic activity (d-form), and the other with predominant antimuscarinic activity (1-form). The net activity for any given analogue would depend on the relative contributions of these two components. Furthermore, for benztropine and homatropine the in vivo potency of the antimuscarinic activity is greater than the a-adrenolytic activity by only three times. In addition, benztropine has a wide range of activities and is regarded as one of the most potent inhibitors of dopamine uptake (Horn et al., 1971; Iversen, 1973). One, therefore, may wonder about the antimuscarinic selectivity of these analogues, and should not ignore their adrenolytic properties when assessing their pharmacological effects.
Acknowledgements We wish to thank Ms. Diane Gentile for excellent and devoted technical assistance with the in vitro receptor assays.
References Abraham, S., E.H. Cantor and S. Spector, 1980, Hypotensive effect of atropine: increased responsiveness of hypertensive rats, Pharmacologist 22, 162. Abraham, S., E.H. Cantor and S. Spector, 1981a, Atropine lowers blood pressure in normotensive rats through blockade of a-adrenergic receptors, Life Sci. 28, 315. Abraham, S., E.H. Cantor, E.A. Marcum and S. Spector, 1981b, Structure-activity relationship among analogues of atropine: hypotension and a-adrenergic blockade, Fed. Proc. 40, 659. Abraham, S., E.H. Cantor and S. Spector, 1981c, Studies on the hypotensive response to atropine in hypertensive rats, J. Pharmacol. Exp. Ther. 218, 662. Buckett, W.R. and C.G. Haining, 1965, Some pharmacological studies on the optically active isomers of hyoscine and hyoscyamine, Br. J. Pharmacol. 24, 138. Burn, J.H. and N.K. Dutta, 1948, The action of antagonists of
acetylcholine on the vessels of the rabbit's ear, Br. J. Pharmacol. 3, 354. Bussell, L.J., 1940, The relation of atropine to adrenaline and the sympathetic system, J. Pharmacol. Exp. Ther. 60, 128. Cantor, E.H., S. Abraham and S. Spector, 1980, Specific interaction of atropine with a-adrenergic receptors in rat brain, Soc. Neurosci. Abstrs. 6, 794. Cantor, E,H., S. Abraham and S. Spector, 1981, Central neurotransmitter receptors in hypertensive rats, Life Sci. 28, 519. Cheng, H.C., O.K. Reavis, Jr., J.M. Grisar, G.P. Claxton, D.L. Weiner and J.K. Woodward, 1980, Antihypertensive and adrenergie receptor blocking properties of the enantiomers of medroxalol, Life Sci. 27, 2529. Constantine, J.W., W.K. McSbane, A. Scriabine and H.-J. Hess, 1973, Analysis of the hypotensive action of prazosin, in: Hypertension: mechanisms and management, eds. G. Onesti, K.E. Kim and J.H. Moyer (Grune & Stratton, New York) p. 429. Fleckenstein, A., 1952, A quantitative study of antagonists of adrenaline on the vessels of the rabbit's ear, Br. J. Pharmacol. 7, 553. Furchgott, R.F., 1955, The pharmacology of vascular smooth muscle, Pharmacol. Rev. 7, 183. Greenberg, D.A. and S.H. Snyder, 1978, Pharmacological properties of [3H]dihydroergokryptine binding sites associated with alpha noradrenergic receptors in rat brain membranes, Mol. Pharmacol. 14, 38. Horn, A.S,, J.T. Coyle and S.H. Snyder, 1971, Catecholamine uptake by synaptosomes from rat brain: Structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons, Mol. Pharmacol. 7, 66. Inch, T.D. and R.W. Brimblecombe, 1974, Acetylcholine drugs: chemistry, stereochemistry and pharmacology, Int. Rev. Neurobiol. 16, 67. Iversen, L.L., 1973, Neuronal and extraneuronal catecholamine uptake mechanisms, in: Frontiers in catecholamine research, eds. E. Usdin and S.H. Snyder (Pergamon Press, New York) p. 403. Long, J.P., F.P. Luduena, B.F. Tullar and A.M. Lands, 1956, Stereochemical factors involved in cholinolytic activity, J. Pharmacol. Exp. Ther. 117, 29. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurements with the folin phenol reagent, J. Biol. Chem. 193, 265. Marshall, P.B., 1955, Antagonism of acetylcholine by ( + )- and (-)-hyoscyamine, Br. J. Pharmacol. 10, 354. Nedergaard, O.A. and J. Schrold, 1977, Effect of atropine on vascular adrenergic neuroeffector transmission, Blood Vessels 14, 325. Oates, H.F., R.M. Graham, L.M. Stoker and G.S. Stokes, 1976, Hemodynamic effects of prazosin, Arch. Int. Pharmacodyn. 224, 239. Rehavi, M., B. Yavetz, O. Ramot and M. Sokolovsky, 1980, Regional heterogeneity of two high affinity binding sites for 3H-WB-4101 in mouse brain, Life Sci. 26. 615. Sakanashi, M., T. Furukawa and Y. Horio, 1979~ Inhibition of constrictor responses of dog coronary artery by atropine. A
83 possible effectiveness of atropine On variant form of angina pectoris, Jap. Heart J. 20, 75. Snedecor, G.W. and W.G. Cochran, 1967, Statistical methods, sixth edition (The Iowa State University Press, Ames, Iowa) p. 419. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454.
Weiner, N., 1980, Atropine, scopolamine and related antimuscarinic drugs, in: The pharmacological basis of therapeutics, eds. A.F. Gilman, L.S. Goodman and A. Gilman (Macmillan Publishing Co., Inc., New York) p. 120. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Natl. Acad. Sci. 71, 1725.
75
Elsevier
STRUCTURE-ACTIVITY R E Q U I R E M E N T S FOR H Y P O T E N S I O N AND et-ADRENERGIC R E C E P T O R BLOCKADE BY ANALOGUES OF A T R O P I N E E L I N O R H. C A N T O R , S H L O M O A B R A H A M *, E U G E N E A. M A R C U M and SYDNEY SPECTOR **
Roche Institute of Molecular Biology, Roche Research Center, Department of Physiological Chemistry and Pharmacology, Nutley, New Jersey 07110, U.S.A. Received 10 November 1982, revised MS received 9 February 1983, accepted 24 February 1983
E.H. CANTOR, S. ABRAHAM, E.A. MARCUM and S. SPECTOR, Structure-activity requirements for hypotension and a-adrenergic receptor blockade by analogues of atropine, European J. Pharmacol. 90 (1983) 75-83. The hypotensive action of various antimuscarinic compounds structurally related to atropine was studied in conscious, unanesthetized rats. The a-adrenolytic activity of these agents was assessed both in vivo (blockade of norepinephrine-induced pressor response) and in vitro (displacement of [3 H]WB-4101 binding). Benztropine, homatropine and hyoscyamine caused hypotension and produced a-adrenergic receptor blockade similar to atropine. Other analogues were either inactive (atroscine, scopolamine, tropic acid and tropine) or evoked nonspecific changes in blood pressure and lacked a-adrenolytic activity (benactyzine, eucatropine, methylatropine, methylhomatropine and methylscopolamine). Based on these data, we propose the following structure-activity relationship for hypotension and c~-adrenolytic activity: (a) the tropine moiety is inactive unless it is attached to another group by an ester linkage, (b) chemical modification of the tropine moiety, including quaternization, decreases potency, (c) the d-stereoisomer appears to be more potent than the corresponding 1-form. Hypotension
a-Adrenergic receptor blockade
Atropine
1. Introduction
Several lines of evidence indicate that atropine may act as an a-adrenergic receptor blocking agent in addition to its commonly recognized action as a muscarinic cholinergic antagonist. Such effects had previously been demonstrated in isolated vascular tissue (Bussell, i 940; Furchgott, 1955; Nedergaard and Schrold, 1977, Sakanashi et al., 1979) and perfused rabbit ear vessels (Burn and Dutta, 1948; Fleckenstein, 1952). We recently reported that this phenomenon is manifested in vivo as a decrease in blood pressure both in normotensive (Abraham et al., 1981a) and hypertensive rats with the hypertensive animals showing a greater sensitivity to * Present address: Department of Pharmacology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, Israel. ** To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
atropine (Abraham et al., 1980; 1981c). In support of these observations, radioligand binding studies have confirmed that atropine interacts with ~ladrenergic receptors, but is virtually ineffective in binding to a2-adrenergic receptors (Abraham et al., 1981a; Cantor et al., 1980). Although the induced hypotension was mediated via a peripheral mechanism, presumably at vascular ~-adrenergic sites, the peripherally active antimuscarinic agent methylatropine, a quaternary analogue of atropine, was devoid of c~-adrenolytic activity in vivo (Abraham et al., 1981a) and failed to displace a-adrenergic receptor ligands in vitro (Cantor et al., 1980). Thus, the a-adrenolytic activity of atropine may be dissociated from its antimuscarinic activity. We have, therefore, undertaken a systematic evaluation of several structurally-related analogues of atropine, the majority of which have established antimuscarinic activity, to elucidate the structural requirements for decreas-
76
ing blood pressure and blocking al-adrenergic receptors. The hypotensive activity of these compounds was characterized in vivo. The interaction of these analogues with a]-adrenergic receptors was evaluated both in vivo (blockade of norepinephrine-induced pressor response) and in vitro (displacement of [3H]WB-4101) and compared to their activity in corresponding cholinergic systems (a preliminary note of these results has already been published (Abraham et al., 1981 b)).
2. Materials and methods 2.1. Animals
Male Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats were purchased from Taconic Farms, Germantown, NY and maintained on Purina Chow CHz--CH--CH 2 I J CH$ C H - - O R
i
I
CH2--CH--CH 2 TROPINE
and tap water ad libitum. At the time of the experiments, all animals were between 12-20 weeks of age.
2.2. Drugs Atropine and the structurally related compounds were obtained from the following sources: Atropine sulfate, atropine methyl nitrate, eucatropine HC1, dl-homatropine HBr, dl-homatropine methyl bromide, 1-hyoscyamine HC1, l-scopolamine HC1, 1-scopolamine methyl nitrate, tropic acid and tropine were purchased from Sigma Chemical Co., St. Louis, MO. Atroscine HBr and benactyzine HC1 were purchased from Adams Chemical Co., Round Lake, IL and Aldrich Chemical Co., Inc., Milwaukee WI, respectively. Benztropine mesylate was generously supplied by Merck Sharp & Dohme Research Laboratories, Rahway,
0
HO--C--CH
,/CH2OH
~C6Rs
TROPIC ACID(dL)
GENERAL FORMULA
COMPOUND
CH2--CH--CHo I I ~ ~ */CH2OH R--NCH$ C H - - 0 - - C - - C H I ~C6H5 CH2--CH--CH 2
R
HYOSCYAMINE ATROPINE METHYLATROPINE
STEREOISOMER
--
t
--
dl dt
CH3
CHz--CH--CH 2 -
I
I
0
./OH
R--NCH~
CH--O--C--CH I ~C6H5 CH2--CH--CH 2
CH
HOMATROPINE METHYLHOMATROPINE
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dt dt
SCOPOLAMINE METHYLSCOPOLAMINE
---
dL L
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t
--
' CH--CH 2 R--
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-
I
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I
I
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CH2--CH--CH 2 BENZTROPINE
zc,.,
~c6.5
CL>H5 iCH~ CH--O--CH'--CH I ~C6H 5 CH3--CH--CH2 EUCATROPINE((:It)
~N-C2H5~"
0
CHz--CH2--O--C--C
EIENACTYZINE
Fig. 1. Structural formulae of analogues of atropine tested in this study. * Indicates optically active carbon.
C6H5 / OH ~C6H5 /
77 NJ. The structural formulae of these analogues are illustrated in fig. 1. Other compounds used in this study were acetylcholine iodide (Sigma), 1-norepinephrine HC1 (Sigma), pentolinium tartrate (Sigma), phentolamine HC1 (generously supplied by Ciba-Geigy Corp., Summit, NJ), [3H]QNB (12 Ci/mmol, Amersham Corp., Arlington Heights, IL) and [ 3H]WB-4101 (30-50 Ci/mmol, Amersham Corp., Arlington Heights, IL.).
2.3. Measurement of blood pressure and i.v. administration of drugs Blood pressure was measured in conscious, freely-moving WKY rats as previously described (Abraham et al., 1981a). Drugs were dissolved in saline and, with the exception of pentolinium, were administered via a venous catheter in a volume of 0.1 ml/kg body weight. Pentolinium was injected i.m. in a volume of 0.05 ml/kg body weight. Doses of all analogues of atropine are expressed as #mol/kg. Unless otherwise specified, doses of other compounds are expressed on a weight basis in terms of the salt. Blood pressure and heart rate were monitored for 30-60 min after the administration of the various analogues of atropine. In some experiments, animals were pretreated with a ganglionic (pentolinium, 30 mg/kg) or a-adrenergic (phentolamine, 5 mg/kg) blocking agent to differentiate a direct effect on vascular smooth muscle from an effect on sympathetic function (Constantine et al., 1973). In preliminary experiments, it was established that the above doses of blocking agents were sufficient to fully block the pressor responses elicited by l,l-dimethyl-4-phenyl piperazinium iodide (DMPP, 0.1 mg/kg) or norepinephrine (0.001 mg/kg), respectively. Analogues of atropine were administered 15 min after pentolinium or 5 min after phentolamine, at which times blood pressure had stabilized.
2.4. Evaluation of a-adrenergic receptor blockade by analogues of atropine 2.4.1. In vivo: blockade of norepinephrine-induced pressor response The effects of the analogues of atropine on the
norepinephrine-induced pressor response were assessed in WKY rats by generating dose-response curves to norepinephrine (0.05-5 nmol/kg) at the time of the maximal change in mean arterial blood pressure elicited by the analogues. The extent of blockade was expressed as a percent of the pressor response elicited by a given dose of norepinephrine in the absence of the blocker. The blocking dose 50 (BDs0) was defined as the dose of the analogue which blocked the pressor response produced by norepinephrine (0.5 nmol/kg) by 50%. BDs0 values were calculated by the linear regression analyses of log-probability plots of the response curves obtained with each analogue.
2.4.2. In vitro: displacement of ct-adrenergic ligand binding Rat brain was used for in vitro displacement studies as this tissue is rich in both a-adrenergic and muscarinic receptors. The brains from SD rats were removed following decapitation, dissected free of membranes, and the cerebella discarded. Tissues were then homogenized with a Brinkmann Polytron in 20 vol of ice cold Tris-HCl buffer (50 mM, pH 7.7). The homogenates were centrifuged at 40000 × g for 10 min at 4°C and the pellets resuspended in the buffer at a concentration of 50 mg wet weight/ml. Binding studies were performed according to U'Prichard et al. (1977) and as previously described by Cantor et al. (1981). Displacement curves were generated with 0.2 nM [3H]WB-4101 using 1.5-2.5 mg protein per reaction. Protein concentration was measured by the method of Lowry et al. (1951), using bovine serum albumin as the standard. IC50 values were determined by linear regression of the log probability plots of the displacement curves obtained with each analogue. Apparent K~ values were calculated according to the formula K i = I C s 0 / ( C / K D ) , where C is the concentration of the ligand. The K D value for [3H]WB-4101 was previously determined in our laboratory to be 0.5 nM. 2.5. Evaluation of muscarinic receptor blockade by analogues of atropine Although most of the analogues of atropine included in this study have well-defined anti-
78 muscarinic activity we have, for comparative purposes, included an evaluation of the antimuscarinic properties of these compounds using methods equivalent to those used for the assessment of adrenoceptor blocking activity. 2.5.1. In vivo: blockade of acetylcholine-induced hypotension Acetylcholine was administered to WKY rats in a dose (36 nmol/kg) which caused a submaximal reduction in mean arterial blood pressure. Animals were pretreated with different doses of the analogues of atropine and at various time intervals thereafter the animals were challenged with acetylcholine. The ability of the analogues to block the hypotension produced by acetylcholine was expressed as the BDs0 (for definition, see above). 2.5.2. In vitro: displacement of muscarinic ligand binding Rat brain was prepared as previously described using sodium phosphate buffer (50 mM, pH 7.2). Binding studies were performed according to the procedure described by Yamamura and Snyder (1974) as modified by Cantor et al. (1981). The reaction mixture contained 0.06 nM of [3H]QNB and 0.2-0.4 mg protein. The K D value for [3H]QNB was previously determined in our laboratory to be 0.1 nM. Analysis of the results was performed as described above. 2.6. Statistical analysis Data are presented as mean ± S.E.M. Statistical analysis were performed using Student's t-test (two-tailed). Slopes of regression lines were compared by an F-test for analysis of covariance (Snedecor and Cochran, 1967), the parameters of the lines having first been determined by a computer-assisted linear regression analysis using the method of least squares. 3. Results
3.1. Effects on blood pressure and heart rate Analogues of atropine were evaluated at doses ranging from 1 to 150 /xmol/kg except in cases
where the appearance of behavioral responses typical of anticholinergic toxicity (ataxia, convulsions) precluded testing the higher doses. The changes in blood pressure produced by the analogues of atropine could be grouped into three categories. Analogues included in Category I were atroscine, scopolamine, tropic acid and tropine. These agents were inactive at doses as high as 100 /~mol/kg (tropine) or 150 /~mol/kg (atroscine, scopolamine and tropic acid). When tropine was administered at a dose of 150 /~mol/kg, a transient fall in blood pressure of about 20 mmHg was observed, but the blood pressure returned to control levels within the first minute following injection. This decrease in blood pressure was not affected by pentolinium pretreatment. Category II included those analogues which produced mixed responses as follows: (a) benactyzine (3 and 15/~mol/kg) and eucatropine (30 and 75 /~mol/kg) produced slight pressor responses of 10-20 mmHg. (b) Methylatropine, methylhomatropine, and methylscopolomine, at doses up to 15 /~mol/kg, also produced slight pressor responses of 10-20 mmHg. At 30//mol/kg, however, the pressor response was immediately followed by a 10-20 mmHg reduction in blood pressure below control levels. Neither the increase nor the decrease in blood pressure were fully blocked by pretreatment with pentolinium. Analogues in Category III (benztropine, homatropine, hyoscyamine) were those which produced hypotensive responses with characteristics similar to that elicited by the reference compound, atropine. As shown, in fig. 2, the decrease in blood pressure was dose-dependent. The order of potency for these agents for producing hypotension was benztropine > atropine > homatropine > hyoscyamine. As was the case with atropine, the hypotensive response was blocked by pentolinium. Like atropine, the analogues included in this study, except tropic acid and tropine, produced tachycardia within the first minute. The magnitude of the increase in heart rate (40-80 beats/min) was not dose-dependent. At the highest doses tested, benztropine (30 #mol/kg), eucatropine, and hyoscyamine (150 /~mol/kg) lowered heart rate 30-50 beats/min. This bradycardia was of short duration (< 5 min) and then reverted to tachycardia as above.
79 a w
HOMATROPINE
o
6O to w~
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/ATROPINE
z
L~
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8ENZTROPINE
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rr Lu 75 "i"(/) wO tel
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~ o
~//F
/j /
/
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~:Q: 50 SCYAMINE
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75
15 30 DOSE (/Jrnol/kg)
75
150
Fig. 2. Blood pressure response to i,v. administration of atropine and Category III analogues. Only one dose of any given c o m p o u n d was tested in each rat. Blood pressure was measured one minute after injection, at which time the magnitude of the response was maximum. Each point represents the mean responses of 5 - 6 animals. Control mean arterial blood pressure was 129+ 1 m m H g (n = 98).
3.2. c~-Adrenergic receptor blockade Atropine, benztropine, hyoscyamine, and homatropine caused a shift to the right of the norepinephrine dose-response curve (data not shown). This inhibition was dose-dependent when tested against a single concentration of norepinephrine (0.5 nmol/kg) (fig. 3). The analogues in Categories I and II failed to block the pressor response to norepinephrine over the range of doses tested. BDs0 values were calculated for each active compound (table 1). The effects of the analogues in Category III on the binding of [3H]WB-4101 are shown in fig. 4. The relative potency of these analogues in displacing the ligand paralleled that for the hypotensive action and the blockade of the norepinephrine-induced pressor response. Apparent K i values for all the analogues included in this study are given in table 2. These data indicate that the tertiary analogues are more potent than the corresponding quaternary N-methyl compounds e.g. (methylatropine vs. atropine) and that the dl-isomers were more active than the 1-forms (atropine vs. hyoscyamine and atroscine vs. scopolamine). Furthermore, replacing the tropic acid moiety of atropine with mandelic acid to form homatropine resulted in a decrease in potency. In view of the a-adrenolytic activity exhibited
~
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50
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150
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Fig. 3. Dose-related blockade of the norepinephrine-induced pressor response by atropine and Category III analogues, Pressor responses were elicited by i.v. administration of a single dose of norepinephrine (0.5 n m o l / k g ) which increased blood pressure to 85% of the m a x i m u m attainable increase. Comp o u n d s were administered 0.5-1 min prior to norepinephrine. Each point is the mean of 3 - 5 separate experiments.
by the analogues of Category III, the hypotensive action of these compounds was evaluated following an a-adrenergic blockade. The analogues were administered to phentolamine-pretreated rats. Un-
• '~'
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[M] Fig. 4, Displacement of the a-adrenergic receptor ligand [3H]WB-4101, by atropine and Category IIl analogues. Binding was measured in rat brain homogenates in the presence of the indicated concentrations of the various compounds as described in Materials and methods. Control specific binding was 4 4 + 2 f m o l / m g protein ( n = l l ) . Each point is the mean of 3-5 separate experiments performed in duplicate.
80 TABLE 1 Blockade of norepinephrine-induced pressor and acetylcholine-induced depressor responses by atropine and Category III analogues. The BDs0 value was defined as the dose needed to block the indicated responseby 50%. Values were calculated as described in Materials and methods. Acetylcholine (36 nmol/kg) or norepinephrine (0.5 nmol/kg) was administered i.v. at the time of the maximum change in blood pressure elicited by the test compound. Compound
Benztropine Atropine Homatropine Hyoscyamine
BDs0 (~tmol/kg) vs. norepinephrineinduced pressor response
vs. acetylcholineinduced depressor response
9 35 45 85
4.0 0.3 17 0.4
der this condition, the hypotensive action of these analogues was fully blocked (data not shown).
3.3. Muscarinic cholinergic receptor blockade Acetylcholine (36 n m o l / k g ) decreased blood pressure by 61 +__2 m m H g (n = 31). All analogues TABLE2 K i values for displacement of aradrenergic and muscarinic cholinergic ligand binding in rat brain by analogues of atropine. K i values were determined as described in Materials and methods. Each value is the mean :t:S.E.M. of 3-4 separate determinations. Compound
K i value a 1-Adrenergic receptor (~.M)
Benztropine Atropine Homatropine Hyoscyamine Atroscine Benactyzine Scopolamine Eucatropine Methylscopolamine Methylatropine Methylhomatropine Tropic acid Tropine
0.7 + 0. I 2.05:0.3 6.0± 0.9 14 5:2.4 33 ± 6.0 33 5:1.0 75 + 23 130 5:20 160 5:30 190 5:30 720 5:80 > 10-3 > 10-3
Muscarinic cholinergic receptor (nM) 8.6 5:0.4 7.05:0.5 310 +40 3.2+ 0.4 7.25:0.9 65 + 6.0 4.2 5:0.5 780 5:30 4.6 5:0.8 9.3___ 1.0 350 5:40 > 10 -3
> 10-4
except for tropic acid and tropine blocked the acetylcholine-induced hypotension in a dose-dependent manner. None of the compounds caused hypotension at the dose which blocked acetylcholine-induced hypotension. K i values for the displacement of [3H]QNB are shown in table 2. In contrast to the data for anti-adrenergic properties, neither the BDs0 nor the K i values for antimuscarinic activity discriminate among the categories into which these analogues were grouped. For example, dl- and 1-stereoisomers or quaternary and tertiary analogues exhibited similar potency. There was, however, fairly good agreement in both the cholinergic and adrenergic systems between the relative potencies of the compounds in vivo and in vitro.
4. Discussion In the present study, we evaluated the hypotensive and a-adrenolytic activities of several structural analogues of atropine and demonstrated that, like atropir~e, some of these agents possessed both anticho!inergic and a:adrenolytic activity~ The hypotensive activity produced ~ by benztropine, homatropine, and hyoscyamine resembled that reported for atropine (Abrahamet aL, 1980; 1981a,c). That this hypotensive action wasclue to a-adrenergic blockade was confirmed by the' findings that these analogues 'blocked the increase in 'blood pressure caused by norepinephrine and displaced the binding of the a~-adrenergic ligand [3H]WB4101. Furthermore, the hypotension produced by these analogues was itself blocked by phentolamine. The total loss of hypotensive activity after blockade of sympathetic ganglia by pentolinium precluded a papaverine-like action of the analogues directly on the smooth muscle (Constantine et al., 1973). The persistence of the hypotensive effect when hexamethonium was used as the ganglionic blocking agent in the study of prazosin (Oates et al., 1976) or of phentolamine and atropine (Abraham et al., 1981a), could be attributed to incomplete ganglionic blockade by hexamethonium (Oates et al., 1976). The relative potency for a-adrenergic blockade in vivo and in vitro was comparable to that ob-
81 served by Nedergaard and Schrold (1977) who evaluated the ability of four antimuscarinic compounds to block the contractile response of rabbit pulmonary artery to electrical stimulation. They also found, as in this study, that methylatropine and scopolamine were relatively inactive. Greenberg and Snyder (1978) and Rehavi et al. (1980) r e p o r t e d K i values for atropine and benztropine vs ~t-adrenergic receptor ligands which are consistent with our data. In contrast to analogues assigned to Category III, those grouped in Categories I and II were devoid of a-adrenolytic activity in vivo and displayed considerably lower activity in vitro. Although the analogues in categories I and II produced some changes in blood pressure, the changes were not specific in nature as they persisted after ganglionic blockade. Since these compounds did not produce the pattern of response typical of atropine, we did not pursue further the mechanism of their action on blood pressure. With the exception of tropic acid and tropine, the analogues included in this study have established antimuscarinic activity (Weiner, 1980) and the structural and conformational requirements for antimuscarinic activity have been defined (Inch and Brimblecombe, 1974). Nevertheless, in the course of this investigation, we re-evaluated this parameter in order to compare the relative potencies of the antimuscarinic and a-adrenolytic activities of these analogues using similar pharmacological approaches. As was the case for antimuscarinic properties, neither tropic acid nor tropine exhibited hypotensive or ct-adrenolytic activity. When these two components were linked by an ester or ether bond, the compound was active. Replacing the tropic acid moiety of atropine with mandelic acid to form homatropine did not markedly decrease a-adrenolytic activity although antimuscarinic potency was reduced. On the other hand, the tropine portion of the molecule was essential for a-adrenolytic activity. Substituting scopine for tropine to yield atroscine resulted in a loss of activity at the c~-adrenergic receptor with no change in antimuscarinic potency. This was further demonstrated in the case of eucatropine in which one of the carbon-carbon bonds in the tropine moiety is cleaved, rendering the compound inactive on the a-adrenergic system. In addition, benactyzine,
which has no tropine structure, has no ct-adrenolytic activity. It is interesting to note that the tropine structure contained in cocaine probably contributes to the reported a-adrenergic blocking activity of that compound (Bussell, 1940). The quaternary N-methyl analogues of atropine, homatropine and scopolamine were inactive as ct-adrenolytic agents in vivo. This lack of effect is not due to the inability of these agents to enter the central nervous system, but to their failure to interact with ct-adrenergic receptors as was demonstrated in vitro. Furthermore, centrally acting aadrenergic antagonists would be expected to produce a hypertensive response. Thus, it is imperative for an action on the a-adrenergic receptor that the nitrogen atom be tertiary, while tertiary and quaternary analogues were equipotent on the muscarinic receptor both in vivo and in vitro. This preservation of full muscarinic activity in quaternary derivatives has been previously demonstrated (Weiner, 1980). Due to the presence of an asymetric carbon atom in tropic and mandelic acids, the corresponding analogues were examined for stereochemical specificity. In line with our results, it has been reported that the l-isomers (scopolamine and hyoscyamine) are more potent antimuscarinic agents than the respective dl-forms (atroscine and atropine) (Buckett and Haining, 1965; Long et al., 1956; Marshall, 1955). In contrast to the stereospecific requirements for antimuscarinic activity, we found that for a-adrenergic activity, the dlforms were more potent than the corresponding l-forms. A preliminary evaluation of d-atropine (generously supplied by Dr. Russell Miller, Howard University College of Medicine, Washington, D.C.) indicated that, compared to dl-atropine, datropine was 10 times less active as an antimuscarinic agent whereas its a-adrenolytic effects were fully preserved both in vivo and in vitro. Thus, the d-stereoisomers appear to be more potent as hypotensive agents than the corresponding 1forms. There are other instances in which the d-stereoisomer has a greater effect than the 1-form on the t~-adrenergic receptor. For example, Cheng et al. (1980) reported that the d-enantiomers of medroxalol were 10 times more potent than the corresponding 1-enantiomers as c~-adrenergic blockers in vitro.
82
The structural requirements outlined above for the a-adrenolytic and hypotensive activities of the analogues of atropine are entirely different from those for their antimuscarinic activity. The total loss of activity of scopolamine is therefore attributed to both replacing tropine with scopine and to the fact that scopolamine is the l-stereoisomer. One can look at racemic mixtures as having two components, one with predominant ct-adrenolytic activity (d-form), and the other with predominant antimuscarinic activity (1-form). The net activity for any given analogue would depend on the relative contributions of these two components. Furthermore, for benztropine and homatropine the in vivo potency of the antimuscarinic activity is greater than the a-adrenolytic activity by only three times. In addition, benztropine has a wide range of activities and is regarded as one of the most potent inhibitors of dopamine uptake (Horn et al., 1971; Iversen, 1973). One, therefore, may wonder about the antimuscarinic selectivity of these analogues, and should not ignore their adrenolytic properties when assessing their pharmacological effects.
Acknowledgements We wish to thank Ms. Diane Gentile for excellent and devoted technical assistance with the in vitro receptor assays.
References Abraham, S., E.H. Cantor and S. Spector, 1980, Hypotensive effect of atropine: increased responsiveness of hypertensive rats, Pharmacologist 22, 162. Abraham, S., E.H. Cantor and S. Spector, 1981a, Atropine lowers blood pressure in normotensive rats through blockade of a-adrenergic receptors, Life Sci. 28, 315. Abraham, S., E.H. Cantor, E.A. Marcum and S. Spector, 1981b, Structure-activity relationship among analogues of atropine: hypotension and a-adrenergic blockade, Fed. Proc. 40, 659. Abraham, S., E.H. Cantor and S. Spector, 1981c, Studies on the hypotensive response to atropine in hypertensive rats, J. Pharmacol. Exp. Ther. 218, 662. Buckett, W.R. and C.G. Haining, 1965, Some pharmacological studies on the optically active isomers of hyoscine and hyoscyamine, Br. J. Pharmacol. 24, 138. Burn, J.H. and N.K. Dutta, 1948, The action of antagonists of
acetylcholine on the vessels of the rabbit's ear, Br. J. Pharmacol. 3, 354. Bussell, L.J., 1940, The relation of atropine to adrenaline and the sympathetic system, J. Pharmacol. Exp. Ther. 60, 128. Cantor, E.H., S. Abraham and S. Spector, 1980, Specific interaction of atropine with a-adrenergic receptors in rat brain, Soc. Neurosci. Abstrs. 6, 794. Cantor, E,H., S. Abraham and S. Spector, 1981, Central neurotransmitter receptors in hypertensive rats, Life Sci. 28, 519. Cheng, H.C., O.K. Reavis, Jr., J.M. Grisar, G.P. Claxton, D.L. Weiner and J.K. Woodward, 1980, Antihypertensive and adrenergie receptor blocking properties of the enantiomers of medroxalol, Life Sci. 27, 2529. Constantine, J.W., W.K. McSbane, A. Scriabine and H.-J. Hess, 1973, Analysis of the hypotensive action of prazosin, in: Hypertension: mechanisms and management, eds. G. Onesti, K.E. Kim and J.H. Moyer (Grune & Stratton, New York) p. 429. Fleckenstein, A., 1952, A quantitative study of antagonists of adrenaline on the vessels of the rabbit's ear, Br. J. Pharmacol. 7, 553. Furchgott, R.F., 1955, The pharmacology of vascular smooth muscle, Pharmacol. Rev. 7, 183. Greenberg, D.A. and S.H. Snyder, 1978, Pharmacological properties of [3H]dihydroergokryptine binding sites associated with alpha noradrenergic receptors in rat brain membranes, Mol. Pharmacol. 14, 38. Horn, A.S,, J.T. Coyle and S.H. Snyder, 1971, Catecholamine uptake by synaptosomes from rat brain: Structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons, Mol. Pharmacol. 7, 66. Inch, T.D. and R.W. Brimblecombe, 1974, Acetylcholine drugs: chemistry, stereochemistry and pharmacology, Int. Rev. Neurobiol. 16, 67. Iversen, L.L., 1973, Neuronal and extraneuronal catecholamine uptake mechanisms, in: Frontiers in catecholamine research, eds. E. Usdin and S.H. Snyder (Pergamon Press, New York) p. 403. Long, J.P., F.P. Luduena, B.F. Tullar and A.M. Lands, 1956, Stereochemical factors involved in cholinolytic activity, J. Pharmacol. Exp. Ther. 117, 29. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurements with the folin phenol reagent, J. Biol. Chem. 193, 265. Marshall, P.B., 1955, Antagonism of acetylcholine by ( + )- and (-)-hyoscyamine, Br. J. Pharmacol. 10, 354. Nedergaard, O.A. and J. Schrold, 1977, Effect of atropine on vascular adrenergic neuroeffector transmission, Blood Vessels 14, 325. Oates, H.F., R.M. Graham, L.M. Stoker and G.S. Stokes, 1976, Hemodynamic effects of prazosin, Arch. Int. Pharmacodyn. 224, 239. Rehavi, M., B. Yavetz, O. Ramot and M. Sokolovsky, 1980, Regional heterogeneity of two high affinity binding sites for 3H-WB-4101 in mouse brain, Life Sci. 26. 615. Sakanashi, M., T. Furukawa and Y. Horio, 1979~ Inhibition of constrictor responses of dog coronary artery by atropine. A
83 possible effectiveness of atropine On variant form of angina pectoris, Jap. Heart J. 20, 75. Snedecor, G.W. and W.G. Cochran, 1967, Statistical methods, sixth edition (The Iowa State University Press, Ames, Iowa) p. 419. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454.
Weiner, N., 1980, Atropine, scopolamine and related antimuscarinic drugs, in: The pharmacological basis of therapeutics, eds. A.F. Gilman, L.S. Goodman and A. Gilman (Macmillan Publishing Co., Inc., New York) p. 120. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Natl. Acad. Sci. 71, 1725.