Prediction of the non-specific cardiodepressant effects of β-adrenoceptor blocking agents in vitro and in vivo by means of the Hansch analysis

European Journal of Pharmacology 29 (1974) 223-235 © North-Holland Publishing Company

PREDICTION OF THE NON-SPECIFIC CARDIODEPRESSANT

EFFECTS OF

/ 3 - A D R E N O C E P T O R B L O C K I N G A G E N T S IN V I T R O A N D I N V I V O B Y M E A N S O F THE HANSCH ANALYSIS D. HELLENBRECHT, K.-F. MLILLER and H. GROBECKER Zentrum der Pharmakologie der Universitfft, 6000 Frankfurt/Main, Theodor-Stern-Kai 7, Germany

Received 25 October 1973, accepted 24 July 1974 D. HELLENBRECHT, K.-F. MiJLLER and H. GROBECKER, Prediction o f the non-specific cardiodepressant effects of ~-adrenoceptor blocking agents in vitro and in vivo by means o f the Hansch analysis, Ettropean J. Pharmacol. 29 (1974) 223-235. Hydrophobicity (n-octanol/buffer partition coefficients) of six l-alkylamino-3-(2-nitrilophenoxy)-propan-2-ol derivatives with different substituents at the amino group (K6 1439, K6 1561, K6 1560, Ki5 1313, Kb 1366, Ki5 1500), and of five l-isopropylamino-3-(alkylphenoxy)-propan-2-ol derivatives with different ring substituents (Ki5 592, Ki5 707, K6 1030, Kb 1124, K6 1292), with #-adsenoceptor blocking properties, was correlated with the non-specific cardiodepressant effects of the drugs. Influence on the conduction velocity of the frog heart was studied in vitro and influence of the drugs on dp/dtma x in anaesthetized cats after #-adrenoceptor blockade was measured in vivo. The physicochemical and pharmacological data were analysed by the method of Hansch. The partition coefficients of the #-adrenoceptor blocking drugs ranged more than three orders of magnitude. The differences in hydrophobicity were solely dependent on the sum of the hydrophobic substituents of the drug molecules at the phenyl ring and at the amino group. Slowing of conduction velocity in vitro was more pronounced the higher the partition coefficients of the two series of the K/5 compounds investigated; multiple regression analysis revealed parabolic correlation equations between hydrophobicity and pharmacological effects, depending on the experimental conditions (incubation time, mode of stimulation of the preparation) used. A parabolic correlation was also observed between hydrophobicity and decrease of dp/dtma x in vivo. Similar correlation equations were obtained when the hydrophobic properties of the compounds were calculated according to the method of Hansch. From the physicochemical, pharmacological, and analytical data it is concluded that the non-specific cardiodepressant effects of #-adrenoceptor blocking drugs can be predicted for given pharmacological systems by determination or by calculation of the hydrophobicity of the drug molecules, irrespectively of the site of the hydrophobie substituents (whether at the ring system or at the amino group). Non-specific cardiodepressio n #-Adrenocep[or blocking drugs Partition coefficients

Contractility measurement (dp/dt) Conduction velocity Hansch analysis

1. Introduction &Adrenoceptor blocking drugs possess at least two pharmacologically relevant actions. First specific ~adrenoceptor antagonistic activity, often together with partial agonistic activity, and second a nonspecific membrane affinity which appears as 'quinidine-like' cardiodepressant effects. While structural requirement relevant to the /5-

Ring substitution N-Substitution

adrenoceptor blocking potencies of the compounds is based largely on empirical evidence, the non-specific membrane affinity of these compounds can be adequately predicted by investigation of their physicochemical properties (Pratesi et al., 1966; Lemmer et al., 1972; Hellenbrecht et al., 1973; Wiethold et al., 1973). These conclusions were based predominantly on compounds which differed only in the chemical substituents at the aromatic ring system, but there is

224

D. Hellenbrecht et al., Cardiodepressant effects of #-adrenoceptor blocking compounds

now evidence from the investigations of Mylecharane and Raper (1971) that the degree of alkylation of the amino group of/3-adrenoceptor antagonists is also correlated with the non-specific cardiodepressant activity of this kind of compounds. It was the aim of this investigation to study the relationship between the physicochemical properties and the cardiodepressant effects of two rather homologous series of phenoxypropanolamines under identical conditions: 5 /3adrenoceptor blocking drugs which differed only in the number or length of the alkyl substituents at the ring system, and 6 compounds which differed only with respect to the alkyl substituents at the amino group. From the data of these two series of compounds and of further data derived from the additive principle of the Hansch analysis it was tested how far physicochemical and non-specific pharmacological properties of/3-adrenoceptor blocking drugs were correlated in vivo and in vitro. If there were strong correlations it should be possible to predict the potency of the non-specific pharmacological actions merely from the chemical structure of this group of compounds.

2. Materials and methods

2.1. Octanol buffer partition coefficients (Pob s] These were determined as previously described (Hellenbrecht et al., 1973). There were some difficulties determining the partition coefficients of the extremely polar compounds e.g. K6 1439, K5 1561, K6 1560: though the volume of octanol taken was 200 times that of the buffer phase, the extinction differences of the drug solution before and after extraction of the buffer phase with octanol were very small. An additional factor of inaccuracy was the inevitable emulsification of the buffer phase with one of the most polar compounds, K6 1560.

2.2. Calculation of Err values The log P values for the unionized form of the investigated drugs (= ~n) were calculated by using the additivity rule of Hansch and Fujita (1964); from the literature values of log P (= 70 of various nuclei and n for substituents were taken and summed (cf. Tute, 1971). For the /3-adrenoceptor blocking drugs this

was done with data described previously (Hellenbrecht et al., 1973). In addition, some approximations were used because of lack of references in the literature. The following approximated terms were used: rr3-CN instead of rr2-CN for the nitrilo-substituted compounds; rr3-t-butyl instead of rr4-t-butyl for K6 1292.

2.3. Calculation of the pK a values The pK a for Ki5 592 was kindly supplied by Drs. Engelhardt and K6ppe (Boehringer Sohn, Ingelheim). This value was also assumed for the ring-alkylated compounds Ki3 707, K6 1030, Ki5 1124, K~5 1292 and propranolol. It was likewise taken for K6 1313 because of the same isopropylamino group. For the other N-alkylated K/5 compounds corrections relative to the value of K~5 592 were applied which were based on the pK a data given by Pratesi and Grana (1965) for N-substituted noradrenaline derivatives.

2.4. Calculation of P' (= P'calc) The partition coefficients for the ionized form at a definite pH, P', were calculated from the partition coefficients of the unionized molecule (= P = Nrr) and from the calculated pK a values by means of the following formula (cf. Tute, 1971): P/P'= 1 + antilog (PKa-PH).

2.5. Measurement of myocardial conduction velocity of the frog heart in vitro Extracellular 'pre'- and 'post'-anaesthetic action potentials in vitro were recorded from heart muscle strips of Rana esculenta with the method previously described in detail (Hellenbrecht et al., 1973). In the present investigation a slight modification was used: the first stimulus of the muscle strips was followed by a second one after a fixed time interval of 1400 msec. The sweep rate of the oscilloscope was 200 msec/cm. Two groups of experiments were carried out. In the first group, time-response curves were studied with each drug at 2 - 3 different submaximal concentrations over a period of 60 min. In another group of experiments dose-response curves were compiled cumulatively, with incubation periods of l0 min for

D. Hellenbrecht et al., Cardiodepressant effects of #-adrenoceptor blocking compounds N-substituted

each drug concentration. The ICs o's were determined graphically. 2.6. Measurement o f contractility in vivo (dp/dt) in the anaesthetized cat Changes in maximum rate of rise of intraventricular pressure (dp/dtmax) as an index of the inotropic state of the heart muscle were measured in 24 male cats, (2.3-4.2 kg) anaesthetized with pentobarbital • Na (30 mg/kg i.p.) and heparinized with 300 I.U. heparin/kg. The cats breathed spontaneously through a tracheal cannula and were atropinized (3 mg/kg). A 210 mm steel cannula (inner diameter 1.04 mm)was inserted into the left ventricle through the right carotid artery, according to the method of Heeg (1967), and attached to a Statham P23 Db transducer. The signal was differentiated by an analogue differentiating circuit, preamplified through a carrier amplifier and recorded with a Helcoscriptor (Fa. Hellige, Freiburg). Blood pressure was measured from the femoral artery. Heart rate was recorded by a tachometer triggered by the blood pressure signal. The drugs were injected through the femoral vein in volumes less than 1.5 ml. Two groups of experiments were carried out: 8 cats served for time-response curves. 10 /amoles/kg of each K~5 compound were given successively to each cat, in order of increasing hydrophobicity of the compounds (see Results). Each of the drugs was applied only after the level of dp/dtma x of the preceding drug effect had reached the level of the period before application of the drug. The second group of experiments was carried out with one drug per cat. To each animal, 2--4 submaximal doses of the respective drug were given. The maximal cardiodepressant effects, observed within the first 2 0 - 6 0 sec, were used to obtain the EDs o'S from these dose-response curves. 2. 7. Statistics Regression lines were calculated by the method of the least squares with a computer program. Results are expressed in terms of the equation for the best fitting line, together with the standard error of the estimates, s, standard deviations of the calculated terms, S.D., correlation coefficients, r, F-test values, and level of significance, p. In some results the stand-

225 phenyl - s u b s t i t u t e d

,Crt3 OH

R=

KO 1439

- H

KO 1561

-CH 3

Ko

1560

- CHTCH 3

Ko

1313

,,.CH3 -CH

KO 1366

R- O- CH2-(~H-CH2-NH- CH OH \Ell 3 R=

"C1% CH

- C"-~"CFt3 "ell 3

Ko

592

C<~

KO

707

C~>_ CH3

Ko 1030

Ch~

,,CH'zCH.~'CH3 KO 1500

-CH ",CH 3

CH~ CH3"CH2~H,'CH3

Kti

1124

CH3~

Fig. 1. Structural formulas of the #-adrenoceptor blocking K6 compounds studied. ard error of the mean, S.E.M. is given. The paired t-test was used for combined data. 2.8. Drugs used Atropine sulphate; heparin (Liquemin, Roche); pentobarbital • Na (Nembutal, Roche); (-+)-proprano1ol • HCI (Inderal, I.C.I.); ( -)-penbutolol sulphate (Hoechst; for chemical constitution see discussion); the structural formulas of the 11 /3-adrenergic blocking K6 compounds (Boehringer Sohn, lngelheim), used as the racemates, are shown in fig. 1. 2.9. List o f abbreviations P

octanol partition coefficient for the unionized molecule p' octanol partition coefficient for the ionized molecule at a definite pH pKa ionization constant hydrophobic constant of chemical substituent, usually calculated according to the equation nx = log Px - log PH where Px and PH are the partition coefficients of the substituted and the parent molecule equivalent to P, obtained by addition of 1r-constants Pobs octanol buffet partition coefficient obtained experimentally

P'ealc octanol

buffer partition coefficient calculated by use of Zrr and pK a

226

D. ltellenbrecht et al., Cardiodepressant effects of a-adrenoceptor blocking compounds

3. Results

3.1. Partition coefficients and pK a values o f the Ko compounds There was a gradual increase of the partition coefficient, Pobs, in the series of the phenoxypropanolamines with increasing size of the alkyl substituents, table 1. The most polar amino-substituted compounds K/5 1439, K/5 1561 and Ki5 1560 partitioned into the hydrophobic octanol phase only to approximately 0.01 ; there was a considerable increase in Pobs with increasing N-alkyl chain: for the N-t-butyl form, Ki5 1366, it was more than 0.1; K/5 1500, due to the N-isopentyl substituent, partitioned more into the hydrophobic medium than into the aqueous phase, indicated by a partition coefficient higher than 1.0. For the ring substituted N-isopropylphenoxypropanolamines a similar increase of Pobs was found. While Ki5 592 was partitioned predominantly into the aqueous phase (Pobs < 1.0), the more alkylated compounds K/5 707 and K~ 1030 were found to a greater extent in the hydrophobic medium: K~5 1124 and Ki5 1292 for example, partitioned 1 5 - 4 3 times more into octanol. The logarithms of the partition coefficients (log Pobs, table 1) reflect the wide range o f the partition coefficients of the members of the two series of compounds investigated. It is of interest that Ki5 1500 (N-isopentyl) despite its polar nitrilo sub-

stituent at the ring, showed a higher partition coefficient than did the ring-methylated compound K/5 592 (N-isopropyl). The octanol partition coefficient for propranolol, Pobs = 5.39, was taken from the previous study (Hellenbrecht et al., 1973). For the data of penbutolol see discussion, part 4.3. The logarithmic values of the calculated hydrophobicity of the unionized form of the ~-adrenoceptor blocking compounds are also listed as Err in table 1. These were in general more than two orders of magnitude higher than the octanol partition coefficients obtained experimentally at pH 7.0. The respective pK a values of the drugs investigated (table 1) range from 9.10 to 9.65. For all N-isopropylphenoxypropanolamines, a pK a of 9.50 was assumed. In accordance with Pratesi and Grana (1965) the highest pK a (9.65) was taken for K/5 1366 (N-t-butyl). With a higher degree of N-alkylation, e.g. K6 1500, and also with a lower degree of alkylation of the amino substituents, lower pK a values were assumed: note the lowest value of 9.10 for the primary amine K/5 1439. The calculated data for propranolol were Zn = 3.09 and pK a = 9.50. The results of the calculated partition coefficients t corrected to pH 7.0 (= log P calc) are likewise listed in table 1. These values are similar to the observed partition coefficients (cf. log Pobs)observed experimentally (for further details see discussion). Log r P calc for propranolol was 0.59.

Table 1 Observed partition coefficients (Pobs) of O-adrenoceptor blocking drugs (n-octanol/phosphate buffer, 0.16 M, pH 7.0) and the respective calculated partition coefficients (P'calc) derived from the Xn values and the pKa constants (the data of Pobs refer to x ± S.E.M.). Compound

Pobs

K~5 1439 K/5 1561 K/5 1560 K~5 1313 K~:, 1366 K/5 1500 K/5 592 K~5 707 Ki5 1030 K/3 1124 K/J 1292

0.0087 0.0127 0.0114 0.0892 0.1395 1.4830 0.571 1.789 3.671 15.46 43.24

± 0.0041 z 0.0024 ± 0.0006 ± 0.0062 ± 0.0123 ± 0.1160 ± 0.038 ± 0.104 ± 0.284 t 1.04 ± 1.31

n

log Pobs

X*r a

pKa b

log Pcalc

4 4 4 5 4 4 4 4 4 4 3

-2.0605 -1.8962 -1.9431 -1.0496 -0.8539 0.1712 --0.2426 0.2529 0.5647 1.1895 1.6359

0.25 0.75 1.25 1.55 1.85 2.55 2.36 2.87 3.39 3.66 4.05

9.10 9.43 9.53 9.50 9.65 9.40 9.50 9.50 9.50 9.50 9.50

-1.8539 -1.6819 -1.2815 -0.9503 -0.8002 0.1488 -0.1413 0.3687 0.8891 1.1590 1.5489

a Derived from the data of Tute (1971). b For calculation of the pK a values see Materials and methods.

D. Hellenbrecht et al., Cardiodepressant effects of O-adrenoceptor blocking compounds 3.2. Effects o f the Ki5 compounds on the conduction velocity of frog heart muscle strips The first experimental set was carried out with 22 experiments ( 5 - 6 muscle strips per experiment). The results listed in table 2 show the percentage conduction velocities obtained after 60 min of incubation with two different concentrations of the respective /3-adrenoceptor blocking compounds (the initial value of conduction velocity before application of the drugs, 13.1 ± 2.1 cm/sec, ~ -+ S.D., was taken as

100%). From table 2 it can be seen that, for example, K6 1439 (3 X l0 -3 M) decreased conduction velocity to 65% of the control period. The higher concentration of K6 1439 (4.5 × 10 -3 M) produced a decrease to 33%. The resulting IC5o for K6 1439, determined graphically by means of probability paper, was 3.6 × 10-3 M. With increasing N-alkylation of the nitrilophenoxypropanolamines, the ICso'sdecreased progressively from 3 × 10 -3 M for K6 1561 to 3.5 × 10-4 M for K6 1500. The different ring-alkylated compounds (cf. fig. 1) showed similar increase in potency of the reduction of conduction velocity: while the ICso for Ki5 592 was practically identical with that of K~ 1500, this decreased stepwise with each additional CH3 group at the phenyl ring. The maximum inhibitory activity of the compounds was

227

observed for K5 1124, and Ki5 1292. Ki5 1292, despite its longest alkyl substituent, was slightly less active than Ki5 1124. For propranolol (not shown in table 2) the ICs o was 1.2 × 10 -4 M. In the second experimental procedure, in which cumulative dose-response curves were obtained, the results cited above were confirmed (table 3). 55 experiments were carried out in which the drug concentrations, beginning from 10 -s M, were cumulatively increased every 10 min by a factor of 3 to 3.33, up to 10-2 M. In table 3 the results of the conduction velocity of these experiments are listed as % of the initial conduction velocity, 13.1 -+ 1.5 cm/sec (,~ -+ S.D.), of the control period before addition of the drugs. The first and second values in the table refer to the first (I) and to the second (II) stimulus, respectively (cf. Materials and methods). Several facts can be deduced from table 3 (for reasons of brevity the results of the lowest concentrations, 10 -s to 10 -4 M, are not depicted): (1) The sooner block of conduction (=0% conduction velocity) occurred, the higher was the degree of alkylation of the compounds. (2) The respective ICs0 values, depicted in the table, decreased gradually, with increasing hydrophobic character of both series of compounds. (3) Conduction velocities after the second stimulus (!I) were in general lower than those observed after the first stimulus (I). This is reflected by the occurrence of block of

Table 2 Cardiodepressant effects of #-adrenoceptor blocking drugs in vitro (decrease of conduction velocity of frog heart muscle strips), and in vivo (50% decrease of dp/dtma x in anaesthetized cats after #-adrenoceptor blockade with propranolol). Compound

Ki5 1439 K6 1561 K6 1560 K6 1313 Ki~ 1366 Ki5 1500 K6 592 K6 707 K6 1030 K5 1124 K/5:1292

Frog heart muscle strips Inhibitory concentrations (X 10-4 M)

% conduction velocity after drug exposure a,b

30 10 9 10 4.5 3.0 3.0 2.0 0.9 0.9 0.9

65 71 76 74 62 70 69 62 87 69 65

45 30 30 30 7.5 4.5 6.0 4.5 2.0 1.2 1.2

33 50 23 10 50 23 25 11 29 32 49

IC50 q t'Xl0 M)

dp/dtma x, cat c EDso (X 10-6 tamoles/kg i.v.)

36 30 16 13 7.5 3.5 4.1 2.4 1.5 1.0 1.2

130 I10 90 35 23 14 13 8 6 4.2 7.4

a In'cubation time 60 min. b % conduction velocity of the control period; 5-6 muscle strips per drug concentration. c EDso values calculated from the results of fig. 3.

D. Hellenbrecht et al.. Cardiodepressant effects of #-adrenoceptor blocking compounds

228

Table 3 Effects of O-adrenoceptor blocking drugs on the conduction velocity of frog heart muscle strips after cumulative application of the drugs. Incubation time of each drug concentration: 10 min. The values represent the % of conduction velocity of the control period before addition of the drug (x- ± S.E.M. from 5 experiments per compound). The interval between the first (1) and second (II) stimulus was 1400 msec. 0 = block of conduction. Compound

Drug concentration (M) 3 × 10 -4

10 -3

I

I

II

1I

3 X 10 -3

10 -2

I

I

II

1I

ICso (I)

IC50 (II)

(X 10 -4 M)

(× 10-4M

1

II

ICso (I)/ICso (II)

Ki51439

91.1 94.2 86.3 86.5 76.6 69.6 24.1 11.4 -*2.4 -*2.3 ±0.9 -+2.7 -*2.2 *-3.0 -+10.4 -+9.0

68

50

1.36

K~i1561

97.8 91.1 92.7 83.5 78.9 66.7 4.9 0 ± 1.8 -* 2.7 ±3.2 -.2.3 -.4.5 -.5.0 -* 2.0 '-

60

43

1.39

K~ 1560

97.6 94.1 91.1 83.4 75.9 49.1 7.0 -+ 1.7 -+ 1.5 -* 1.8 -+ 2.7 -+ 1.8 -+6.1 • 7.0

0

61

28

2.18

K5 1313

91.6 86.6 80.4 62.4 42.4 8.5 -+2.9 ±2.0 ~5.5 -+6.2 ±4.7 -.5.2

0

0

27

13

2.08

K~i 1366

89.7 84.6 -+2.7 -.3.1

0

0

20

11

1.82

K/5 1500

89.2 68.0 49.8 * 3.8 ±5.5 ±9.1

0

0

10

4.4

2.27

K~; 592

88.2 81.1 62.9 8.6 3.5 -+ 2.3 -+ 7.3 -* 2.3 -+4.5 ± 1.7

0

0

0

13

5.8

2.24

K~ 707

86.3 61.4 41.7 4.3 -*2.8 *4.8 ±10.5-+4.3

0

0

0

0

8.2

3.9

2.10

K5 1030

91.2 62.9 26.8 3.0 * 4.3 -.12.4 -+10.5 -+ 0.2

0

0

0

0

7.0

3.8

1.84

K~J 1124

76.4 52.5 18.4 2.2 -+9.5 -*8.3 -+8.1 ±2.2

0

0

0

0

5.0

3.2

1.56

K~ 1292

84.0 75.1 27.8 18.6 -+4.2 -+5.1 -.12.5 -*9.5

0

0

0

0

6.5

5.0

1.30

72.0 53.8 36.3 8.2 -.1.6 *-2.4 -+9.0 -+6.4 15.1 13.2 8.2 -.15.1 -+13.2 ~ . 2

c o n d u c t i o n at lower drug concentrations, and by lower IC5o values. The ratios o f the two 1Cso'S ( I C s o ( I ) / I C s o ( I I ) ) are also listed in the table and ranged from 1.30 to 2.27.

3.3. Influence o f the KO compounds on the contractility o f the cat heart in vivo (dp/dtmax) in the first group o f experiments, the time course o f the changes o f the m a x i m u m isometric d p / d t (= d p / d t m a x ) after a single dose ( 1 0 / a m o l e s / k g ) o f each drug was studied in 8 cats (fig. 2). To eliminate the initial decrease o f d p / d t m a x due to the specific [3a d r e n o c e p t o r blocking activity o f the c o m p o u n d s investigated, the cats were pretreated with a standard

dose o f propranolol (1 mg/kg i.v.; the dose was repeated w h e n s y m p a t h o m i m e t i c actions due to intrinsic agonistic activity o f some c o m p o u n d s was observed). This procedure was assumed to be sufficient to block 3-adrenoceptors in the heart. A steady state level o f 'basic c o n t r a c t i l i t y ' was observed 30 min later which lasted for at least 1 - 2 hr. d p / d t m a x during the reference period was 3230 ± 95 m m Hg/sec (n = 8). As can be seen f r o m fig. 2, there were only slight decreases o f d p / d t m a x under the influence o f 10 /amoles/kg o f the highly polar substances K6 1439, K6 1561, K6 1560 and o f K6 1313. However, K6 1366, K6 592 and K6 1500 p r o d u c e d consistent decreases o f d p / d t m a x which lasted for at least 5 - 1 0 min (fig. 2). Very m a r k e d and prolonged depressions

D. llellenbrecht et aL, Cardiodepressant effects of #-adrenoceptor blocking compounds

229

decreose of dp/dtmox,COt (ram Hglsec) 0 KO1561

500

'~ "J" (o',' ~ ,i¢'

_Ko1313

KO1366 W'

100C K, 5 .

V ,;

KO 1500

'11'

.Ar"~a~~" .o ~"

.a

~

/~

~50C KO 707 KO 1030 Ko 1292 KO 112/.

2OO( i

i

i

0

5

|0

i

15

I

1

20

25

I

30 min

Fig. 2. T i m e - r e s p o n s e curves of the effects of the #-adrenoceptor blocking K6 compounds (10 pmoles/kg i.v.) on d p / d t m a x in 8 anaesthetized cats alter #-adrenoceptor blockade with propranolol (1 mg/kg i.v.). Ordinate: decrease o f dp/dtma x (mm Hg/sec; reference value K t S.E.M. = 3220 ± 95 mm Hg/sec). Abscissa: time in min. • . . . . . o, N-substituted K6 compounds; • •, ring-substituted K6 compounds.

of contractility were observed with the hYdrophobic compounds K~ 707, K6 1030 and K8 1124. The cardiodepressant effect of K6 1292 did not exceed that of K6 1124 in spite of the more hydrophobic character of the former compound (statistical significance of the difference p < 0.05). The differences in non-specific cardiodepressant activity observed in experiments after one fixed dose, were also seen when the dose-response relationships were studied by application of increasing doses of one Ko compound to one animal. Before ~-adrenoceptor blockade, maximum isometric dp/dt of 11 cats was 4800 -+ 235 mm Hg/sec; this decreased significantly to 3140 -+ 160 (p < 0.001) after complete ~-adrenoceptor blockade with propranolol (I mg/kg = 3 /anoles/kg). As can be seen from the results (fig. 3), the decrease of dp/dtma x (% of control period) was dose dependent for all compounds investigated (the EDso's obtained from these curves are given in table 2 as tanoles/kg). While the cardiodepressant potency of the very polar compounds Kb 1439, K5 1561 and K8 1560 showed ICso'S near 100/amoles/ kg, the following less polar drugs K6 1313, K6 1366, K8 1500 and K8 592 ranged between 35 and 13 #moles/kg. EDs o's more than one order of magnitude

lower were obtained for the hydrophobic ~-adrenoceptor blocking drugs Ki5 707, Ki5 1030, Ki5 1124 and K6 1292. Again the most hydrophobic compound K6 1292 was less cardiodepressant than its congener K~J 1124. For the reference drug proprano1ol, the EDs o under identical experimental conditions was 7.6 × 10-6 pmoles/kg. % dp/dtmo~ , cot 90 1030 --

707

,

1500

/

1560

1366

/,.,

,,,

i

3

I

10

I

30

|

J

100 3O0 ,un~.es/kg i.v Fig. 3. Dose-response curves of the effects o f the/3-adrenoceptor blocking K6 compounds on d p / d t m a x in 11 anaesthetized cats after 0-adrenoceptor blockade with propranolol (1 mg/kg i.v.). Ordinate: % decrease of dp/dtma x from individual controls (reference value ~ ± S.E.M. = 3140 ± 160 mm Hg/sec). Abscissa: drug dosage, pmoles/kg i.v.

D. Hellenbrecht et al., Cardiodepressanteffects of f-adrenoceptor blocking compounds

230 4. Discussion

conduction vetocity, frog heart muscle

1 log I - ~

4. I. Hydrophobicity of ~adrenoceptor blocking drugs The partition coefficient in n-octanol/buffer, pH 7.0, of the drugs investigated ranged 5000-fold between the most polar substance K6 1439 (Pobs --0.0087) to the most hydrophobic compound Ki5 1292 (Pobs = 43.24). These differences can be attributed to the degree of alkylation of the phenyl ring and to the size of the alkyl substituent at the amino group because these are the only differences in the chemical structure of the two series of K/5 compounds investigated. According to the additivity rule of Hansch (cf. Leo et al., 1971) any additional CH2 or CH3 group should increase the log partition coefficient P of the unionized molecule (= ~n) by a value of 0.50 for unbranched substitutents since n-CH2 and n-CH3 are approxmately 0.50 (cf. Leo et al., 1971; Tute, 1971; Hansch and Dunn, 1972); for branched CI-13 groups n is only 0.30. This simple principle was used for calculation of the Xn values given in table !. Because the ~-adrenoceptor antagonists were a l m ~ t completely ionized at pH 7.0, log P' values were obtained by correcting Zn for the degree of dissociation with the pK a of the compounds. These predicted parr tition coefficients P calc agreed with the partition coefficients found experimentally (Pobs) except for compounds Ki5 1439, K6 1561, Ki5 1560 and Ki5 1030. How can these deviations be explained? As was already pointed out under Materials and methods, the difficulties in estimating the most polar substances were considerable. The low experimental value of the 3 ',4',5 '-methylated com pound K5 1030, corn pare d to the calculated value, indicates that intramolecular folding and/or strong hydrogen bonding is probably involved (cf. Tute, 1971). Another explanation for the deviations between calculated and found partition coefficients has been proposed by Davis (1973): n-values are more probably additive on the mole fraction concentration scale than on the molar scale which has been used in this study:

4.2. Correlation between hydrophobicity of the Ki5 compounds and depression of myocardial conduction velocity of frog heart muscle To test the hypothesis that the physicochemical

K6~30 1~112&K61292 K6S~01~

~ ~'~r

-

.L-,/*'-° K01560 /

I~ 9 Po = 5.0

o

0 ~ ' 0 "~

K61&39 ,~-0K61SG1/~"0

/ ' ~

-2

log Po

i

i

I

i

-1

0

1

2 log Pabsd

Fig. 4a. Parabolic correlations of the non-specific pharmacological effects of the /~-adrenoceptor blocking drugs with the respective octanol/buffer partition coefficients. Double logarithmic plots of the reciprocal ICs0'S of the K6 compounds on the conduction velocity of the frog heart muscle in vitro, ordinate, against the partition coefficients Pobs (octanol/buffer, pH 7.0), abscissa, e, data obtained with incubation periods of 60 min. including propranolol; the indicated parabola refers to equation (2) in the text. o, data obtained with incubation periods of 10 min; the parabola refers to equation (4) in the text. The apex of the parabola, log Po, indicates the calculated hydrophobicity of #Jadrenoceptor blocking drugs with expected maximum biological activity. properties of the /3-adrenoceptor blocking drugs are the determinants of their non-specific pharmacological actions, the octanol/buffer partition coefficients of the Ko compounds investigated were plotted against the respective ICs o'S of the inhibitory potency of the drugs on the excitability of the frog myocardium. As can be seen from fig. 4a, there was a clear realtionship between the two parameters investigated. Regression analysis revealed that the best linear fit of the data (K6 compounds and propranolol) was obtained by the following equation (the numbers in brackets are the standard deviations, S.D.; the Fvalues refer to the statistical significance of the introduced terms; the symbols are * p < 0.05; * * p < 0.01 ; *** p < 0.001): log (1/IC50) = 3.45 + 0.43 (-+ 0.032) log Pobs

(l)

n = 12; s = 0.133; r = 0.973; F = 183 *** A slightly better correlation was obtained by including a quadratic term of the observed partition coefficient:

D. Hellenbrecht et al., Cardiodepressant effects of #-adrenoceptor blocking compounds

log (1/IC50) = 3.50 + 0.40 (+- 0.037) log Pobs - 0.04 (+- 0.029)[1og Pobs] 2

log (I/ICs0(II)) = 3.15 + 0.32 (+- 0.046) log Pobs (2)

n = 12; s = 0.128; r = 0.978; F = 1.83

(3)

n= ll;s=0.128;r=0.958;F=101*** log (1/IC50(I)) = 3.01 + 0.26 (-+ 0.02) log Pobs -

0.08 (-+ 0.016) log [Pobs]2

(5)

n = 1 I; s = 0.188; r = 0.915; F = 46.6 *** log (1/ICs0(II)) = 3.32 + 0.23 (-+ 0.022) log Pobs

The parabolic shape of this regression line implies an optimum P value of biological activity. This P value (termed Po, cf. Tute, 1971; Hansch and Clayton, 1973) obtained from the regression equation, log Po = 5.00, might be interpreted that drugs with partition coefficients up to 100.000 produce suboptimal depression of conduction velocity of the frog heart muscle. In practice, this conclusion is too speculative (the quadratic term was not statistically significant: F = 1.83; p > 0.05) and can only be supported after more extensive investigation with more hydrophobic compounds than those used in the present study. As stated by Hansch and Clayton (1973), there are a variety of reasons for expecting parabolic relationships, among them time dependency of drug action. This phenomenon was actually observed in the present study when the data of the shorter incubation period (10 min, stimulus I) were correlated with hydrophobic properties (cf. fig. 4a). The equations of the linear and of the parabolic regression functions of the K6 compounds were log (I/ICso(I)) = 2.91 + 0.32 (-+ 0.032) log Pobs

231

(4)

n = 11; s = 0.068; r =0.989; F = 23.4 *** The (log Pobs) 2 term of equation (4) was significant (F = 23.4; p < 0.001) and optimum hydrophobicity Po in this case was considerably lower (log Po = 1.73). Therefore it must be assumed that the short incubation period of lO min was not sufficient to establish equilibrium conditions. For the more sensitive system of the data obtained after the stimulus II this limiting factor became even more prominent since the correlation coefficients for the linear and the quadratic regression equations were 0.915 and 0.988, respectively. The quadratic term was highly significant, and Po was much lower (log Po = 1.04) that that of equation (4):

- 0.12 (-+ 0.018)[log Pobs] 2

(6)

n = 11; s = 0.076; r = 0.988; F = 47.4 *** The mechanism of action of the K6 compounds on the depression of the excitability of the frog myocardium is comparable to the local anaesthetic effect of the/3-adrenoceptor blocking drugs on the nerve action potential, as was previously shown for 9 therapeutically used ~adrenoceptor blocking drugs (Hellenbrecht et al., 1973). This means that slowing of conduction of the action potential of the myocardium is a rather non-specific impairment of activation and inactivation of the sodium carrier system. This probable mechanism of action would also explain the increased effective refractory period of isolated rabbit atria treated with the K5 compounds 1439, 1561, 1560, 1313 and 1366, as was observed by Mylecharane and Raper (1971). In the present investigation similar properties of the K/5 compounds may be .substantiated by the observation that the second stimulus applied to the frog heart muscle revealed a 1.4-2.3-fold higher sensitivity of the system (see table 3, ICso(I)/ICso(lI)). This higher sensitivity is probably due to a prolongation of the functional refractory period of the frog heart muscle cells under the non-specific influence of the drugs. Further aspects on the non-specific mechanism of action of ~-adrenoceptor blocking agents came from the investigations of Wiethold et al. (1973). It was shown that a highly significant correlation exists between the physicochemical properties of/3-adrenoceptot blocking drugs (partition coefficient, CHC13/buffer) and their ability to protect red blood cells against hemolysis, i.e. by 'membrane-stabilization' (Seeman, 1972). Furthermore, there was a similar correlation between these two parameters and the ability of the drugs to increase the number of binding sites for the fluorophore l-anilino-8-naphthalene sulfonate (ANS). It was concluded that membrane impairment by adrenoceptor blocking agents can be explained by confofmational changes of biological membranes. A similar non-specific mechanism of action of the 13adrenoceptor blocking agents was assumed for the

232

D. tlellenbrecht et al., Cardiodepressant effects of ~-adrenoceptor blocking compounds

inhibition of serotonin transport by human blood platelets in vitro (Lemmer et al., 1972) and in vivo (Grobecker et al., 1973). Previously, Pratesi et al. (1966) had shown a highly significant correlation between the hydrophobic properties of a series of ringsubstituted /3-adrenoceptor blocking drugs and their non-specific spasmolytic action on the small intestine. Levy (1968), however, suggested from the data obtained from chloroform/water partition coefficients of some/3-adrenoceptor blocking drugs and from their non-specific negative inotropism on isolated rabbit atria, that there was no correlation between these two parameters. Most likely Levy's discrepancies can be explained by the fact that the CHC13/water partition coefficients obtained for Ki5 592 (0.096) and for K~5 1124 (0.099) were far too low. The CHCla/buffer partition coefficients (pH 7.0) found in our laboratory were 1.3 for Ki5 592, and 30.0 for Ki5 1124 (cf. Wiethold et al., 1973). 4.3. Cardiodepressant activity of the ~adrenoceptor blocldng drugs in vivo and their correlation with hydrophobic properties Our experiments are based on the assumption that the non-specific activities of 13-adrenoceptor blocking agents can be studied if all ~3-adrenoceptors are blocked by 'pure' /3-adrenoceptor blocking drugs. It was found that propranolol, 1 mg/kg i.v., was sufficient to block the ~adrenoceptors of the heart completely; during the first minutes after injection of this dose a transient non-specific depression of dp/dtma x was observed in addition to the specific 13-adrenolytic decrease of dp/dt. This dose is comparable with that used by Fitzgerald et al. (1972) in anaesthetized dogs. The correlation of the cardiodepressant effects of the K6 compounds with their respective hydrophobic properties can be seen from the results of the experiments after a fixed dose of the/3-adrenoceptor blocking drugs (fig. 2). Increasing cardiodepression was obtained with drugs of increasing octanol partition coefficients, with a maximum biological activity for Ki5 1124. The cardiodepressant effects were solely dependent on the sum of the hydrophobic substituents of the K8 compounds, independent of the site of the substitution at the amino group (KS 1439 to Ki5 1500) or at the phenyl ring system (K/5 592 to Ki~ 1292). These rather qualitative conclusions were

5[

log ~ 5 0 cclrdiodepression, cot

K61030 K61121, • K61292 K0?07 t ) ~ . ~ _ . ® . /p~but

K* s g L ~ e - - - - - - -Propr

K6= ~ , ~ "

K~143g

3 • -3

i

i

I

i

i

-2

- I

0

1

2 log Po~s~

Fig. 4b. Parabolic correlations of the non-specific phamaco-

logical effects of the #-adrenoceptor blocking drugs with the respective octanol/buffer partition coefficients. Double logarithmic plot of the reciprocal EDso's of the 13-adrenoceptor blocking drugs (K~5compounds, propranolol, and penbutolol) producing non-specific decreases of dp/dtma x in anaesthetized cats after 13-adrenoceptorblockade, ordinate, against the partition coefficients Pobs (octanol/buffer, pH 7.0), abscissa. The indicated parabola refers to equation (9) in the text. Log Po indicates the calculated maximum hydrophobicity of #-adrenoceptor blocking drugs with expected maximum cardiodepressant effects. strongly confirmed by the results obtained with the second group of in vivo experiments. When the values of log 1/EDso of the decreased dp/dtmax (cf. fig. 3) were correlated with the respective octanol/buffer partition coefficients of the K6 compounds and propranolol, the following linear and parabolic equations were calculated (fig. 4b). log ~

1

= 4.84 + 0.40 (-+ 0.039) log Pobs

(7)

n = 12;s =0.161;r =0.955; F = 105 *** 1

= 4.97 + 0.33 (-+ 0.029) log Pobs log -EDs0 - 0.10 (-+ 0.023)[log Pobs] 2

(8)

n=12;s=0.100;r=0.984;F=16.7*** Since addition of the (log Pobs) 2 term to the linear equation in log Pobs was significant at the 0.999 level of significance, an optimum P-value should exist. This Po-value was calculated as log Po = 1.72, indicating that maximum biological activity will be obtained with drugs showing octanol/buffer partition coefficients of about 50 at pH 7.0. It was therefore of

233

D. Hellenbrecht et al., Cardiodepressant effects of #-adrenoceptor blocking compounds

interest to add the data of another very hydrophobic /3-adrenoceptor blocking drug, penbutolol, (-)-l-tbutylamino-3-(cyclopentylphenoxy)-propan-2-ol-HCl, cf. H~trtfelder et al. (1972), to a correlation procedure. The data of penbutolol (Pobs = 39; Zn = 4.12; pK a = 9.65; EDs0 in vivo = 9.4 X 10-6 #moles/ kg) together with the data used in equation (8) revealed the following correlation (cf. fig. 4b):

,3

log (1/ED50) = 4.97 + 0.30 (-+ 0.027) log Pobs - 0.11 (-+ 0.023)[1og Pobs] 2 n=13;s=O.l10;r=0.980;F=24.1

(9) ***

e~ o

Equation (9) does not differ significantly from equation (8). However, the Po-value of equation (9) was somewhat lower (Po = 1.33) than that of equation (8), probably due to the fact that more highly hydrophobic compounds were subject to the statistical procedure. Therefore, it can be assumed that the maximum of the parabola, Po = 1.33, is a better estimation of the maximum hydrophobicity of/3-adrenoceptor blocking drugs producing acute decreases of dp/dtma x under the experimental conditions used in this in vivo study. The data of the correlation equations do not allow consistent conclusions about the nature of the parabolic relationship between the physicochemical and pharmacological parameters, because of the complexity of the parabolic phenomena (Hansch and Clayton, 1973). It seems reasonable to assume, however, that in the living animal pharmacokinetic factors, such as binding to plasm.a proteins, distribution into other body compartments, and metabolism in the liver, are additional determinants of the acute non-specific cardiodepressant effects of the/3-adrenoceptor blocking drugs. The parabolic behaviour of the acute non-specific cardiodepressant effects of the/3-adrenoceptor blocking drugs in vivo suggests that this therapeutically undesirable component of action may be reduced by the election of compounds either with very polar or with highly hydrophobic character.

O

The highly significant correlation between the octanoi/buffer partition coefficients of the K5 com-

+~

~.

I

I

+1

+l

-

v

--

I

'F~ I

~

o

d

~

V

E +1

x-,

+1

+l

+l

~

+{

o

o

~

V

•~

g

0

0

~ II

0

II

II

II

II

II

II

II

eL

,~

,,~

4.4. Prediction o f the non-specific effects o f the ~adrenoceptor blocking drugs

+I

~

0

~.

0

0

0

0

8.~ ~ ' ~ ' ~

0

~s 0

""

= e~

e-

~*

*

e-

=o_

¢-

=o

'~

_

"d

"d

234

D. Hellenbrecht et al., Cardiodepressant effects of a-adrenoceptor blocking compounds

pounds determined experimentally, and the values obtained by mere calculation, log P~:alc= 0.1 1 + 0.90 (-+ 0.042) log Pobs n=ll;s=0.173;r=0.989;F=441

Dr. Kfppe, Boehringer Sohn, lngelheim, for providing the K6 compounds. We also thank Prof. Fitzgerald, I.C.I., for gifts of propranolol, and Prof. Vogel, Hoechst, for gifts of penbutoIol.

***

implies that the hydrophobic properties o f the drugs investigated can sufficiently well be predicted by using the additive principle of Hansch and coworkers. It was therefore o f interest to correlate these calculated P-values, P'calc, with the pharmacological data obtained in vitro and in vivo. Table 4 shows the resuits o f these correlation equations (equation ( 1 ' ) refers to equation (1) o f the text, etc.): It is evident t that the terms o f these equations, using P calc, are very close to the corresponding terms obtained with the measured partition coefficients. Interestingly, the addition of the quadratic term (log Pcalc) ' 2 to the linear correlation equations is significant for each set of results obtained from the different experimental conditions investigated. Furthermore, the optimum P-values, Po, o f the equations (2') and (6') are even somewhat lower than the corresponding values o f the equations (2) and (6), respectively. From the experimental results obtained in this study and from the data obtained by the Hansch analysis it is concluded that the following predictions can be made for the non-specific cardiodepressant effects of /3-adrenoceptor blocking drugs: (1) Non-specific cardiodepression increases parabolically with increasing hydrophobicity o f the drug molecule. The apex of the parabola, i.e. the hydrophobicity of the compound with maximum biological activity, is characteristic for the given pharmacological system. (2) Nonspecific cardiodepression is solely dependent on the sum o f the hydrophobic substituents of the drug molecule, irrespectively of the site of the substituents (aromatic ring system and/or amino group). (3) Nonspecific cardiodepressant activity of a/3-adrenoceptor blocking drug in a given pharmacological system can be predicted by determination or by calculation of the hydrophobic property o f the drug molecule.

Acknowledgements For technical advice in measuring dp/dt we are indebted to Prof. E. Heeg, Braunschweig. We thank Dr. Engelhardt and

References Davis, S.S., 1973, Use of substituent constants in structure activity relations and the importance of the choice of standard state, J. Pharm. Pharmacol. 25,293. Fitzgerald, J.D., Janet L. Wale and M. Austin, 1972, The hemodynamic effects of (±)-propranolol, dextroproprano1ol, oxprenolol, practolol and solatol in anaesthetised dogs, European J. Pharmacol. 17, 123. Grobecker, H., B. Lemmer, D. Hellenbrecht and G. Wiethold, 1973, Inhibition by antiarrhythmics and a-sympatholytic drugs of serotonin uptake by human platelets: Experiments in vitro and in vivo, European J. Clin. Pharmacol. 5, 145. H/irtfelder, G., H. Lessenich und K. Schmitt, 1972, Penbuto1ol (Hoe 893 d), ein neues, stark wirksames a-Sympatholyticum mit langer Wirkungsdauer, Arzneim. Forsch. (Drug Res.) 22,930. Hansch, C. and T. Fujita, 1964, p - o - n analysis. A method for the correlation of biological activity and chemical structure, J. Amer. Chem. Soc. 86, 1616. Hansch, C. and W.J. Dunn, 1972, Linear relationships between lipophilic character and biological activity of drugs, J. Pharm. Sci. 61, 1. Hansch, C. and J.M. Clayton, 1973, Lipophilic character and biological activity of drugs. II: The parabolic case, J. Pharm. Sci. 61, 1. Heeg, E., 1967, Untersuchungen iiber den Einfluss herzwirksamer Pharmaka auf den Druckablauf in der linken Herzkammer der Katze, Habilitationsschrift, Diisseldorf. Hellenbrecht, D., B. Lemmer, G. Wiethold and H. Grobecker, 1973, Measurement of hydrophobicity, surface activity, local anaesthesia, and myocardial conduction velocity as quantitative parameters of the non-specific membrane affinity of nine a-adrenergic blocking agents, NaunynSchmiedeb. Arch. Pharmacol. 277, 211. Lemmer, B., G. Wiethold, D. Hellenbrecht, l.J. Bak and H. Grobecker, 1972, Human blood platelets as cellular models for investigation of membrane active drugs: #adrenergic blocking agents, Naunyn-Schmiedeb. Arch. Pharmacol. 275,299. Leo, A., C. Hansch and D. Elkins, 1971, Partition coefficients and their uses, Chem. Rev. 71,525. Levy, J.V., 1968, Myocardial and local anesthetic actions of a-adrenergic receptor blocking drugs: Relationship to physicochemieal properties, European J. Pharmacol. 2, 250. Mylecharane, E.J. and C. Raper, 1971, a-Receptor blocking and cardiodepressant actions of 2-nitrilophenoxypropanolamines, European J. Pharmacol. 16, 14.

D. Hellenbrecht et al., Cardiodepressant effects of ~-adrenoceptor blocking compounds Pratesi, P. and E. Grana, 1965, Structure and activity at adrenergic receptors of cate'cholamines and certain related compounds, Advan. Drug. Res. 2, 127. Pratesi, P., E. Grana and L. Villa, 1966, Molecular properties and biological activity of catecholamines and certain related compounds, Proc. Intern. Pharmacol. Meet. 3rd, Vol. 7, ed. E.J. Ari(~ns (Pergamon Press, Oxford) p. 283. Seeman, P., 1972, The membrane actions of anesthetics and tranquilizers, Pharmacol. Rev. 24,583.

235

Tute, M.S., 1971, Principles and practice of Hansch analysis: A guide to structure activity correlation for the medicinal chemist, Advan. Drug Res., 6, 1. Wiethold, G., D. Hellenbrecht, B. Lemmer and D. Palm, 1973, Membrane effects of #-adrenergic blocking agents: investigations with the fluorescence probe l-anilino-8naphtalene sulfonate (ANS) and antihemolytic activities, Biochem. Pharmacol. 22, 1437.