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Beta-receptor blocking and cardiodepressant actions of 2-nitrilophenoxypropanolamines
EUROPEAN JOURNAL O17 PHARMACOLOGY 16 (1971) 14-20. NORTH-HOLLAND PUBLISttING COMPANY
BETA-RECEPTOR BLOCKING AND CARDIODEPRESSANT ACTIONS OF 2-NITRILOPHENOXYPROPANOLAMINES E.J. MYLECHARANE and C. RAPER Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Received 2 November 1970
Accepted 28 June 1971
E.J. MYLECHARANE and C. RAPER, Beta-receptor blocking and cardiodepressant actions of 2-nitrilophenoxypropanolamines, European J. Pharmacol. 16 (1971) 14-20. The #-adrenoreceptor blocking and cardiodepressant activities of a series of 1-alkylamino-3-(2-nitrilophenoxy)-2propanols have been studied in isolated rabbit atria. Increasing the amine substituent through the series --H, C1t3, C2 Hs, CH(CH3)2, and -C(CH3) 3 increases both the #-receptor blocking and the cardiodepressant activity of the compounds. In the above series pA 2 values against isoprenaline ranged from 5.83 to 8.65. Cardiodepressant activity, as judged by negative inotropic and chronotropic actions and by increases in effective refractory period, occurred at much higher concentrations than those required for/3-receptor blockade. The potency ratio of the -C(CH3) 3 to the -H derivative was 661 for #-receptor blockade, and ranged from 7 to 14 for the cardiodepressant actions. Despite the same rank order of potency of the compounds in producing cardiodepressant actions and #-receptor blockade, no correlation exists between the two effects. l-Alkylamino-3-(2-nitrilophenoxy)-2-propanols
1.INTRODUCTION In addition to their effects on t3-adrenoreceptors, /3-receptor antagonists have been shown to possess cardiodepressant activity (Kuschinsky and Rahn, 1965; Morales-Aguilerff and Vaughan Williams, 1965 ; Levy and Richards, 1966; Raper and Jowett, 1967; Levy, 1968; Raper and Wale, 1968; Nayler et al., 1969). S t r u c t u r e - a c t i v i t y relationships with respect to /3-receptor blocking actions of compounds have been extensively studied, however, little work has been done in attempting to relate chemical structure to depressant activity. In the present experiments the cardiodepressant and /3-receptor blocking activities of a series of 1-alkylamino-3-(2-nitrilophenoxy)-2-propanols have been studied in isolated atrial preparations. The isopropyl and t-butyl substituted compounds have previously been shown to possess /3-receptor blocking
#-Receptor antagonism
Cardiodepressant activity
activity against isoprenaline-induced cardiac inotropic effects in anaesthetized dogs (Engelhardt and Traunecker, 1969).
2. METHODS Rabbits weighing 2 to 3.5 kg were killed by a blow on the neck and the hearts removed. Left and right atrial preparations were suspended in McEwen's (1956) solution maintained at 37°C and gassed with 5% CO2 in oxygen. A 1 hr equilibration period was allowed in all experiments before the addition of drugs. In experiments where isoprenaline was used, ascorbic acid (20/ag/ml) was included in the physiological solution. 2.1. (3-Receptor blockade Cumulative c o n c e n t r a t i o n - e f f e c t curves for isometric tension were obtained at hourly intervals in
E.J.Mylecharane, C.Raper, N-alkyl substituted #-receptorantagonists driven left atrial preparations using isoprenaline as an agonist. The atria were stimulated at a frequency of 1 Hz using square wave pulses of 1 msec duration and a voltage which was 10 to 25% greater than the threshold required for driving (1.0 to 3.5 V). After the establishment of control curves, further curves were obtained in the presence of varying concentrations of the 1-alkylamino-3-(2-nitrilophenoxy)-2propanols. An antagonist contact time of 30 rain was allowed; further increases in contact time did not increase the degree of antagonism obtained. 2.2. Cardiodepressant actions Inotropic activity and changes in refractory period produced by the compounds were assessed from isometric tension records obtained from driven left atrial preparations. Doses were administered cumulatively without washout, a 15 rain contact time being allowed at each concentration. Inotropic activity was determined in preparations stimulated at 1 Hz. When monitoring refractory period changes, the preparations were stimulated at a frequency of 1 Hz using paired stimuli. The pulse-separation of the paired stimuli was increased from an initial 10 msec separation until potentiated contractions were observed. The pulse interval at which this occurred was taken as a measure of the effective refractory period. Chronotropic activity of the compounds was
CN ~ -
OH o - e l l = - / H - C H=-NH-R • HCI R
'r
- H
Kt5 1439
]1
-- CH 3
K6 1561
l"n
- c 2 Hs
K~ 1560
I CH3 -- CH
K~ 1313
" CH 3
'V"
CIH3 -- C - CH 3
K~ 1366
CH 3
Fig. 1. Molecular structures of the N-alkyl substituted compounds used in the present study.
15
assessed in spontaneously beating right atrial preparations. Preliminary studies showed that after the initial equilibration period, the rate of spontaneous beating slowly declined over a period of an hour or more. This decline was not apparent in tissues where the bathing solution was replaced at intervals of 15 to 30 min. In these experiments, therefore, cumulative drug additions were made at 15 min intervals and the bath fluid was replaced after observations had been made at each individual concentration. The drugs used were (+)-isoprenaline hydrochloride (Winthrop); propranolol hydrochloride (Imperial Chemical Industries) and the hydrochloride salts of the five 1-alkylamino-3-(2-nitrilophenoxy)-2-propanols (Boehringer Ingelheim) shown in fig. 1.
3. RESULTS 3.1. fi-Receptor blockade In driven left atrial preparations, preliminary studies in which cumulative concentration-effect curves were established for the positive inotropic activity of isoprenaline, it was shown that repeated exposure of the tissues to high concentrations of the amine produced changes in the resting contractility and in the maximal responses attained. However, when the results from successive isoprenaline exposures were expressed as percentages of the maximal response (Emax) in each individual curve, the concentration-effect curves were superimposable. In the presence of the nitrilo-compounds the cumulative concentration-effect curves for isoprenaline were moved to the right in a parallel fashion. The mean values for the maximal evoked tension in the presence of the nitrilo-compounds expressed as a percentage of those obtained in the control isoprenaline curves are shown in table 1. The mean result (+- S.E.) for all the experiments with the nitrilo-compounds was 100% +10%. These results suggest that competitive antagonism might be involved. Table 1 also shows the mean pA2 values (-+ S,E.) of the compounds calculated from dose-ratios obtained at the 50% Ema x level, and their relative potencies with respect to K/5 1439. In each individual experiment the slope of the relationship between log (dose-ratio - 1) and log molar concentration of antagonist was calculated. The mean slopes (-+ S.E.) (table 1) fall close to the theoretical
16
E.J.Mylecharane, CRaper, N-alkyl substituted g-receptor antagonists
Table 1 Cardiac ¢~-receptorblockade Antagonistic activity of the N-alkyl substituted compounds expressed as pA 2 values using isoprenaline as an agonist in driven left atrial preparations. Each value represents the mean (-+ S.E.) from 8 to 10 determinations. The relative potencies of the compounds as ¢~-receptor antagonists are shown (KS 1439 = 1). The mean values (-+ S,E.) from 8 to 10 determinations of the maximal isoprenaline evoked tension in the presence of the compounds expressed as a percentage of controls is shown. The mean values (-+ S.E.) of the slope obtained in 4 to 5 experiments from plots of log (dose-ratio - l) against log molar concentration of antagonist are also shown. Compound
pA 2 (isoprenaline)
Relative potency
Maximal response (% control)
Slope log (DR - 1)/log [M]
Ki5 1439 K~5 1561 Ki5 1560 K~5 1313 K8 1366
5.83 +_0.15 5.96 +- 0.08 7.06 +_0.04 8.03-+0.07 8.65-+0.17
1.0 1.3 17 159 661
98 _+ 10 94 + 9 137 _+ 18 84± 7 87-+ 4
1.11 -+ 0.05 1.10 -+ 0.07 0.91 -+ 0.08 0.97-+0.11 1.05 + 0.10
value of unity, characteristic of competitive antagonism (Arunlakshana and Schild, 1959).
3.2. Cardiodepressant activity Figs. 2a,b and 3 show the effects of the compounds on the contractility, the rate of spontaneous contractions and the effective refractory period of isolated a t r i a . The results are expressed as the mean percentage changes (+ S.E.) in the parameters from initial values obtained in each experiment in the absence of drugs. In 15 control experiments performed under identical conditions but without addition of the nitrilocompounds there was little change from initial control levels in inotropic or chronotropic activity or in the effective refractory period of the atrial preparations. The maximum duration of any experiment was 3 hr after the initial equilibration period. Control studies showed that at this time the mean values (+ S.E.) for the developed tension, the spontaneous rate and the effective refractory period were 95 +-3%, 96 +-2% and 92 +-6% respectively of the initial control values. At the lower concentrations ( 1 0 -9 to 10 -6 M), K6 1313 and Ki5 1366 possessed positive inotropic and chronotropic actions (fig. 2a,b). In a further series of 10 experiments in which the atria were exposed to single doses (4 X 10-8 M) o f the cornpounds, it was found that these effects were abolished by the administration of the t3-receptor antagonist propranolol in a concentration of 3.3 X 10 -8 M.
This concentration of propranolol produced no changes in contractility or rate of spontaneous beating of the atria, but antagonized responses to isoprenaline (7.5 X 10 -8 M). Significant depression of inotropic and chronotropic activity and increases in effective refractory period occurred at concentrations greater than 10-SM with all the compounds. The nitrilo-compounds fell into two groups in this respect. Those with hydrogen, methyl and ethyl substituents (K6 1439, K6 1561 and K6 1560) possessed less depressant activity than those with isopropyl or tertiary butyl substituents (K6 1313 and K6 1366). The mean rank order for depressant potency was K6 1439 < K6 1561 < K~5 1560 < K6 1313 < Ki5 1366, which is the same rank order as that found for j3-receptor blockade. However, the range of the relative potencies of the compounds as depressants is much smaller than that found for antagonistic activity at /3-receptors, and the threshold concentrations of the nitrilocompounds required to produce depressant activity were much larger than those required for/3-receptor antagonism. Table 2 shows the concentrations of the compounds required to produce a 25% depression of inotropic and chronotropic activity and a 25% increase in effective refractory period of atrial preparations. For ease of comparison with pA2 values, these results are expressed as the negative log of the molar concentrations required to produce the effects. The relative depressant potencies of the compounds are also shown together with the approximate dose-ratios
E.J.Mylecharane, CRaper, N-alkyl substituted H-receptorantagonists
(a)
INOTROPIC
17
ACTIVITY
T s
J
O
°
~
T
O
100
.
~
~
~
T
i~ K ~ . ~
Response
.iT
%
Control
50,
O.
,
1~9
!
i
|
i
1o-'
i
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16 ~ Concentration ( M )
(b)
CHRONOTFIOPIC
ACTIVITY
g -'-'-'-'-'---.-~ a ~
~
"
:
~
""-- i
1
to i -9
10
IL, |
~_
ii
7
115
10
Concentration (M)
|
2
|
10- 3
Fig. 2. Graphs showing the effects of Ki5 1439 (=), K~i 1561 (v), K6 1560 (a), K5 1313 (e) and K6 1366 (o) on (a) inotropic activity in driven left atrial preparations and (b) chronotropic activity in spontaneously beating right atrial preparations. Each point represents the mean (+ S.E.) of the results from 6 to 9 experiments expressed as a percentage of the initial response in the absence of the compounds. Points without an error bar represent means where the S.E. was < 2%.
18
E.J.Mylecharane, CRaper. N-alkyl substituted fX-receptorantagonists
20(
Response
%
Control
10C
w'-8
•
10
w--6
10 Concentration
=
=--4
10
(M)
Fig. 3. Effects of the N-alkyl substituted c o m p o u n d s (see fig. 2) on the effective refractory period of driven left atria preparations. Each point represents the mean (_+ S.E.) of the results from 5 to 8 experiments.
Table 2 Cardiodepressant activity Concentrations of the c o m p o u n d s producing a 25% increase in the effective refractory period and 25% decreases in inotropic and chronotropic activity o f rabbit atria expressed as the - v e log of the molar concentration producing these effects. The relative potencies of the c o m p o u n d s (Kt5 1439 = 1) are shown for each effect. The dose-ratios for the t3-receptor blockade produced by tile c o m p o u n d s at 10 s M concentration are also shown.
Compound
Effective refractory period
Relative potency
- v e inotropic effect
Relative potency
- r e chronotropic effect
Relative potency
Dose ratio (#-blockade at 10 -s M)
K~5 1439 K~5 1561 K6 1560 Ki5 1313 K6 1366
3.67 3.72 3.87 4.36 4.53
1.0 1.1 1.6 4.9 7.2
3.39 3.85 3.65 4.47 4.53
1.0 2.8 1.8 12 14
3.44 3,60 3,63 4,13 4.26
1.0 1.5 1.6 4.9 6.6
9 12 76 900 7300
E.J.Mylecharane, C.Raper, N.alkyl substituted H-receptorantagon&ts
for the expected/3-receptor blockade found at concentrations of 10-s M. The latter results were obtained by extrapolation of graphs relating log (doser a t i o - 1) to the log of the molar concentration of the antagonists. This concentration is just below the threshold for the cardiodepressant actions of the drugs.
4. DISCUSSION The 1-alkylamin o-3-(2-nitrilophenoxy)-2-propanols used in the present study, like other ring-substituted N-isopropyl-phenoxypropanolamines (e.g., propranolol, oxprenolol and practolol), possess competitive antagonistic actions at cardiac/3-adrenoreceptors. The nature of the N-alkyl substituents has a marked effect on the potency of the compounds as /3-receptor antagonists. The rank order of potency of the compounds in the series increases with increasing size of the substituent, this being especially marked with the branched chain isopropyl and t-butyl derivatives. In this respect the findings with the nitrilocompounds are similar to those found by other authors who have studied the f3-receptor blocking potencies of N-alkyl derivatives of compounds such as sotalol (UIoth et al., 1966), pronethalol (Howe et al., 1968), propranolol (Crowther and Smith, 1968)and 3,4-dichloro, 3,4-difluoro, and 3,4-dimethoxy phenylethanolamine (Pratesi and Grana, 1965). As with other /3-receptor antagonists the nitrilocompounds used in the present experiments display cardiodepressant activity only in concentrations which produce a substantial degree of 13-receptor blockade. Previous workers have shown that ring substituents affect the depressant activity of both N-isopropylphenylethanolamines (e.g., dichloroisoprenaline, pronethalol, sotalol, and INPEA) and N-isopropylphenoxypropanolamines (e.g., propranolol, oxprenolol and practolol). Compounds with electron-withdrawing substituents such as methanesulphonanilide, nitro and acetanilide, as found in sotalol, INPEA and practolol, respectively, have only weak depressant activity (Lish et al., 1965; Somani and Lum, 1965; Schmid and Hanna, 1967; Wale et al., 1969), in comparison with fl-receptor antagonists such as propranolol and pronethalol (Morales-Aguilera" and
19
Vaughan Williams, 1965; Raper and Jowett, 1967). Although compounds with the /3-hydroxyl group in the (-)-configuration have a greater/3-receptor blocking potency than their corresponding (+)- or (+)-isomers, (+)-, (-)- and (+)-isomers have similar potencies as cardiodepressants (Levy and Richards, 1966; Ari~ns, 1967; Parmley and Braunwald, 1967; Raper and Jowett, 1967; Barrett and Cullum, 1968; Belliveau and Covino, 1969). Little work has been done on the effects of N-alkyl substitution on the cardiodepressant actions of /3-receptor antagonists. Within the nitrilo-compounds studied, increasing the bulk of the alkyl substituent on the amine group increased the cardiodepressant activity of the compounds. As with the /3-receptor blocking actions of the drugs, the greatest depressant activity occurred with the branched-chain derivatives (K6 1313 and K6 1366). However, the increase in cardiodepressant activity with increasing alkyl substitution was much Jess than the increase in /3-receptor blocking activity produced with the same molecular changes. The fact that threshold doses are similar for the production of negative inotropic and chronotropic actions and increases in effective refractory period by the compounds, suggests that a single underlying mechanism may be involved in these depressant effects. Ari~ns (1960), when assessing the 13-receptor blocking and 'papaverine-like' activities of the 3,4-dichloro derivatives of noradrenaline, adrenaline and isoprenaline in isolated tracheal preparations, showed that increasing alkyl loading increased both types of activity. This is in accord with the present findings with the nitrilo-compounds. However, Ari~ns (1960) found that depressant activity as assessed by pD'2 values increased to a greater extent than did the pA2 values for j3-receptor antagonistic activity, and on this basis he suggested that the noradrenaline derivative would give the most specific /3-receptor blocking activity without involvement of a depressant action. This finding may be due to the comparatively low potencies of the dichloro derivatives as /3-receptor antagonists and to the relatively small change in potency of the compounds with increasing alkyl substitution. The sympathomimetic actions of the compounds might also give a false impression of 'papaverine-like' activity in tracheal preparations.
?0
E.J.Mylecharane, CRaper, N-alkyl substituted ~-receptor antagonists
In conclusion, although increasing the basicity of the amine nitrogen by increasing the bulk of the alkyl loading increases b o t h the ~3-receptor blocking and the cardiodepressant activity of the n i t r i l o - c o m p o u n d s , no correlation exists b e t w e e n the two activities. The depressant actions only occur at m u c h higher c o n c e n t r a t i o n s than those required for j3-receptor blockade and the relative potencies o f the c o m p o u n d s for the two types o f activity are m a r k e d l y different. A similar lack of correlation has been f o u n d by o t h e r workers w h o have studied the relative cardiodepressant and /3-receptor blocking activities of N - i s o p r o p y l - s u b s t i t u t e d 13-receptor antagonists (Levy and Richards, 1966; Raper and J o w e t t , 1967; Levy, 1968). ACKNOWLEDGEMENTS We would like to thank Miss J. Baird for technical assistance. We are grateful to the Medical Directors of Boehringer Ingelheim (Australia) for their interest and for the supply of the nitrilo-compounds. This work was supported by a grant from the National Heart Foundation of Australia.
REFERENCES Ari6ns, E.J., 1960, Various types of receptc " for sympathomimetic drugs, in: Ciba Symposium on Adrenergic Mechanisms, eds. Vane, Wolstenholme and O'Connor, p. 253. Arigns, E.J., 1967, The structure-activity relationships of beta-adrenergic drugs and beta-adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139, 606. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Brit. J. Pharmacol. Chemotherap. 14, 48. Barrett, A.M. and V.A. Cullum, 1968, The biological properties of the optical isomers of propranolol and their effects on cardiac arrhythmias, Brit. J. Pharmacol. 34, 43. Belliveau, R.E. and B.G. Covino, 1969, Effects of a new f3-adrenergic receptor blocking agent, alprenolol, and its optical isomers, Arch. Intern. Pharmacodyn. 180,341. Crowther, A.I:. and L.H. Smith, 1968, ~3-Adrenergic blocking agents. 11. Propranolol and related 3-amino-l-naphthoxy2-propanols, J. Med. Chem. 11, 1009. Engelhardt, A. and W. Traunecker, 1969, Pharmakologie einiger Phenoxypropanolamin-Derivate mit /3-adrenolytischer Wirkung, Arch. Exptl. Pathol. Pharmakol. 263, 203. ttowe, R., A.I,. Crowther, J.S. Stephenson, B.S. Rao and L.tl. Smith, 1968, ~-Adrenergic blocking agents. 1. Pronethalol and related N-alkyl and N-aralkyl derivatives
of 2-amino-l-(2-naphthyl) ethanol, J. Med. Chem. 11, 1000. Kuschinsky, G. and K.H. Rahn, 1965, Untersuchungen i~ber die Beziehungen zwischen chinidinartigen und j3-adrenolytischen Wirkungen von 1-(3-methylphenoxy)-2-hydroxy-3-isopropylaminopropan (K6592), Arch. Exptl. Pathol. Pharmakol. 252, 50. Levy, J.V., 1968, Myocardial and local anesthetic actions of /3-adrenergic receptor blocking drugs: relationship to physicochemieal properties, European J. Pharmacol. 2. 250. Levy, J.V. and V. Richards, 1966, Inotropic and chronotropic effects of a series of t3-adrenergic blocking drugs: some structure-activity relationships, Proc. Soc. Exptl. Biol. Med. 122, 373. Lish, P.M., J.H. Weikel and K.W. Dungan, 1965, Pharmacological and toxicological properties of two new t3-adrenergic receptor antagonists, J. Pharmacol. Exptl. Therap. 149, 161. McEwen, L.M., 1956, The effect on the isolated rabbit heart of vagal stimulation, and its modification by cocaine, hexamethonium and ouabain, J. Physiol. (London) 131, 678. Morales-Aguilerd, A. and E.M. Vaughan Williams, 1965, The effects on cardiac muscle of t3-receptor antagonists in relation to their activity as local anaesthetics, Brit. J. Pharmacol. Chemotherap. 24,332. Nayler, W.G., D. Chipperfield and T.E. Lowe, 1969, The negative inotropic effect of adrenergic beta-receptor blocking drugs on human heart muscle, Cardiovasc. Res. 3, 30. Parmley, W.W. and E. Braunwald, 1967, Comparative myocardial depressant and anti-arrhythmic properties of d-propranolol, dl-propranolol and quinidine, J. Pharmacol. Exptl. Therap. 158, 11. Pratesi, P. and E. Grana, 1965, Structure and activity at adrenergic receptors of catecholamines and certain related compounds, Advan. Drug Res. 2, 127. Raper, C. and A. Jowett, 1967, Anti-fibrillary and anti-adrenaline activity of ~3-receptor blocking drugs, European J. Pharmacol. 1, 353. Raper, C. and J. Wale, 1968, Propranolol, MJ 1999 and Ciba 39089Ba in ouabain and adrenaline induced cardiac arrhythmias, European J. Pharmacol. 4, 1. Schmid, J.R. and C. Hanna, 1967, A comparison of the anti-arrhythmic actions of two new synthetic compounds, iproveratril and MJ 1999, with quinidine and pronethalol, J. Pharmacol. Exptl. Therap. 156, 331. Somani, P. and B.K.B. Lum, 1965, The anti-arrhythmic actions of ¢3-adrenergic blocking agents, J. Pharmacol. Exptl. Therap. 147, 194. Uloth, R.tI., J.R. Kirk, W.A. Gould and A.A. Larsen, 1966, Sulfonanilides. I. Monoalkyl- and arylsulfonamidophenethanolamines, J. Med. Chem. 9, 88. Wale, J., L.Q. Pun and M.J. Rand, 1969, The effects of ICI 50172, propranolol, pronethalol and MJ 1999 on t3-receptots, European I. Pharmacol. 8, 25.
BETA-RECEPTOR BLOCKING AND CARDIODEPRESSANT ACTIONS OF 2-NITRILOPHENOXYPROPANOLAMINES E.J. MYLECHARANE and C. RAPER Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Received 2 November 1970
Accepted 28 June 1971
E.J. MYLECHARANE and C. RAPER, Beta-receptor blocking and cardiodepressant actions of 2-nitrilophenoxypropanolamines, European J. Pharmacol. 16 (1971) 14-20. The #-adrenoreceptor blocking and cardiodepressant activities of a series of 1-alkylamino-3-(2-nitrilophenoxy)-2propanols have been studied in isolated rabbit atria. Increasing the amine substituent through the series --H, C1t3, C2 Hs, CH(CH3)2, and -C(CH3) 3 increases both the #-receptor blocking and the cardiodepressant activity of the compounds. In the above series pA 2 values against isoprenaline ranged from 5.83 to 8.65. Cardiodepressant activity, as judged by negative inotropic and chronotropic actions and by increases in effective refractory period, occurred at much higher concentrations than those required for/3-receptor blockade. The potency ratio of the -C(CH3) 3 to the -H derivative was 661 for #-receptor blockade, and ranged from 7 to 14 for the cardiodepressant actions. Despite the same rank order of potency of the compounds in producing cardiodepressant actions and #-receptor blockade, no correlation exists between the two effects. l-Alkylamino-3-(2-nitrilophenoxy)-2-propanols
1.INTRODUCTION In addition to their effects on t3-adrenoreceptors, /3-receptor antagonists have been shown to possess cardiodepressant activity (Kuschinsky and Rahn, 1965; Morales-Aguilerff and Vaughan Williams, 1965 ; Levy and Richards, 1966; Raper and Jowett, 1967; Levy, 1968; Raper and Wale, 1968; Nayler et al., 1969). S t r u c t u r e - a c t i v i t y relationships with respect to /3-receptor blocking actions of compounds have been extensively studied, however, little work has been done in attempting to relate chemical structure to depressant activity. In the present experiments the cardiodepressant and /3-receptor blocking activities of a series of 1-alkylamino-3-(2-nitrilophenoxy)-2-propanols have been studied in isolated atrial preparations. The isopropyl and t-butyl substituted compounds have previously been shown to possess /3-receptor blocking
#-Receptor antagonism
Cardiodepressant activity
activity against isoprenaline-induced cardiac inotropic effects in anaesthetized dogs (Engelhardt and Traunecker, 1969).
2. METHODS Rabbits weighing 2 to 3.5 kg were killed by a blow on the neck and the hearts removed. Left and right atrial preparations were suspended in McEwen's (1956) solution maintained at 37°C and gassed with 5% CO2 in oxygen. A 1 hr equilibration period was allowed in all experiments before the addition of drugs. In experiments where isoprenaline was used, ascorbic acid (20/ag/ml) was included in the physiological solution. 2.1. (3-Receptor blockade Cumulative c o n c e n t r a t i o n - e f f e c t curves for isometric tension were obtained at hourly intervals in
E.J.Mylecharane, C.Raper, N-alkyl substituted #-receptorantagonists driven left atrial preparations using isoprenaline as an agonist. The atria were stimulated at a frequency of 1 Hz using square wave pulses of 1 msec duration and a voltage which was 10 to 25% greater than the threshold required for driving (1.0 to 3.5 V). After the establishment of control curves, further curves were obtained in the presence of varying concentrations of the 1-alkylamino-3-(2-nitrilophenoxy)-2propanols. An antagonist contact time of 30 rain was allowed; further increases in contact time did not increase the degree of antagonism obtained. 2.2. Cardiodepressant actions Inotropic activity and changes in refractory period produced by the compounds were assessed from isometric tension records obtained from driven left atrial preparations. Doses were administered cumulatively without washout, a 15 rain contact time being allowed at each concentration. Inotropic activity was determined in preparations stimulated at 1 Hz. When monitoring refractory period changes, the preparations were stimulated at a frequency of 1 Hz using paired stimuli. The pulse-separation of the paired stimuli was increased from an initial 10 msec separation until potentiated contractions were observed. The pulse interval at which this occurred was taken as a measure of the effective refractory period. Chronotropic activity of the compounds was
CN ~ -
OH o - e l l = - / H - C H=-NH-R • HCI R
'r
- H
Kt5 1439
]1
-- CH 3
K6 1561
l"n
- c 2 Hs
K~ 1560
I CH3 -- CH
K~ 1313
" CH 3
'V"
CIH3 -- C - CH 3
K~ 1366
CH 3
Fig. 1. Molecular structures of the N-alkyl substituted compounds used in the present study.
15
assessed in spontaneously beating right atrial preparations. Preliminary studies showed that after the initial equilibration period, the rate of spontaneous beating slowly declined over a period of an hour or more. This decline was not apparent in tissues where the bathing solution was replaced at intervals of 15 to 30 min. In these experiments, therefore, cumulative drug additions were made at 15 min intervals and the bath fluid was replaced after observations had been made at each individual concentration. The drugs used were (+)-isoprenaline hydrochloride (Winthrop); propranolol hydrochloride (Imperial Chemical Industries) and the hydrochloride salts of the five 1-alkylamino-3-(2-nitrilophenoxy)-2-propanols (Boehringer Ingelheim) shown in fig. 1.
3. RESULTS 3.1. fi-Receptor blockade In driven left atrial preparations, preliminary studies in which cumulative concentration-effect curves were established for the positive inotropic activity of isoprenaline, it was shown that repeated exposure of the tissues to high concentrations of the amine produced changes in the resting contractility and in the maximal responses attained. However, when the results from successive isoprenaline exposures were expressed as percentages of the maximal response (Emax) in each individual curve, the concentration-effect curves were superimposable. In the presence of the nitrilo-compounds the cumulative concentration-effect curves for isoprenaline were moved to the right in a parallel fashion. The mean values for the maximal evoked tension in the presence of the nitrilo-compounds expressed as a percentage of those obtained in the control isoprenaline curves are shown in table 1. The mean result (+- S.E.) for all the experiments with the nitrilo-compounds was 100% +10%. These results suggest that competitive antagonism might be involved. Table 1 also shows the mean pA2 values (-+ S,E.) of the compounds calculated from dose-ratios obtained at the 50% Ema x level, and their relative potencies with respect to K/5 1439. In each individual experiment the slope of the relationship between log (dose-ratio - 1) and log molar concentration of antagonist was calculated. The mean slopes (-+ S.E.) (table 1) fall close to the theoretical
16
E.J.Mylecharane, CRaper, N-alkyl substituted g-receptor antagonists
Table 1 Cardiac ¢~-receptorblockade Antagonistic activity of the N-alkyl substituted compounds expressed as pA 2 values using isoprenaline as an agonist in driven left atrial preparations. Each value represents the mean (-+ S.E.) from 8 to 10 determinations. The relative potencies of the compounds as ¢~-receptor antagonists are shown (KS 1439 = 1). The mean values (-+ S,E.) from 8 to 10 determinations of the maximal isoprenaline evoked tension in the presence of the compounds expressed as a percentage of controls is shown. The mean values (-+ S.E.) of the slope obtained in 4 to 5 experiments from plots of log (dose-ratio - l) against log molar concentration of antagonist are also shown. Compound
pA 2 (isoprenaline)
Relative potency
Maximal response (% control)
Slope log (DR - 1)/log [M]
Ki5 1439 K~5 1561 Ki5 1560 K~5 1313 K8 1366
5.83 +_0.15 5.96 +- 0.08 7.06 +_0.04 8.03-+0.07 8.65-+0.17
1.0 1.3 17 159 661
98 _+ 10 94 + 9 137 _+ 18 84± 7 87-+ 4
1.11 -+ 0.05 1.10 -+ 0.07 0.91 -+ 0.08 0.97-+0.11 1.05 + 0.10
value of unity, characteristic of competitive antagonism (Arunlakshana and Schild, 1959).
3.2. Cardiodepressant activity Figs. 2a,b and 3 show the effects of the compounds on the contractility, the rate of spontaneous contractions and the effective refractory period of isolated a t r i a . The results are expressed as the mean percentage changes (+ S.E.) in the parameters from initial values obtained in each experiment in the absence of drugs. In 15 control experiments performed under identical conditions but without addition of the nitrilocompounds there was little change from initial control levels in inotropic or chronotropic activity or in the effective refractory period of the atrial preparations. The maximum duration of any experiment was 3 hr after the initial equilibration period. Control studies showed that at this time the mean values (+ S.E.) for the developed tension, the spontaneous rate and the effective refractory period were 95 +-3%, 96 +-2% and 92 +-6% respectively of the initial control values. At the lower concentrations ( 1 0 -9 to 10 -6 M), K6 1313 and Ki5 1366 possessed positive inotropic and chronotropic actions (fig. 2a,b). In a further series of 10 experiments in which the atria were exposed to single doses (4 X 10-8 M) o f the cornpounds, it was found that these effects were abolished by the administration of the t3-receptor antagonist propranolol in a concentration of 3.3 X 10 -8 M.
This concentration of propranolol produced no changes in contractility or rate of spontaneous beating of the atria, but antagonized responses to isoprenaline (7.5 X 10 -8 M). Significant depression of inotropic and chronotropic activity and increases in effective refractory period occurred at concentrations greater than 10-SM with all the compounds. The nitrilo-compounds fell into two groups in this respect. Those with hydrogen, methyl and ethyl substituents (K6 1439, K6 1561 and K6 1560) possessed less depressant activity than those with isopropyl or tertiary butyl substituents (K6 1313 and K6 1366). The mean rank order for depressant potency was K6 1439 < K6 1561 < K~5 1560 < K6 1313 < Ki5 1366, which is the same rank order as that found for j3-receptor blockade. However, the range of the relative potencies of the compounds as depressants is much smaller than that found for antagonistic activity at /3-receptors, and the threshold concentrations of the nitrilocompounds required to produce depressant activity were much larger than those required for/3-receptor antagonism. Table 2 shows the concentrations of the compounds required to produce a 25% depression of inotropic and chronotropic activity and a 25% increase in effective refractory period of atrial preparations. For ease of comparison with pA2 values, these results are expressed as the negative log of the molar concentrations required to produce the effects. The relative depressant potencies of the compounds are also shown together with the approximate dose-ratios
E.J.Mylecharane, CRaper, N-alkyl substituted H-receptorantagonists
(a)
INOTROPIC
17
ACTIVITY
T s
J
O
°
~
T
O
100
.
~
~
~
T
i~ K ~ . ~
Response
.iT
%
Control
50,
O.
,
1~9
!
i
|
i
1o-'
i
l o=-3
16 ~ Concentration ( M )
(b)
CHRONOTFIOPIC
ACTIVITY
g -'-'-'-'-'---.-~ a ~
~
"
:
~
""-- i
1
to i -9
10
IL, |
~_
ii
7
115
10
Concentration (M)
|
2
|
10- 3
Fig. 2. Graphs showing the effects of Ki5 1439 (=), K~i 1561 (v), K6 1560 (a), K5 1313 (e) and K6 1366 (o) on (a) inotropic activity in driven left atrial preparations and (b) chronotropic activity in spontaneously beating right atrial preparations. Each point represents the mean (+ S.E.) of the results from 6 to 9 experiments expressed as a percentage of the initial response in the absence of the compounds. Points without an error bar represent means where the S.E. was < 2%.
18
E.J.Mylecharane, CRaper. N-alkyl substituted fX-receptorantagonists
20(
Response
%
Control
10C
w'-8
•
10
w--6
10 Concentration
=
=--4
10
(M)
Fig. 3. Effects of the N-alkyl substituted c o m p o u n d s (see fig. 2) on the effective refractory period of driven left atria preparations. Each point represents the mean (_+ S.E.) of the results from 5 to 8 experiments.
Table 2 Cardiodepressant activity Concentrations of the c o m p o u n d s producing a 25% increase in the effective refractory period and 25% decreases in inotropic and chronotropic activity o f rabbit atria expressed as the - v e log of the molar concentration producing these effects. The relative potencies of the c o m p o u n d s (Kt5 1439 = 1) are shown for each effect. The dose-ratios for the t3-receptor blockade produced by tile c o m p o u n d s at 10 s M concentration are also shown.
Compound
Effective refractory period
Relative potency
- v e inotropic effect
Relative potency
- r e chronotropic effect
Relative potency
Dose ratio (#-blockade at 10 -s M)
K~5 1439 K~5 1561 K6 1560 Ki5 1313 K6 1366
3.67 3.72 3.87 4.36 4.53
1.0 1.1 1.6 4.9 7.2
3.39 3.85 3.65 4.47 4.53
1.0 2.8 1.8 12 14
3.44 3,60 3,63 4,13 4.26
1.0 1.5 1.6 4.9 6.6
9 12 76 900 7300
E.J.Mylecharane, C.Raper, N.alkyl substituted H-receptorantagon&ts
for the expected/3-receptor blockade found at concentrations of 10-s M. The latter results were obtained by extrapolation of graphs relating log (doser a t i o - 1) to the log of the molar concentration of the antagonists. This concentration is just below the threshold for the cardiodepressant actions of the drugs.
4. DISCUSSION The 1-alkylamin o-3-(2-nitrilophenoxy)-2-propanols used in the present study, like other ring-substituted N-isopropyl-phenoxypropanolamines (e.g., propranolol, oxprenolol and practolol), possess competitive antagonistic actions at cardiac/3-adrenoreceptors. The nature of the N-alkyl substituents has a marked effect on the potency of the compounds as /3-receptor antagonists. The rank order of potency of the compounds in the series increases with increasing size of the substituent, this being especially marked with the branched chain isopropyl and t-butyl derivatives. In this respect the findings with the nitrilocompounds are similar to those found by other authors who have studied the f3-receptor blocking potencies of N-alkyl derivatives of compounds such as sotalol (UIoth et al., 1966), pronethalol (Howe et al., 1968), propranolol (Crowther and Smith, 1968)and 3,4-dichloro, 3,4-difluoro, and 3,4-dimethoxy phenylethanolamine (Pratesi and Grana, 1965). As with other /3-receptor antagonists the nitrilocompounds used in the present experiments display cardiodepressant activity only in concentrations which produce a substantial degree of 13-receptor blockade. Previous workers have shown that ring substituents affect the depressant activity of both N-isopropylphenylethanolamines (e.g., dichloroisoprenaline, pronethalol, sotalol, and INPEA) and N-isopropylphenoxypropanolamines (e.g., propranolol, oxprenolol and practolol). Compounds with electron-withdrawing substituents such as methanesulphonanilide, nitro and acetanilide, as found in sotalol, INPEA and practolol, respectively, have only weak depressant activity (Lish et al., 1965; Somani and Lum, 1965; Schmid and Hanna, 1967; Wale et al., 1969), in comparison with fl-receptor antagonists such as propranolol and pronethalol (Morales-Aguilera" and
19
Vaughan Williams, 1965; Raper and Jowett, 1967). Although compounds with the /3-hydroxyl group in the (-)-configuration have a greater/3-receptor blocking potency than their corresponding (+)- or (+)-isomers, (+)-, (-)- and (+)-isomers have similar potencies as cardiodepressants (Levy and Richards, 1966; Ari~ns, 1967; Parmley and Braunwald, 1967; Raper and Jowett, 1967; Barrett and Cullum, 1968; Belliveau and Covino, 1969). Little work has been done on the effects of N-alkyl substitution on the cardiodepressant actions of /3-receptor antagonists. Within the nitrilo-compounds studied, increasing the bulk of the alkyl substituent on the amine group increased the cardiodepressant activity of the compounds. As with the /3-receptor blocking actions of the drugs, the greatest depressant activity occurred with the branched-chain derivatives (K6 1313 and K6 1366). However, the increase in cardiodepressant activity with increasing alkyl substitution was much Jess than the increase in /3-receptor blocking activity produced with the same molecular changes. The fact that threshold doses are similar for the production of negative inotropic and chronotropic actions and increases in effective refractory period by the compounds, suggests that a single underlying mechanism may be involved in these depressant effects. Ari~ns (1960), when assessing the 13-receptor blocking and 'papaverine-like' activities of the 3,4-dichloro derivatives of noradrenaline, adrenaline and isoprenaline in isolated tracheal preparations, showed that increasing alkyl loading increased both types of activity. This is in accord with the present findings with the nitrilo-compounds. However, Ari~ns (1960) found that depressant activity as assessed by pD'2 values increased to a greater extent than did the pA2 values for j3-receptor antagonistic activity, and on this basis he suggested that the noradrenaline derivative would give the most specific /3-receptor blocking activity without involvement of a depressant action. This finding may be due to the comparatively low potencies of the dichloro derivatives as /3-receptor antagonists and to the relatively small change in potency of the compounds with increasing alkyl substitution. The sympathomimetic actions of the compounds might also give a false impression of 'papaverine-like' activity in tracheal preparations.
?0
E.J.Mylecharane, CRaper, N-alkyl substituted ~-receptor antagonists
In conclusion, although increasing the basicity of the amine nitrogen by increasing the bulk of the alkyl loading increases b o t h the ~3-receptor blocking and the cardiodepressant activity of the n i t r i l o - c o m p o u n d s , no correlation exists b e t w e e n the two activities. The depressant actions only occur at m u c h higher c o n c e n t r a t i o n s than those required for j3-receptor blockade and the relative potencies o f the c o m p o u n d s for the two types o f activity are m a r k e d l y different. A similar lack of correlation has been f o u n d by o t h e r workers w h o have studied the relative cardiodepressant and /3-receptor blocking activities of N - i s o p r o p y l - s u b s t i t u t e d 13-receptor antagonists (Levy and Richards, 1966; Raper and J o w e t t , 1967; Levy, 1968). ACKNOWLEDGEMENTS We would like to thank Miss J. Baird for technical assistance. We are grateful to the Medical Directors of Boehringer Ingelheim (Australia) for their interest and for the supply of the nitrilo-compounds. This work was supported by a grant from the National Heart Foundation of Australia.
REFERENCES Ari6ns, E.J., 1960, Various types of receptc " for sympathomimetic drugs, in: Ciba Symposium on Adrenergic Mechanisms, eds. Vane, Wolstenholme and O'Connor, p. 253. Arigns, E.J., 1967, The structure-activity relationships of beta-adrenergic drugs and beta-adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139, 606. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Brit. J. Pharmacol. Chemotherap. 14, 48. Barrett, A.M. and V.A. Cullum, 1968, The biological properties of the optical isomers of propranolol and their effects on cardiac arrhythmias, Brit. J. Pharmacol. 34, 43. Belliveau, R.E. and B.G. Covino, 1969, Effects of a new f3-adrenergic receptor blocking agent, alprenolol, and its optical isomers, Arch. Intern. Pharmacodyn. 180,341. Crowther, A.I:. and L.H. Smith, 1968, ~3-Adrenergic blocking agents. 11. Propranolol and related 3-amino-l-naphthoxy2-propanols, J. Med. Chem. 11, 1009. Engelhardt, A. and W. Traunecker, 1969, Pharmakologie einiger Phenoxypropanolamin-Derivate mit /3-adrenolytischer Wirkung, Arch. Exptl. Pathol. Pharmakol. 263, 203. ttowe, R., A.I,. Crowther, J.S. Stephenson, B.S. Rao and L.tl. Smith, 1968, ~-Adrenergic blocking agents. 1. Pronethalol and related N-alkyl and N-aralkyl derivatives
of 2-amino-l-(2-naphthyl) ethanol, J. Med. Chem. 11, 1000. Kuschinsky, G. and K.H. Rahn, 1965, Untersuchungen i~ber die Beziehungen zwischen chinidinartigen und j3-adrenolytischen Wirkungen von 1-(3-methylphenoxy)-2-hydroxy-3-isopropylaminopropan (K6592), Arch. Exptl. Pathol. Pharmakol. 252, 50. Levy, J.V., 1968, Myocardial and local anesthetic actions of /3-adrenergic receptor blocking drugs: relationship to physicochemieal properties, European J. Pharmacol. 2. 250. Levy, J.V. and V. Richards, 1966, Inotropic and chronotropic effects of a series of t3-adrenergic blocking drugs: some structure-activity relationships, Proc. Soc. Exptl. Biol. Med. 122, 373. Lish, P.M., J.H. Weikel and K.W. Dungan, 1965, Pharmacological and toxicological properties of two new t3-adrenergic receptor antagonists, J. Pharmacol. Exptl. Therap. 149, 161. McEwen, L.M., 1956, The effect on the isolated rabbit heart of vagal stimulation, and its modification by cocaine, hexamethonium and ouabain, J. Physiol. (London) 131, 678. Morales-Aguilerd, A. and E.M. Vaughan Williams, 1965, The effects on cardiac muscle of t3-receptor antagonists in relation to their activity as local anaesthetics, Brit. J. Pharmacol. Chemotherap. 24,332. Nayler, W.G., D. Chipperfield and T.E. Lowe, 1969, The negative inotropic effect of adrenergic beta-receptor blocking drugs on human heart muscle, Cardiovasc. Res. 3, 30. Parmley, W.W. and E. Braunwald, 1967, Comparative myocardial depressant and anti-arrhythmic properties of d-propranolol, dl-propranolol and quinidine, J. Pharmacol. Exptl. Therap. 158, 11. Pratesi, P. and E. Grana, 1965, Structure and activity at adrenergic receptors of catecholamines and certain related compounds, Advan. Drug Res. 2, 127. Raper, C. and A. Jowett, 1967, Anti-fibrillary and anti-adrenaline activity of ~3-receptor blocking drugs, European J. Pharmacol. 1, 353. Raper, C. and J. Wale, 1968, Propranolol, MJ 1999 and Ciba 39089Ba in ouabain and adrenaline induced cardiac arrhythmias, European J. Pharmacol. 4, 1. Schmid, J.R. and C. Hanna, 1967, A comparison of the anti-arrhythmic actions of two new synthetic compounds, iproveratril and MJ 1999, with quinidine and pronethalol, J. Pharmacol. Exptl. Therap. 156, 331. Somani, P. and B.K.B. Lum, 1965, The anti-arrhythmic actions of ¢3-adrenergic blocking agents, J. Pharmacol. Exptl. Therap. 147, 194. Uloth, R.tI., J.R. Kirk, W.A. Gould and A.A. Larsen, 1966, Sulfonanilides. I. Monoalkyl- and arylsulfonamidophenethanolamines, J. Med. Chem. 9, 88. Wale, J., L.Q. Pun and M.J. Rand, 1969, The effects of ICI 50172, propranolol, pronethalol and MJ 1999 on t3-receptots, European I. Pharmacol. 8, 25.