Synthesis and adrenolytic activity of 1-(1H-indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethyl]amino}propan-2-ol and its enantiomers. Part 1

Article abstract of DOI:10.1016/j.ejmech.2008.05.019

The synthesis of (2RS)-1-(1H-indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethyl]amino}propan-2-ol ((RS)-9) and its enantiomers has been described and tested for electrocardiographic, antiarrhythmic, hypotensive and spasmolytic activities as well as for α₁-, α₂- and β₁-adrenoceptors' binding affinities. All compounds significantly decrease systolic and diastolic blood pressure, and possess antiarrhythmic activity and affinity to α₁-, α₂- and β₁-adrenoceptors. The results suggest that the antiarrhythmic and hypotensive effects of these compounds are related to their adrenolytic but not spasmolytic properties.

Full text of DOI:10.1016/j.ejmech.2008.05.019

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 809e817
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and adrenolytic activity of 1-(1H-indol-4-yloxy)-3-{[2-(2-
methoxyphenoxy)ethyl]amino}propan-2-ol and its enantiomers. Part 1
a,
b
c
*
b,**
_
Grazyna Groszek , Marek Bednarski , Ma1gorzata Dyba1a , Barbara Filipek
a
´
Faculty of Chemistry, Rzeszo´w University of Technology, 6 Powstanco´w Warszawy Avenue, 35-959 Rzeszo´w, Poland
b Laboratory of Pharmacological Screening, Jagiellonian University Medical College, 9 Medyczna, 30-689 Krako´w, Poland
c Department of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna, 30-689 Krako´w, Poland
Received 18 February 2008; received in revised form 21 May 2008; accepted 22 May 2008
Available online 2 July 2008
Abstract
The synthesis of (2RS )-1-(1H-indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethyl]amino}propan-2-ol ((RS )-9) and its enantiomers has been de-
scribed and tested for electrocardiographic, antiarrhythmic, hypotensive and spasmolytic activities as well as for a1-, a2- and b1-adrenoceptors’
binding affinities. All compounds significantly decrease systolic and diastolic blood pressure, and possess antiarrhythmic activity and affinity to
a1-, a2- and b1-adrenoceptors. The results suggest that the antiarrhythmic and hypotensive effects of these compounds are related to their adre-
nolytic but not spasmolytic properties.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: a1-, a2-, b1-Adrenoceptor antagonist; Antiarrhythmic, hypotensive and spasmolytic activities; Synthesis
1. Introduction
propan-2-ol] [2] (Fig. 1) is a selective a1- and non-selective
b-adrenoceptor antagonist, which decreases peripheral vascular
resistance, systolic and diastolic blood pressure, moderately
slows down the heart rhythm but does not have a significant
effect on cardiac output. Carvedilol possesses antioxidant and
antiproliferative effects on vascular smooth muscle, decreases
insulin-resistance without influence on lipid and glucose metab-
olism and improves endothelial function [3e6]. Hence it is
favourable for carvedilol, hemodynamic and metabolic profiles
and great efficacy in treatment of hypertension, and heart
failure encouraged us to look for new drugs with similar proper-
ties and mechanism of action. As a result of research we
designed and obtained a new compound of chemical name 1-
(1H-indol-4-yloxy)-3-{2-(2-methoxyphenoxy)ethyl]amino}
propan-2-ol, (RS )-9 [7]. This compound has structure fragments
of carvedilol and pindolol’s structures that are linked by amino-
propanol moiety. Most of the clinically available b-adrenergic
blocking agents possess in their structures aminopropanol moi-
ety A (Fig. 2).
Stimulation of b-adrenoceptors in brain and periphery
promotes the release of neurotransmitters which increase sym-
pathetic nervous system activity. Stimulation of b1-adrenocep-
tors in the sinoatrial node and in the myocardium increases
heart rate and force of contraction. Stimulation of b-adrenocep-
tors in the kidney promotes renin release and the activity in-
crease of renineangiotensinealdosterone system. The overall
effect of stimulation of these receptors increases cardiac output,
peripheral vascular resistance and causes an increase in aldoste-
rone-mediated sodium and water retention [1].
Treatment with b-adrenergic blocking agents antagonizes all
theseeffects resultinginbloodpressurereductionandcardiacout-
put decrease. Some b-blockers, such as labetalol and carvedilol
also block effects of peripheral a-adrenoceptors. Carvedilol: [1-
(9H-carbazol-4-yloxy)-3-{[2-(2-methoxyphenoxy)ethyl]amino}
* Corresponding author. Tel.: þ48 17 8651751; fax: þ48 17 8543655.
** Corresponding author. Tel.: þ48 12 6588224; fax: þ48 12 6570262.
E-mail addresses: ggroszek@prz.edu.pl (G. Groszek), mffilipe@cyf-kr.
edu.pl (B. Filipek).
This paper reports the synthesis and results of preliminary
pharmacological testing of a new analogue of carvedilol and
its enantiomers. The newly synthesized compounds were tested
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.019

810
G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
O
*
O
*
O
N
H
N
H
O
OH
OH
N
H
N
H
Carvedilol
Pindolol
Fig. 1. Chemical structure of carvedilol and pindolol.
for electrocardiographic, antiarrhythmic and hypotensive activ-
ities as well as for a1-, a2- and b1-adrenoceptors’ binding
affinities.
3. Pharmacological results
3.1. Radioligand receptor binding assay for a- and
b-adrenergic receptors
The affinity of compound (RS )-9 and its enantiomers to the
catecholamines’ binding side of a1-, a2- and b1-adrenoceptors
was measured as a rate of specific displacement of [3H]prazo-
sin, [3H]clonidine and [3H]CGP12177 at the concentration of
0.2, 2 and 0.2 nM, respectively. Compound (RS )-9 and their
enantiomers inhibited [3H]prazosin binding with Ki ranging
from 56.7 to 89.8 nM, [3H]clonidine binding with Ki ranging
from 365.5 to 1400 nM and [3H]CGP12177 binding with Ki
ranging from 2.0 to 143 nM to cortical a1-, a2- and b1-adreno-
ceptors, respectively. The shape of curves suggests competi-
tive binding. The results are given in Table 1.
2. Chemistry
The synthesis of target compound is outlined in Figs. 3 and 4.
The synthesis of compound (RS )-9 was achieved with the
use of commercially available cheap substrate, guaiacol 1
and 3-nitrophenol 4. The substrate 1 was transformed into bro-
moderivative 2 by method described elsewhere [8]. After that,
appropriate primary amine 3 was obtained through known Ga-
briel synthesis [9]. The amine 3 is sensitive to air and that is
why it should be stored under nitrogen or argon. For the syn-
thesis of indole derivative 7 a method described by Ma˛kosza
et al. was used [10]. The derivative 7 was obtained by applying
vicarious nucleophilic substitution (VNS) of hydrogen to aro-
matic nitro derivative 5. Then compound 6 was subjected to
reductive 1,2-elimination and compound 7 was prepared.
Deprotection of hydroxyl group took place in that condition.
Phenol derivative 7 was converted to epoxide 8 using proce-
dure described by Tejani-Butt and Brunswick [11]. The ring-
opening addition reaction of epoxide 8 to amine 3 was per-
formed according to our original procedure and the free base
of compound (RS )-9 as a racemic mixture was afforded.
Also, enantiomers of compound (RS )-9 were obtained. The
R(ꢀ) or S(þ)-epichlorohydrin was used to obtain enantiomers
of epoxide 8 in procedure similar to described for racemic
mixture. Then, enantiomers of epoxide 8 in addition reaction
to amine 3 were converted to enantiomer S or R of compound
(RS )-9. Configuration was established according to the Cahne
IngoldePrelog convention [12]. Finally, three compounds:
racemic mixture (RS )-9 and its enantiomers (R)-9 and (S )-9
were obtained.
3.2. Effect on normal electrocardiogram (ECG)
in vivo in rats
Effects on ECG intervals and heart rate were determined for
all compounds in the same dose of 1 mg kgꢀ1. Only compound
(S )-9 administered iv decreased heart rate by 5.6e9.6% in 1, 5
and 15 min after administration with no significant influence
on PeQ, QeT intervals and QRS complex (Table 2). Com-
pounds (RS )-9 and (R)-9 did not significantly affect the nor-
mal ECG in anesthetized rats.
3.3. Effect on adrenaline-induced arrhythmia in rats
In anesthetized rats, iv injections of adrenaline (20 mg kgꢀ1
)
caused sinus bradycardia (100%), atrioventricular distur-
bances, ventricular and supraventricular extrasystoles (100%)
which led to death of approximately 50% of animals. The
tested compounds administered 15 min prior to adrenaline in-
jection decreased the number of premature ventricular and
supraventricular beats and reduced mortality.
The ED50 values (a dose producing a 50% inhibition of pre-
mature ventricular beats) and therapeutic indexes of prophy-
lactic antiarrhythmic activity in the adrenaline-induced
arrhythmia are listed in Table 3. These compounds adminis-
tered 15 min before adrenaline prevented and/or reduced in
a statistically significant manner the number of premature ven-
tricular beats. Compound (RS )-9 and its enantiomers exhibited
R2
R1
*
N
H
O
OH
Fig. 2. A moiety.

G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
811
Br
NH2
OH
O
O
O
O
O
(a)
(b),(c)
1
2
3
Fig. 3. (a) 1,2-Dibromoethane, NaOH; (b) potassium phthalimide, DMF; (c) NH2eNH2$H2O.
important antiarrhythmic effects with ED50 values ranging
between 0.059 and 0.43 mg kgꢀ1 and with therapeutic indexes
ranging 47e613.
period and diastolic blood pressure (8e16%) only in the first
few minutes after administration. The compound (RS )-9 after
iv administration significantly decreased systolic and diastolic
blood pressure (10e32%) in the range of doses 0.25e
1 mg kgꢀ1. Compound (R)-9 after iv administration showed
hypotensive activity only in dose of 1 mg kgꢀ1 (Fig. 5).
After po administration all tested compounds maintained
their hypotensive activity (Fig. 6). (RS )-9 and (S )-9 decreased
systolic and diastolic blood pressure in doses ranging between
0.125e1 and 0.062 mg kgꢀ1, respectively. Compound (R)-9
did not show hypotensive activity after po administration.
All tested compounds retained antiarrhythmic activity after
po administration. The range of their ED50 values was 0.44e
1.28 mg kgꢀ1
.
3.4. Influence on blood pressure in rats
Hypotensive activity of compound (RS )-9 and both enan-
tiomers was determined after iv and po administrations in
normotensive anesthetized rats. After iv administration, the
highest hypotensive activity was observed for compound
(S )-9. It significantly decreased the systolic (15e34%) and di-
astolic (14e40%) blood pressure throughout the whole obser-
vation period in the range of doses 0.125e1 mg kgꢀ1. In the
lowest dose (0.0625 mg kgꢀ1), compound (S )-9 decreased sys-
tolic blood pressure (8e13%) throughout whole observation
3.5. Influence on blood pressor response in rats
To examine the mechanism of the hypotensive effects of
these compounds, we studied their influence on the pressor
responses to epinephrine, norepinephrine and methoxamine
(Figs. 7e9). All tested compounds, given in the dose of
OH
OBn
OBn
(a)
CN
(b)
NO2
NO2
NO2
6
4
5
OH
O
O
(c)
(d)
N
H
N
H
7
8
8
10
O
*
11
N
H
O
9
12
13
O
14
4
3
18
19
4a
OH
amine 3
5
6
2
15
(e)
17
N
7a
16
H
7
(RS)-9
Fig. 4. Synthetic pathway: (a) benzyl chloride, K2CO3, CH3CN, KI; (b) t-BuOK, p-ClePheOCH2CN, DMF, ꢀ20 ꢁC; (c) H2, Pd/C, EtOH/AcOH; (d) (ꢂ)-epichlo-
rohydrin, 1 N NaOH aq., dioxane; (e) amine 3, CH3CN.

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G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
Table 1
Affinity for different adrenoceptor types in the rat cerebral cortex
Table 3
Prophylactic antiarrhythmic activity in anesthetized rats
Compound
[3H]Prazosin
[3H]Clonidine
[3H]CGP12177
Compound ED50 iv
(mg kgꢀ1
LD50 iv
(mg kgꢀ1
IT iv ED50 po
(mg kgꢀ1
)
Ki [nM] ꢂ SEM
Ki [nM] ꢂ SEM
Ki [nM] ꢂ SEM
)
)
(RS )-9
(R)-9
(S )-9
Carvedilol
89.8 ꢂ 9.5
88.3 ꢂ 5.8
56.7 ꢂ 2.1
2.2 ꢂ 0.2a
1.4 ꢂ 0.4 mM
467.3 ꢂ 32.3
365.5 ꢂ 28.2
3.4 ꢂ 0.9 mM
3.0 ꢂ 0.6
143 ꢂ 16.5
2 ꢂ 0.08
(RS )-9
(R)-9
(S )-9
0.34 (0.23e0.51) 20.0 (19.2e20.8) 59 0.44 (0.18 ꢂ 1.10)
0.43 (0.28e0.65) 20.4 (16.7e24.5) 47 1.28 (0.56 ꢂ 2.93)
0.059 (0.038e0.093) 36.2 (28.9e45.4) 613 0.61 (0.30 ꢂ 1.27)
0.81 ꢂ 0.06a
Carvedilol 0.25 (0.12e0.53) 25a
100
39 (33.6e45.2)b 37
Propanolol 1.05 (0.64e1.73)
a
Ref. [5].
a
Ref. [13].
Ref. [14].
b
1 mg kgꢀ1, significantly antagonized the pressor response eli-
cited by epinephrine and methoxamine. Compounds (RS )-9,
(S )-9 and carvedilol significantly reduced the pressor response
to norepinephrine (Fig. 10).
according to Litchfield and Wilcoxon [15]. The LD50 values
are given in Table 3. Compounds (RS )-9 and (R)-9 showed
imperceptibly higher toxicity than carvedilol, while (S )-9
showed a little lower toxicity than the reference compound.
3.6. Influence on isolated rabbit ileum
Only compound (RS )-9 gave at the highest dose (10ꢀ5 M)
statistically significant amplitude decrease of contractions of
isolated rabbit small intestine of about 29%. At the lowest con-
centration (10ꢀ9 M), tested compound significantly increased
frequency of contractions of isolated rabbit small intestine of
about 8%. Compound (R)-9 gave at the highest dose (10ꢀ5 M)
statistically significant amplitude decrease of contractions of
isolated rabbit small intestine of about 31%. Compound (S )-
9 had no influence on the frequency and amplitude of contrac-
tions of isolated rabbit small intestine.
4. Discussion
The aim of our study was to evaluate cardiovascular activ-
ity of a new compound e (RS )-9 and its enantiomers which
are similar to carvedilol that is a non-selective b-antagonist
with a1-adrenolytic activity and antioxidant, antiproliferative
and antiendothelin activities [16e19].
This work includes synthesis and pharmacological studies
of 1-(1H-indol-4-yloxy)-3-{2-(2-methoxyphenoxy)ethyl]ami-
no}propan-2-ol, (RS )-9 and its enantiomers. It examines anti-
arrhythmic and hypotensive activities and a- and b-adrenolytic
mechanisms of action. Pharmacological properties of compound
(RS)-9 and its enantiomers were investigated in comparison
with carvedilol.
3.7. Acute toxicity
The acute toxicity of compound (RS )-9 and its two enantio-
mers was obtained in rats after intravenous administration
The method which can initially determine the direction of
action of newly tested compounds is a radioligand binding
assay. The compound (RS )-9 and both enantiomers had high
affinity to adrenergic receptors in rat cerebral cortex. For a-
adrenoceptors (a1 and a2) there were no essential differences
for enantiomers, but the greatest discrepancy was shown in af-
finity to b1-adrenoceptor. (S )-9 enantiomer had 70-fold greater
affinity to b1-adrenoceptors than (R)-9 enantiomer. Unfortu-
nately, none of the enantiomers had larger affinity to adreno-
ceptors than carvedilol. These results gave evidence of
relationship between spatial configuration and affinity to b-adre-
noceptors, and lack of this relationship in the case of a-adreno-
ceptors. The same results were observed for enantiomers of
carvedilol and other b-adrenergic antagonists [20e22].
The antiarrhythmic effects of novel compounds were exam-
ined on rats using model of adrenaline-induced arrhythmia.
The tested compound (RS )-9 and its enantiomers given iv
15 min before arrhythmogen prevented or attenuated the
symptoms of adrenaline-induced arrhythmia. (S )-9 was the
most active compound. Data reported in Table 3 suggest that
all compounds show more beneficial therapeutic indexes
than propanolol but only (S )-9 has better therapeutic index
than carvedilol. All tested forms of compound (RS )-9 kept
its antiarrhythmic activity after po administration but the dif-
ferences were less noticeable. Carvedilol in dose 12 mg kgꢀ1
po and lower did not prevent the adrenaline-induced rhythm
Table 2
Effects of an iv injection of the investigated compounds in dose 1 mg kgꢀ1 on
heart rate and ECG intervals in anesthetized male Wistar rats (60 mg of thio-
pental kgꢀ1 ip)
Compound Parameters Time of observation (min)
0
1
5
15
(RS )-9
(R)-9
PeQ (ms) 45.7 ꢂ 2.0 47.2 ꢂ 1.6 48.1 ꢂ 2.4 46.2 ꢂ 1.7
QRS (ms) 26.4 ꢂ 1.3 28.4 ꢂ 1.2 24.9 ꢂ 1.3 26.9 ꢂ 1.1
QeT (ms) 72.6 ꢂ 1.7 69.2 ꢂ 1.1 69.0 ꢂ 1.6 72.8 ꢂ 1.3
Beats/min 304.9 ꢂ 8.9 298.7 ꢂ 7.2 294.5 ꢂ 7.7 287.3 ꢂ 7.9
PeQ (ms) 48.5 ꢂ 0.9 53.7 ꢂ 1.7 50.7 ꢂ 1.8 51.7 ꢂ 3.1
QRS (ms) 23.0 ꢂ 1.5 22.3 ꢂ 0.6 21.7 ꢂ 0.8 21.7 ꢂ 0.6
QeT (ms) 61.0 ꢂ 3.2 63.0 ꢂ 1.9 66.0 ꢂ 3.2 62.8 ꢂ 1.6
Beats/min 299.3 ꢂ 12.3 307.8 ꢂ 10.8 310.2 ꢂ 8.9 298.0 ꢂ 9.0
PeQ (ms) 45.0 ꢂ 3.5 52.3 ꢂ 3.4 49.7 ꢂ 3.3 48.0 ꢂ 2.9
QRS (ms) 26.0 ꢂ 1.5 24.3 ꢂ 2.2 24.3 ꢂ 2.0 22.7 ꢂ 1.3
QeT (ms) 69.7 ꢂ 3.7 72.3 ꢂ 2.3 76.3 ꢂ 2.3 72.0 ꢂ 2.0
Beats/min 322.0 ꢂ 4.7 297.2 ꢂ 5.4b 291.3 ꢂ 5.3b 304.2 ꢂ 3.6a
(S )-9
Carvedilol PeQ (ms) 50.0 ꢂ 3.2 50.0 ꢂ 3.2 55.0 ꢂ 5.5 54.6 ꢂ 3.0
QRS (ms) 21.2 ꢂ 0.8 22.0 ꢂ 0.6 22.8 ꢂ 1.0 23.2 ꢂ 0.8
QeT (ms) 72.0 ꢂ 3.1 74.4 ꢂ 2.8 68.0 ꢂ 3.7 74.0 ꢂ 2.4
Beats/min 356.7 ꢂ 21.0 345.2 ꢂ 17.7 340.3 ꢂ 14.8 320.4 ꢂ 10.2
Values are the mean ꢂ SEM of six experiments. Statistical analyses were per-
formed using a one-way ANOVA test.
a
p < 0.02.
p < 0.01.
b

G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
813
Time (min)
0
-5
0
1
2
3
5
15
20
30
40
50
60
10
-10
-15
-20
-25
-30
*
*
**
**
***
***
**
***
***
***
***
***
***
***
**
***
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
(RS)-9
(R)-9
(S)-9
Carvedilol
-35
Fig. 5. Changes in mean blood pressure after iv administration of the tested compounds in the dose 0.25 mg kgꢀ1. Statistical analyses were performed using a one-
way ANOVA test: *p < 0.05, **p < 0.02, ***p < 0.01, ****p < 0.001.
disturbances. Antiarrhythmic activity of pure b-adrenergic
blocking agents is known for years. Carvedilol is known to ex-
ert an antiarrhythmic effect too. The electrophysiological
properties of carvedilol include moderate prolongation of po-
tential action duration and effective refractory period. It shows
atrioventricular conduction and reduced dispersion of refracto-
riness. Carvedilol in a concentration range similar to concen-
trations achieved in clinical setting blocks potassium current
such as: IKr, IKur (the rapid and ultra-rapid component of the
delayed rectifier Kþ current) and, in higher concentration, Ito
(the transient outward Kþ current) and L-type Ca2þ current
ICa. Such balanced inhibition of Kþ and Ca2þ channels re-
sulted in a moderate prolongation of action potential duration
with minimal reverse frequency-dependence. This property
would be beneficial in the treatment of ventricular tachyar-
rhythmias in patients with chronic heart failure or post-
myocardial infarction [4,23e25].
Based on our results, we put forward a hypothesis that the
antiarrhythmic effects of the tested compounds are related to
the b1-adrenoceptor blockade in heart tissue and to potassium
and calcium channels’ inhibition.
Hypotensive activity of the investigated compounds was
determined after their iv and po administration to normoten-
sive anesthetized rats. All compounds caused a significant hy-
potensive effect throughout the whole observation period. The
most hypotensive effect was shown by compound (S )-9 which
significantly diminished blood pressure in dose 0.062 mg kgꢀ1
after iv and po administration. In both routes of administration
(iv and po) relationship between spatial configuration and hy-
potensive activity was kept. Carvedilol after iv administration
was the most active one, but after po administration it was less
effective than compound (S )-9.
The hypotensive effect is probably the result of adrenocep-
tors’ blockade (a1- in arteries and b1- in heart). Both
Time (min)
0
0
5
*
10
*
15
20
30
40
50
60
70
80
90
-5
-10
-15
-20
-25
-30
-35
-40
**
*
*
*
***
**
****
***
****
***
***
***
***
****
****
****
****
****
****
****
****
****
****
****
****
****
****
****
(RS)-9
(R)-9
(S)-9
Carvedilol
Fig. 6. Changes in mean blood pressure after po administration of the tested compounds in the dose 0.5 mg kgꢀ1. Statistical analyses were performed using a one-
way ANOVA test: *p < 0.05, **p < 0.02, ***p < 0.01, ****p < 0.001.

814
G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
90
80
70
60
50
40
30
20
10
0
80
70
60
E
NE
MTX
E
NE
MTX
50
40
****
***
*
**** **
30
20
10
0
****
Control
(S)-9
Control
(RS)-9
Fig. 9. Effect of (S )-9 on the blood pressor response to epinephrine, norepi-
nephrine and methoxamine. All values represent the mean ꢂ S.E.M. in 6e8
rats. Statistical analyses were performed using a one-way ANOVA test:
**p < 0.02, ****p < 0.001.
Fig. 7. Effect of (RS )-9 on the blood pressor response to epinephrine, norepi-
nephrine and methoxamine. All values represent the mean ꢂ S.E.M. in 6e8
rats. Statistical analyses were performed using a one-way ANOVA test:
*p < 0.05, ***p < 0.01, ****p < 0.001.
enantiomers and racemic mixture (RS )-9 similarly to carve-
dilol significantly antagonized the pressor responses to epi-
nephrine and methoxamine. Compounds (RS )-9, (S )-9 and
carvedilol also significantly decreased the pressor response eli-
cited by norepinephrine. These experiments with a non-selec-
tive agonist of a1- and a2-adrenoceptors such as epinephrine
or norepinephrine and a selective agonist of a1-adrenoceptor
such as methoxamine suggest that hypotensive effects of the
tested compounds were related to their adrenolytic properties.
It is generally accepted that a1-antagonists inhibit the pressor
responses.
In the experiment on isolated rabbit ileum only compounds
(RS )-9 and (R)-9, in the highest concentrations (10ꢀ5 M), sig-
nificantly decreased the contractions’ amplitude without influ-
ence on the frequency of contractions of isolated rabbit small
intestine. The most hypotensive compound (S )-9 had no influ-
ence on the amplitude and frequency of contractions of
isolated rabbit small intestine. No correlation between spas-
molytic and hypotensive activities confirmed that hypotensive
activity is caused by a-adrenolytic but not spasmolytic
activity.
5. Conclusion
The results confirmed that 1-(1H-indol-4-yloxy)-3-{2-(2-
methoxyphenoxy)ethyl]amino}propan-2-ol and its enantio-
mers possess a1-, a2- and b1-adrenolytic, antiarrhythmic and
hypotensive activities. Research suggests that the antiarrhyth-
mic and hypotensive effects of tested compounds are related to
their adrenolytic properties. Most of the pharmacological ef-
fects of compound (RS )-9 and its enantiomers, especially en-
antiomer (S )-9, were qualitatively similar or weaker to those
of carvedilol.
100
90
90
80
70
80
70
60
*
E
E
NE
MTX
60
50
40
30
20
10
0
50
NE
MTX
40
30
20
10
0
*
***
****
Control
(R)-9
-10
-20
****
Control
Carvedilol
Fig. 8. Effect of (R)-9 on the blood pressor response to epinephrine, norepi-
nephrine and methoxamine. All values represent the mean ꢂ S.E.M. in 6e8
rats. Statistical analyses were performed using a one-way ANOVA test:
*p < 0.05, ****p < 0.001.
Fig. 10. Effect of carvedilol on the blood pressor response to epinephrine,
norepinephrine and methoxamine. All values represent the mean ꢂ S.E.M. in
6e8 rats. Statistical analyses were performed using a one-way ANOVA test:
*p < 0.05, ***p < 0.01, ****p < 0.001.

G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
815
6. Experimental protocol
6.1. Chemistry
6.1.4. 4-(2,3-Epoxypropoxy)-1H-indole (8)
3-Nitrophenol, 4, was converted to benzyl protected deriva-
tive 5 with yield of 96%, and then transformed to intermediate
6 with yield of 53%. M.p. 93e94 ꢁC (EtOH), lit. 92e94 ꢁC
1
6.1.1. General
Melting points were determined on a Boetuis apparatus and
¨
(EtOH) and H NMR spectra for compound 6 were according
to literature [27]. Then, compound 6 was submitted to reductive
cyclization to give 1H-indol-4-ol, 7, with yield of 78%; m.p.
94e97 ꢁC (cyclohexane), lit. m.p. 95e97 ꢁC (cyclohexane)
[28].
1
are uncorrected. H and 13C NMR spectra were recorded on
Varian Gemini (300 MHz) and Bruker (500 MHz) instruments.
Chemical shifts are expressed in ppm (d) referred to TMS, cou-
pling constants (J ) are in Hz. IR and UV spectra were recorded
on Perkin Elmer and Hewlett Packard 8453 instruments, re-
spectively. Mass spectra were obtained on an AMD-604 spec-
trometer. Optical rotations were obtained on a Jasco P-2000
apparatus. TLC plates were silica gel 60 F254 Merck and silica
gel Merck 70e230 mesh was used for column chromatography.
Indole derivative 7 was treated with (ꢂ)-epichlorohydrin in
the presence of stoichiometric quantity of base in dioxane and
water to give the epoxide 8 with yield of 83% as a colourless
solid. Crystallization from ethyl acetate afforded colourless
1
crystals: m.p. 64e66 ꢁC, lit. m.p. 65e67 ꢁC [28]. H NMR
(acetone-d6) d: 2.75 (2d, 1H, J ¼ 2.85 and 5.19, CH2O); 2.84
(2d, 1H, J ¼ 4.26 and 5.19, CH2O); 3.35e3.40 (m, 1H, CHO);
3.99 (2d, 1H, J ¼ 6.15 and 11.34, OCH2CH); 4.40 (2d, 1H,
J ¼ 2.85 and 11.34, OCH2CH); 6.51 (2d, 1H, J ¼ 0.93 and
7.56, CHCHN); 6.54e6.56 (m, 1H, CHCHN); 6.96e7.06 (m,
2H, ArH ); 7.20e7.22 (m, 1H, ArH ); 10.22 (br s, 1H, NH ).
6.1.2. Materials
Guaiacol, 1,2-dibromoethane, (ꢂ)-1-chloro-2,3-epoxypro-
pane, benzyl chloride, and t-BuOK were purchased from
Aldrich Chemicals; >98.5% of S(þ) and R(ꢀ)-1-chloro-2,3-
epoxypropane, potassium phthalimide and 10% Pd/C were
obtained from Fluka Chemicals. 3-Nitrophenol was submitted
by POCH Chemicals.
6.1.5. (2RS )-1-(1H-Indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)
ethyl]amino}propan-2-ol, ((RS )-9)
(4-Chlorophenoxy)acetonitrile was obtained according to
literature [26].
Solvents were distilled and dried if required, and other
materials were commercial.
Reference compound carvedilol was submitted by Pharma-
ceutical Research Institute, Warsaw, Poland.
The epoxide 8 (0.945 g, 5 mmol) was dissolved in acetoni-
trile (5 cm3). After that amine 3 (2.65 g, 15.8 mmol) was
added and the reaction mixture was heated at 80 ꢁC tempera-
ture. The progress of the reaction was monitored by TLC (tol-
uene/methanol 4:1). After 7 h reaction was completed,
salicylic aldehyde (3 cm3) was added and the reaction mixture
was stirred for 10 min at room temperature. Then, the reaction
mixture was subjected to column chromatography on silica gel
(50 g, eluent toluene/methanol 99.5:0.5e97:3). Compound
(RS )-9 was obtained as a solid (1.03 g, 58%). Crystallization
from dichloromethane afforded fine colourless crystals, m.p.
6.1.3. 2-(2-Methoxyphenoxy)ethylamine (3)
Guaiacol 1 (44.25 cm3; 0.4 mol), water (145 cm3), 1,2-dibro-
moethane (69 cm3; 0.8 mol) and NaOH aq. (20%, 70 cm3;
0.48 mol) were heated together for 48 h at 80 ꢁC temperature.
Organic phase was separated; aqueous phase was shaken with
methylene chloride (2 ꢃ 150 cm3). The combined organic phase
was washed with 10% NaOH aq. and water and then solvent was
evaporated. Oily residue was chromatographed on silica gel
using hexane/ethyl acetate mixture as an eluent. Product 2 was
obtained as colourless crystals; 69.8 g (75% yield), m.p. 39e
40 ꢁC. Lit. m.p. 38e41 ꢁC [27].
1
68e70 ꢁC. H NMR (500 Hz, CDCl3) d: 2.53 (br s, 2H, OH,
NH ); 2.90e3.00 (m, 2H, H-10); 3.06e3.09 (m, 2H, H-11),
3.82 (s, 3H, H-19), 4.12e4.18 (m, 5H, H-8, 9 and 12), 6.52
(br d, 1H, J ¼ 7.70, H-3); 6.62e6.63 (m, 1H, H-2); 6.86e
6.88 and 6.90e6.92 (2m, 7H, ArH ), 8.17 (br s, 1H, NH ).
13C NMR (125 Hz, CDCl3) d: 45.5 (C-11), 48.6 (C-10), 52.6
(C-19), 65.3, 65.9, 67.4 (C-8, 9 and 12), 96.7 (C-2), 97.9
(C-3), 101.5 (C-7), 109.1 (C-15), 111.8 (C-18), 115.7 (C-4a),
117.7, 118.6, 119.3, 119.4 (C-5, 6, 16 and 17), 134.2 (C-7a),
145.2 (C-14), 146.9 (C-13), 149.2 (C-4). IR (KBr, cmꢀ1) n:
744.2 (Ar), 1125 (ether, OCH2), 1252.5 (ether, OeAr),
1505.6 and 1589.1 (C]C, Ar), 3005.5 and 3369.8 (NH,
OH). HRMS (ESI) m/z for C20H25N2O4 (M þ H)þ: found
357.1816, calcd 357.1809 UV (96%, EtOH) (nm) lmax: 217
(3 ¼ 27 000); 266 (3 ¼ 11 450), (c ¼ 0.4 mg/10 cm3).
The mixture of bromide 2 (5 g; 0.0216 mol), potassium
phthalimide (4.26 g; 0.023 mol), 18-crown-6 ether (0.15 g;
6.25 ꢃ 10ꢀ4 mol) in DMF (20 cm3) was heated for 5 h at
50 ꢁC temperature. The reaction mixture was poured into water
(170 cm3), stirred for 2 h, precipitate was filtered off, washed
with water and dried on air afforded N-[2-(2-methoxyphenox-
y)ethylo]phthalimide, 5.69 g (88% yield); m.p. 102e108 ꢁC,
lit. m.p. 102e104.5 ꢁC [27].
A mixture of phthalimide derivative (1 g; 3.37 mmol), wa-
ter (10 cm3) and hydrazine hydrate (100%, 1.5 cm3; 0.03 mol)
was stirred for 5 h at room temperature. Product was extracted
with chloroform (2 ꢃ 20 cm3), dried over potassium carbonate
and evaporated to leave colourless solid pure amine 3, 0.48 g
(86% yield), meltable at room temperature. 1H NMR (CDCl3)
d: 1.72 (s, 2H, NH2); 3.01 (t, 2H, J ¼ 5.3, CH2eNH2); 3.76 (s,
3H, OCH3); 3.96 (t, 2H, J ¼ 5.3, O-CH2); 6.84 (s, 4H, ArH ).
6.1.6. (2S )-1-(1H-Indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)
ethyl]amino}propan-2-ol, ((S )-9)
(S )-(þ)-4-(2,3-Epoxypropoxy)-1H-indole was obtained ac-
cording to procedure described in Section 6.1.4 using R(ꢀ)-1-
chloro-2,3-epoxypropane instead their raceme mixture, with
yield of 75% as colourless solid after solvent evaporation:
m.p. 81e83 ꢁC and [a]D24 ¼ þ10.6ꢁ (c ¼ 1.0 in CHCl3).

816
G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
Then, optical active indole derivative was submitted to addi-
tion reaction with amine 3 according to procedure described
for compound (RS )-9, Section 6.1.5, and gave product (S )-9
as a foam solid with yield of 50%, [a]2D3 ¼ ꢀ6.3ꢁ (c ¼ 1.1 in
CHCl3). Very hygroscopic and must be stored in argon
atmosphere.
6.2.4. Adrenoceptor radioligand binding assay
The experiment was carried out on rat cerebral cortex.
[3H]Prazosin (19.5 Ci mmolꢀ1, an a1-adrenergic receptor),
[3H]clonidine (70.5 Ci mmolꢀ1, an a2-adrenergic receptor)
and [3H]CGP12177 (48 Ci mmolꢀ1, a b1-adrenergic receptor)
were used. The brains were homogenised in 20 volumes of an
ice-cold 50 mM Tris/HCl buffer (pH 7.6) and were centrifuged
at 20 000g for 20 min (0e4 ꢁC). The cell pellet was resus-
pended in the Tris/HCl buffer and centrifuged again. Radioli-
gand binding assays were performed in plates (MultiScreen/
Millipore). The final incubation mixture (final volume
300 ml) consisted of 240 ml of the membrane suspension,
30 ml of [3H]prazosin (0.2 nM), [3H]clonidine (2 nM) or
[3H]CGP12177 (0.2 nM) solution and 30 ml of the buffer con-
taining seven to eight concentrations (10ꢀ11 to 10ꢀ4 M) of
tested compounds. For measuring the unspecific binding,
phentolamine, 10 mM (in the case of [3H]prazosin), clonidine,
10 mM (in the case of [3H]clonidine) and propanolol e 1 mM
(in the case of [3H]CGP12177) were applied. The incubation
was terminated by rapid filtration over glass fibre filters
(Whatman GF/C) using a vacuum manifold (Millipore). The
filters were then washed twice with the assay buffer and placed
in scintillation vials with a liquid scintillation cocktail. Radio-
activity was measured in a WALLAC 1409 DSA liquid scintil-
lation counter. All assays were made in duplicate. The
radioligand binding data were analysed using an interactive
curve-fitting routine (GraphPAD/Prism,Version 3.0 e San
Diego, CA, USA). Ki values were calculated from the Cheng
and Prusoff equation [29].
6.1.7. (2R)-1-(1H-Indol-4-yloxy)-3-{[2-(2-methoxyphenoxy)
ethyl]amino}propan-2-ol, ((R)-9)
(R)-(ꢀ)-4-(2,3-Epoxypropoxy)-1H-indole was obtained as
described above, using S(þ)-1-chloro-2,3-epoxypropane,
with yield of 69% as colourless solid after solvent evaporation:
m.p. 83e84 ꢁC and [a]D24 ¼ ꢀ9.8ꢁ (c ¼ 1.0 in CHCl3).
The product (R)-9 was obtained as a foam solid with yield
of 45%, [a]2D3 ¼ þ4.7ꢁ (c ¼ 1.2 in CHCl3). Very hygroscopic
and must be stored in argon atmosphere.
6.1.8. Enantiomeric purity of (R)-9 and (S )-9
Enantiomeric purity of (R)-9 and (S )-9 was assessed by
chiral HPLC, column of Chiracel OD-RH (4.6 ꢃ 150 mm,
5 mm, Daicel Chemical Industries, Tokyo) with isocratic elu-
tion using a mobile phase of acetonitrile/water (35:65) with
0.01% formic acid at flow rate of 600 ml minꢀ1. Detection
was achieved by an Applied Biosystems AMS Sciex (Con-
cord, Ontario) API 2000 triple quadrupole mass spectrometer.
The retention times of (R)-9 and (S )-9 were 8.22 and
12.64 min, respectively. The isomers were well resolved and
the ratio of the peak areas was used to estimate enantiomeric
purity. (R)-9 and (S )-9 were consistently assayed at >98.5%
enantiomeric purity.
6.2.5. Effect on normal electrocardiogram (ECG)
Electrocardiographic measurement was carried out using
the Ascard B5 Eco apparatus, standard lead II, and paper
speed was 50 mm sꢀ1. The tested compounds were adminis-
tered intravenously in dose of 1 mg kgꢀ1. The ECG recording
was carried out immediately before and 1, 5 and 15 min after
administration of the tested compounds. The effect of the com-
pounds on rat ECG recording was calculated according to
Cambridge et al. [30].
6.2. Pharmacology
6.2.1. Animals
The experiment was carried out on male Wistar rats (180e
250 g) and male rabbits (2.5e3 kg). The animals were housed
in constant temperature facilities exposed to 12:12 lightedark
cycle and maintained on a standard pellet diet, and tap water
was given ad libitum. Control and experimental groups con-
sisted of 6e8 animals each. The investigated compounds
were administered intravenously or intragastrically at a con-
stant volume of 1 ml kgꢀ1. Control animals received the equiv-
alent volume of solvent.
All procedures were conducted according to guidelines of
ICLAS (International Council on Laboratory Animals Sci-
ence) and approved by the Local Ethic Committee on Animal
Experimentation.
6.2.6. Prophylactic antiarrhythmic activity in a model of
adrenaline-induced arrhythmia according to Szekeres and
Papp [31]
Arrhythmia was evoked in thiopental (60 mg kgꢀ1, ip)
anaesthetized rats by iv injection of adrenaline (20 mg kgꢀ1).
The tested compounds were administered intravenously
15 min or orally 60 min before adrenaline. The criterion of an-
tiarrhythmic activity was the lack of premature beats and the
inhibition of rhythm disturbances in comparison with control
group (ventricular bradycardia, atrioventricular block, ventric-
ular tachycardia or ventricular fibrillation). The cardiac
rhythm disturbances were recorded for 15 min after adrenaline
injection. ECGs were analysed according to the guidelines of
the Lambeth Convention [32] on ventricular premature beats
(VBs), bigeminy, salvos (less than four successive VBs), ven-
tricular tachycardia (VT, four or more successive VBs) and
ventricular fibrillation (VF).
6.2.2. Reference compound
Carvedilol was used as a reference drug.
6.2.3. Statistical analysis
Data are expressed as the mean ꢂ S.E.M. The statistical
significance was calculated using one-way ANOVA. Differ-
ences were considered significant when p < 0.05.

G. Groszek et al. / European Journal of Medicinal Chemistry 44 (2009) 809e817
817
[3] M. Hirohashi, K. Takasuna, K. Takamura, K. Yamaguchi, K. Moekawa,
M. Yamada, S. Iwasaki, M. Yoshida, M. Nomura, K. Taguchi, Y. Suzuki,
Drug Res. 40 (7) (1990) 735e746.
6.2.7. Influence on blood pressure in rats
Male Wistar normotensive rats were anaesthetized with
thiopental (50e75 mg kgꢀ1, ip). The right carotid artery was
cannulated with a polyethylene tube filled with heparin in sa-
line to facilitate pressure measurement using the Datamax ap-
paratus (Columbus Instruments). The studied compounds were
injected in a single dose of 1e0.003 mg kgꢀ1 into the cadual
vein or given per os in a single dose 1e0.03 mg kgꢀ1 after
[4] C. Karle, V. Kreye, W. Zhang, D. Thomas, K. RTcki, S. KathTfer,
J. Kiehn, Cardiovasc. Res. 49 (2001) 361e370.
[5] K. Ponicke, I. Heinroth-Hoffmann, O. Brodde, J. Pharmacol. Exp. Ther.
¨
301 (2002) 71e76.
[6] Y. Matsuda, H. Akita, M. Terashima, N. Shiga, K. Kenzawa,
M. Yokoyama, Am. Heart J. 140 (5) (2000) 753e758.
[7] M. Bednarski, B. Filipek, G. Groszek, D. Macia˛g, Patent PL 195413,
2002.
5 min stabilization period at a volume equivalent to 1 ml kgꢀ1
.
[8] C.S. Marvel, A.L. Tanenbaum, J. Am. Chem. Soc. 44 (1922) 2647;
Organic Synthesis, coll. vol. I, John Wiley and Sons, Inc., New York, NY,
1956, p. 435.
6.2.8. Influence on blood pressor response in rats
In separate series of experiments on anesthetized normoten-
sive rats, the effect of studied compounds, given intravenously
in dose of 1 mg kgꢀ1, on a pressor response to epinephrine
(2 mg kgꢀ1), norepinephrine (2 mg kgꢀ1) and methoxamine
(150 mg kgꢀ1) was investigated. Pressor response to norepineph-
rine, norepinephrine and methoxamine injected intravenously
was obtained before and 5 min after the tested compounds (iv).
[9] M.S. Gibson, R.W. Bradshaw, Angew. Chem., Int. Ed. 7 (12) (1968)
919e930.
[10] M. Ma˛kosza, W. Danikiewicz, K. Wojciechowski, Liebigs Ann. Chem.
(1988) 203e208.
[11] S.M. Tejani-Butt, D.J. Brunswick, J. Med. Chem. (1986) 1524e1527.
[12] R.S. Cahn, C.K. Ingold, V. Prelog, Angew. Chem., Int. Ed. Engl. 5 (1966)
385.
[13] ChemIDplus e <http://chem.sis.nlm.nih.gov/chemidplus/chemidlite.jsp>.
´
[14] A. Siemieniuk, H. Sza1kowska-Pa˛gowska, S. Lochynski, K. Pia˛tkowski,
´
B. Filipek, J. Krupinska, R. Czarnecki, T. Librowski, S. Bia1a, Pol. J.
Pharmacol. Pharm. 44 (1992) 557e593.
6.2.9. Influence on isolated rabbit ileum
The influence on isolated rabbit ileum of the investigated
compounds was tested by the method according to Magnus
[33], and modified and described by Orisadipe et al. [34].
White rabbits were sacrificed by cervical dislocation and the
small intestine was immediately removed, and cut into strips
about 3e4 cm long. The isolated strips were incubated in
Krebs solution at 37 ꢁC and aerated with carbogen in special
laboratory dishes. The isolated strip of the intestine was placed
in a test glass tube with the Krebs solution constantly aerated
by carbogen. After 1 h incubation period, during which the
physiological saline solution was changed every 15 min, the
influence of the investigated compounds on spontaneous con-
tractions of the rabbit ileum was evaluated. The contraction of
the intestine was recorded on a TZ-4100 line recorder via iso-
metric Harvard transducer, at the muscle load of 1 g. The in-
fluence of every single dose was recorded for 5 min.
[15] J.T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99e113.
[16] H. Huang, J. Shan, X.H. Pan, H.P. Wang, L.B. Qian, Q. Xia, Diabetes
Res. Clin. Pract. 75 (2007) 7e13.
[17] M. Reiter, Prog. Cardiovasc. Dis. 47 (2004) 11e33.
[18] Y.S. Kim, M.S. Kim, H. Ha, J. Park, H.J. Kim, K. Park, Surg. Today 32
(2002) 230e235.
[19] P.A. Poole-Wilson, K. Swedberg, J.G. Cleland, Lancet 362 (2003) 7e13.
[20] O.E. Brodde, M.C. Michel, Pharmacol. Rev. 51 (1999) 651e689.
[21] B. Dulin, W.T. Abraham, Pharmacol. Carvedilol Am. J. Cardiol. 93
(2004) 3Be6B.
[22] R.R. Ruffolo, M. Gellai, J.P. Hieble, R.N. Willette, A.J. Nichols, Eur. J.
Clin. Pharmacol. 38 (1990) 82e88.
[23] J. Cheng, R. Niwa, K. Kamiya, J. Toyama, I. Kodama, Eur. J. Pharmacol.
376 (1999) 189e201.
[24] B. Deng, X. Yu, S. Kuang, W. Zhang, Z. Zhou, K. Zhang, W. Qian,
Z. Shan, M. Yang, S. Wu, S. Lin, Life Sci. 80 (2007) 665e671.
[25] J. Kikuta, M. Ishii, K. Kishimoto, Y. Kurachi, Eur. J. Pharmacol. 529
(2006) 47e54.
[26] E. Grochowski, Z. Eckstein, Bull. Acad. Pol. Sci. Ser. Sci. Chim. 11
(1963) 443e446.
[27] J. Augstein, W.C. Austin, R.J. Boscott, S.M. Green, C.R. Worthing, J.
Med. Chem. 8 (1965) 356e367.
6.2.10. Acute toxicity
The tested compounds were dissolved in a 0.9% saline and
injected into the caudal vein of rat (1 ml kgꢀ1). Each dose was
given to six animals. LD50 values were calculated according to
the method of Litchfield and Wilcoxon [15] after a 24 h obser-
vation period.
[28] A. Rashidbaigi, A.E. Ruoho, J. Pharm. Sci. 71 (1982) 305.
[29] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099.
[30] G.W. Cambridge, J.F. Parsons, R. Stafford, in: R. Budden,
D.K. Detweiler, G. Zbinden (Eds.), The Rat Electrocardiogram in Phar-
macology and Toxicology, Pergamon Press, Oxford, New York, Toronto,
1981, pp. 135e138.
[31] L. Szekeres, G. Papp, in: J. Schmier, O. Eichler (Eds.), Handbook of
Experimental Pharmacology, Springer-Verlag, Berlin, Heidelberg, New
York, 1975.
Acknowledgements
This project wassupportedbyPolishMinistryofScientificRe-
search and Information Technologies, Grant no. 2 P05F 010 28.
[32] M.J. Walker, M.J. Curtis, D.J. Hearse, R.W. Campbell, M.J. Janse,
D.M. Yellon, S.M. Cobbe, S.J. Coker, J.B. Harness, D.W. Harron,
A.J. Higgins, D.G. Julian, M.J. Lab, A.S. Manning, B.J. Northover,
J.R. Parratt, R.A. Riemersma, E. Riva, D.C. Russel, D.J. Sheridan,
E. Winslow, B. Woodward, Cardiovasc. Res. 22 (1988) 447e455.
References
[33] R. Magnus, Versuche am uberlebenden Dunnalarm von Saugetieren,
¨
¨
¨
[1] B. Gomer, Hosp. Pharm. 14 (2007) 119e125.
[2] F. Wiedemann, W. Kampe, M. Thiel, G. Sponer, E. Roesch, K. Dietmann,
Patent DE 2 815 926, 1978; US Patent 4 503 067, 1985.
Pflugers Arch. 102 (1904) 123e151.
¨
[34] A. Orisadipe, S. Amos, A. Adesomoju, L. Binda, M. Emeje, J. Okogun,
C. Wamebe, K. Gamaniel, Biol. Pharm. Bull. 24 (2001) 364e367.