Synthesis, β-adrenoceptor pharmacology and toxicology of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl) ethylamino)propane hydrochloride, a short acting β1-specific antagonist

Article abstract of DOI:10.1016/S0223-5234(02)01399-5

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl) ethylamino)propane hydrochloride (D140S·HCl 6), a novel short acting β₁-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S·HCl 6 has been compared with esmolol, another short acting agent, and other well known β-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S·HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S·HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S·HCl 6 is a highly specific β₁-adrenoceptor antagonist (pA₂=8.15±0.22, β₁/β₂ selectivity>4400). The in vitro studies suggest D140S·HCl is ca. ten times more potent and 60 times more β₁-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg^-1 h^-1 in the rat and 2.5 mg kg^-1 h^-1 in the dog. Both D140S·HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is <15 min for D140S·HCl and 10 min for esmolol). The toxicological tests indicate that D140S·HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.

Full text of DOI:10.1016/S0223-5234(02)01399-5

European Journal of Medicinal Chemistry 37 (2002) 731Á741
/
www.elsevier.com/locate/ejmech
Original article
Synthesis, b-adrenoceptor pharmacology and toxicology of S-(ꢀ
/
)-1-
(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-
dimethoxyphenyl)ethylamino)propane hydrochloride, a short acting
b1-specific antagonist
Graham P. Jackman, Dimitri Iakovidis, Tracy L. Nero, Nagesh S. Anavekar, Linda
A. Rezmann-Vitti, Simon N.S. Louis, Masanori Mori, Olaf H. Drummer, William
J. Louis *
Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin and Repatriation Medical Centre, The University of Melbourne,
Heidelberg, Vic. 3084, Australia
Received 3 April 2002; received in revised form 7 June 2002; accepted 10 June 2002
Abstract
The synthesis of S-(ꢀ
(D140S×HCl 6), a novel short acting b1-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S×
HCl 6 has been compared with esmolol, another short acting agent, and other well known b-adrenoceptor antagonists in isolated rat
tissue preparations. The pharmacokinetics of D140S×HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous
bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced
heart rate increase and the pharmacodynamic half-life of D140S×HCl 6 has been compared with esmolol in a conscious rat model. In
addition, the results of a range of toxicological studies are presented. The results indicate that D140S×HCl 6 is a highly specific b1-
adrenoceptor antagonist (pA2ꢁ8.1590.22, b1/b2 selectivityꢀ4400). The in vitro studies suggest D140S×HCl is ca. ten times more
potent and 60 times more b1-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day
intravenous infusion at toxicological doses above 10 mg kgꢀ1 hꢀ1 in the rat and 2.5 mg kgꢀ1 hꢀ1 in the dog. Both D140S×
HCl 6
and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is B15 min for
HCl 6 shows no unexpected toxicity and none of
/)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride
/
/
/
/
/
/
/
/
/
/
/
D140S×
/
HCl and 10 min for esmolol). The toxicological tests indicate that D140S×
/
´
the tissue irritancy problems reported for esmolol formulations. # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights
reserved.
Keywords: b1-Adrenoceptor antagonist; Short acting antagonist; D140S×
/
HCl; Synthesis; Pharmacology; Toxicology
1. Introduction
Short acting b-blocking drugs represent a special class
of b-adrenoceptor antagonists suitable for parenteral
use with a short half-life (B
/
30 min) in the circulation
[1Á3]. The short acting agents have an advantage over
/
Abbreviations: Co, maximum plasma concentration at time 0;
D140S×HCl, S-(ꢀ)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-
(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride; Homovera-
trylamine, 3,4-dimethoxyphenethylamine; (9)-LK204-
545, (9)-1-(2-(3-(2-cyano-4-(2-cyclopropylmethoxyethoxy)phenoxy)-
oral agents in clinical situations where a quick on/off
effect is desirable. There is considerable parenteral use
of short acting b-blocking drugs in anesthetic proce-
dures, cardiac surgery and in intensive care to control
/
/
/
/
2-hydroxypropylamino)ethyl)-3-(4-hydroxyphenyl)urea; NOEL, no
effect level; t1/2, Half-life; Vd, volume of distribution.
tachycardia, arrhythmias and hypertension [1,2,4Á
/
6,8Á
/
10]. In addition, there is extensive preclinical and clinical
data supporting the early use of b1-adrenoceptor
* Correspondence and reprints
E-mail address: ajenkins@austin.unimelb.edu.au (W.J. Louis).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 1 3 9 9 - 5

732
G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á741
/
antagonists in acute myocardial infarction [1,4,8Á
/
10].
pharmacodynamic t1/2 of D140S×
/
HCl 6 in a conscious
Early introduction of b-blocker therapy following
myocardial infarction would be facilitated by the avail-
ability of a safe non-irritant, short acting parenteral
agent. There is a paucity of available compounds which
have these properties and are significantly b1-adreno-
ceptor specific with minimal negative hemodynamic
effects. The only marketed short acting b-blocker is
rat model, are also presented.
2. Chemistry
The synthesis of D140S×
/
HCl 6 is relatively straight-
forward and achieved with the use of the appropriate
chiral glycidyl-3-nitrobenzenesulfonate as shown in Fig.
2. The hydroxyl group of commercially available 2-
ethoxyethanol 1 was protected using p-toluenesulfonyl
chloride and the resulting compound was reacted with 4-
benzyloxyphenol to give 1-benzyloxy,4-(2-ethoxy-
ethoxy)benzene 3. The benzyl protecting group was
removed by hydrogenation to give the desired phenol 4.
esmolol and its short pharmacodynamic half-life (t1/2
,
ca. 10 min) can be attributed to the ester group in the
para-substituent of the phenoxypropanolamine (Fig. 1),
which undergoes rapid hydrolysis by esterases to a
metabolite with relatively weak activity [4,7]. Esmolol
formulations, however, are tissue irritants and cause
profound hypotension in up to 50% of reported cases
[1].
During our b1-adrenoceptor antagonist design and
synthesis program, we identified a series of antagonists
specific for the b1-adrenoceptor which had very short
pharmacodynamic t1/2 [11]. One of these compounds, S-
The phenol 4 reacted with 2S-(ꢂ
zenesulfonate to produce the S-(ꢂ
opening addition reaction of the epoxide 5 with homo-
veratrylamine, followed by the conversion of the free
base to the hydrochloride salt gave the final compound
/)-glycidyl-3-nitroben-
/)-epoxide 5. The ring
(ꢀ
/
)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-
D140S×
/
HCl 6. The enantiomeric purity of the final
99%, as assessed by chiral HPLC, and
(3,4-dimethoxyphenyl)ethylamino)propane hydrochlo-
product is ꢀ
/
ride (D140S×HCl 6), is highly b1-adrenoceptor selective,
/
essentially dependent on the enantiomeric purity of the
glycidyl-3-nitrobenzenesulfonate. Under the conditions
described in the Section 7.1.4 there appears to be no
‘pseudoracemisation’, which occurs when the phenoxide
anion attacks the oxirane ring rather than directly
displacing the nitrobenzenesulfonate (this phenomenon
was a serious source of racemisation in our initial
synthesis when using chiral glycidyl toluenesulfonate).
non-irritant, minimally cardio- and vasodepressive and
is short acting, making it suitable for parenteral use.
Since D140S×
/
HCl 6 does not contain an ester moiety
(Fig. 2) its short duration of action cannot be due to
esterase hydrolysis, as in the case of esmolol. Unfortu-
nately we have not yet elucidated the exact mechanism
responsible for the short duration of action of D140S×
/
HCl 6. However, the pharmacokinetic and pharmaco-
dynamic studies that we have carried out so far, and
present here, suggest that non-linear kinetics and by
inference saturation of metabolism only occur at the
higher doses used in the toxicology studies. The synth-
The assigned structure of D140S×
/
HCl 6 was fully
confirmed by MS and H-NMR spectra and elemental
analysis. D140S×HCl 6 is very soluble in water and
1
/
stable to treatment with hot acids and bases without
degradation or racemisation.
esis of D140S×/HCl 6 is presented here, along with the in
vitro antagonist potency of this compound in rat atria
(b1-adrenoceptor) and trachea (b2-adrenoceptor) and a
summary of its toxicology. The pharmacokinetics of
3. Pharmacology
D140S×
/
HCl 6 in rats and dogs, as well as the effect on
the isoprenaline-induced heart rate increase and the
3.1. Isolated tissue preparations
The compounds listed in Table 1 were evaluated for
their ability to antagonise b1- and b2-adrenoceptors in
rat atria and tracheal rings, respectively. Cumulative
concentrationÁresponse curves were obtained in each
/
preparation as described by van Rossum [12] and curves
were fitted by computer analysis according to the
method of Zabrowsky et al. [13] using the sigmoidal fit
function of the ORIGIN graphics package (MICROCAL
ORIGIN, MicroCal Software Inc., One Roundhouse
Plaza, Northampton, MA 01060, USA). The antagonist
potencies, or pA2 values, were calculated and represent
Fig. 1. Structures of (a) esmolol, (b) atenolol, (c) metoprolol, (d)
betaxolol, (e) propranolol and (f) LK 204-545. The asterisks denote a
chiral centre.
the mean9
/
S.E.M. from three to five individual experi-
ments.

G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á
/741
733
Fig. 2. Synthetic pathway for D140S×
NaH, DMF; (f) 2S-(ꢂ)-glycidyl-3-nitrobenzenesulfonate, DMF; (g) homoveratrylamine, dioxane; (h) diethylether, ethereal HCl. Note: the brackets
surrounding intermediates 2, 3 and 5 are to indicate that they were not isolated and characterised during the synthesis of D140S×HCl 6.
/HCl 6. Reagents: (a) p-toluenesulfonyl chloride; (b) NaOH; (c) 4-benzyloxyphenol, NaOH; (d) H2, Pd/C; (e)
/
/
3.2. D140S×
/
HCl (6) pharmacokinetics
3.2.2. Four week intravenous bolus injection study in
conscious rats
Forty eight conscious SpragueÁ
males and six females at each dose level, weighing
195Á233 and 162Á193 g, respectively) received intrave-
nous bolus injection doses of 0, 10.0, 20.0 and 30.0 mg
kgꢀ1 bd D140S×
HCl 6 for 4 weeks. Plasma samples
were taken 10 min after the second dose on day 28. The
HCl 6 after the bolus
Plasma concentrations of D140S×
/
HCl 6 for the
/Dawley rats (six
following pharmacokinetic studies were estimated by
HPLC using the assay described in the Section 7.2.3.
The collection of plasma samples was carried out for us
by the commercial laboratories whilst performing the
toxicological studies (Section 4).
/
/
/
plasma concentrations of D140S×
/
injection were calculated and are given in Table 3.
3.2.1. Seven day continuous intravenous infusion in
conscious rats
Plasma samples were taken from 80 conscious
3.2.3. Seven day continuous intravenous infusion in
conscious dogs
SpragueÁ
each dose level, weighing 210Á
respectively), via indwelling catheters, at the end of
D140S×HCl 6 infusions of 7 days duration. Doses
studied were 0, 5.0, 10.0 and 15.0 mg kgꢀ1 hꢀ1. The
plasma concentrations of D140S×HCl 6 on day 7 of the
/
Dawley rats (ten males and ten female at
Plasma samples were taken from 16 conscious beagle
dogs (two males and two females at each dose level,
/
332 and 170Á255 g,
/
weighing 6.8Á
/
9.8 kg) on days 2 and 7 of the study.
/
D140S×HCl 6 intravenous infusion rates were 0, 2.5, 5.0
/
and 10.0 mg kgꢀ1 hꢀ1, via indwelling catheters. The
high dose was reduced to 7.5 mg kgꢀ1 hꢀ1 on day 4
because of side effects developing in all 4 dogs at the
10.0 mg kgꢀ1 hꢀ1 dose (see Sections 5.2.3 and 5.4). The
/
continuous intravenous infusion were calculated and are
given in Table 2.
Table 1
In vitro potency (pA2) and selectivity of D140S×HCl 6 and reference b-adrenoceptor antagonists in rat atria and trachea
a
b
c
Compound
b1-Adrenoceptor atria (pA2)
b2-Adrenoceptor trachea (pA2)
Selectivity b1/b2
D140S×HCl 6
Esmolol
Atenolol
8.1590.22
7.1490.20
7.3090.12
7.6090.17
8.0690.15
8.5390.08
8.4090.32
B4.5
ꢀ4400
76
25
5.2690.16
5.9190.30
6.4390.13
6.3890.13
4.7390.17
8.1390.27
Metoprolol
Betaxolol
15
48
LK 204-545
Propranolol
6300
2
a
Antagonist pA2 value9S.E.M. determined in isolated spontaneously beating atria.
b
c
Antagonist pA2 value9S.E.M. determined in tracheal chain, previously contracted with 1.0 mM carbachol.
Antilog (pA2 atria-pA2 trachea). Nꢁ3Á5 individual experiments.
/

734
G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á741
/
Table 2
Plasma concentrations of D140S×HCl 6 at the end of 7 days of continuous intravenous infusion in conscious rats
Infusion rate of D140S×HCl 6
Plasma concentration of D140S×HCl9S.E.M.
Plasma concentration of D140S×HCl9S.E.M.
a
a
(mg kgꢀ1 hꢀ1
)
(mg mLꢀ1) in males
(mg mLꢀ1) in females
b
5.0
10.0
15.0
0.9190.38
2.6491.22
13.2591.94
1.2290.18
2.6490.25
15.8491.56
b
a
A two-way ANOVA of concentration/dose demonstrates non-linearity with dose, P B0.01.
b
Significantly different from male values, P B0.05, Student’s t-test. Nꢁ80 SpragueÁ
/
Dawley rats, ten of each sex at each dose level. Data for 0
mg kgꢀ1 hꢀ1 infusion rate have not been included in the table, the plasma blank values are given in Section 7.2.2.3.
D140S×
/
HCl 6 plasma concentrations on days 2 and 7 of
experiments, since longer infusion periods are required
to achieve steady state for this compound. The heart
rate and the ability to inhibit the isoprenaline-induced
heart rate increase (expressed as a percent of predrug
response) at the end of the 30 min drug infusion for
the study were calculated and are listed in Table 4.
3.2.4. Four week intravenous bolus injection study in
conscious dogs
D140S×
/
HCl 6, esmolol and atenolol are listed in Table 6.
D140S×
/
HCl 6 intravenous bolus injection doses were
0, 5.0, 10.0 and 15.0 mg kgꢀ1 bd; 6 h apart in 16
conscious beagle dogs (two males and two females at
The pharmacodynamic t1/2, determined from the heart
rate increase in response to bolus doses of isoprenaline
given at 10 min intervals for up to 150 min after the drug
infusion, for the three b-blockers are also given in Table
6.
each dose level, weighing 7.5Á11.0 kg). Plasma samples
/
were taken 1 h before the second dose of the day
(predose) and 0.5, 1, 2, 4, 8 and 12 h after the second
dose on days 1, 14 and 28. The pharmacokinetic t1/2 of
D140S×
/
HCl 6 in dogs on days 1, 14 and 28 of the 4
4. Toxicology
weeks intravenous bolus injection study were deter-
mined and are given in Table 5.
The results of the toxicological studies are sum-
marised in Table 7 and were performed by Hazelton
Laboratories, Madison, WI, USA (HWI), Inveresk
Research International Limited, Tranent, Scotland
(IRI) and Biodevelopment Laboratories, Cambridge,
MA, USA (BDL).
3.3. Pharmacodynamics
Pilot studies have demonstrated that the t1/2 for
HCl 6 at a dose of 20.0 mg kgꢀ1
/
esmolol and D140S×
/
minꢀ1 was ca. 10 min and for atenolol 2Á
5 h in a
conscious cannulated rat model (unpublished results).
The purpose of this study was to compare the pharma-
codynamic t1/2 of these three b-blockers when given by
intravenous infusion for 30 min, i.e. ca. three esmolol
half-lives, at 5.0, 10.0 or 20.0 mg kgꢀ1 minꢀ1. The
degree of b1-adrenoceptor blockade and the t1/2 of the
pharmacodynamic response was assessed by monitoring
the heart rate response to intravenous boluses of
isoprenaline (0.1 mg kgꢀ1) before, during and after a
30 min infusion of each drug. The effect of atenolol is
likely to be greater and the t1/2 longer than seen in these
5. Results and discussion
5.1. Isolated tissue preparations
The functional potency of the compounds (Table 1)
was determined for inhibiting (ꢀ)-isoprenaline-induced:
(i) b1-adrenoceptor mediated chronotropic effects in
spontaneously beating rat atria; and (ii) b2-adrenoceptor
mediated relaxation of rat tracheal chain previously
/
Table 3
Peak plasma concentrations of D140S×HCl 6 10 min after last intravenous bolus injection in conscious rats in a 4 weeks study
D140S×HCl 6 dose
Plasma concentration of D140S×HCl9S.E.M.
Plasma concentration of D140S×HCl9S.E.M.
a
a
(mg kgꢀ1 bd)
(mg mLꢀ1) in males
(mg mLꢀ1) in females
10.0
20.0
30.0
2.6590.31
5.3090.76
9.3491.00
2.8290.24
5.4090.43
8.0791.01
b
Linear regression demonstrates a linearity with dose, for males r2ꢁ0.99 and for females r2ꢁ1.00.
a
b
Significantly different from male value, Pꢁ0.04, Student’s t-test. Nꢁ48 SpragueÁ
/
Dawley rats, six of each sex at each dose level. Data for 0
(mg kgꢀ1 bd) dose have not been included in the table, the plasma blank values are given in Section 7.2.2.3.

G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á
/741
735
Table 4
Plasma concentrations of D140S×HCl 6 during 7 days continuous intravenous infusions in conscious dogs
a
a
Infusion rate of
D140S×HCl 6
Plasma concentration of D140S×HCl9S.E.M. on day 2
Plasma concentration of D140S×HCl9S.E.M. on day 7
(mg mLꢀ1
)
(mg mLꢀ1
)
(mg kgꢀ1 hꢀ1
)
0
0
0
b
b
b
b
2.5
5.0
10.0
0.5490.05
1.4990.19
4.1390.36
0.6790.07
2.1390.51
Á
/
c
7.5
5.0890.61
a
ANOVA of concentration/dose demonstrates non-linearity with dose, P B0.01 for day 2 and P B0.02 for day 7.
b
For both the 2.5 and 5.0 mg kgꢀ1 hꢀ1 dose, the D140S×HCl plasma concentrations were higher on day 7 than on day 2, P B0.03, Paired t-test.
contracted with 1 mM carbachol. In rat atria (b1-
adrenoceptor) D140S×HCl 6 was more potent than
esmolol, atenolol and metoprolol; of similar potency
plasma concentrations increased ca. 5-fold when the
dose was increased from 10.0 to 15.0 mg kgꢀ1 hꢀ1. The
/
relationship between D140S×
concentration was linear to 10.0 mg kgꢀ1 hꢀ1, above
this the relationship is non-linear (P B0.01, paired t-
/HCl 6 dose and plasma
to betaxolol and propranolol; and less potent than LK
204-545 (Table 1). Like LK 204-545 [14], D140S×
was highly cardioselective and the order of selectivity
(i.e. b1/b2) was LK 204-545#D140S×HCl 6ꢀ
esmololꢀbetaxololꢀatenololꢀmetoprololꢀpropra-
nolol. It should be acknowledged that the comparison is
of a single D140×HCl enantiomer with racemic forms of
the other compounds. In the guinea pig the b1/b2 pA2
values for D140S×HCl 6, racemic D140×HCl, racemic
esmolol and racemic propranolol were 7.7/B4.5, 7.25/
4.5, 6.8/5.3 and 8.3/8.0, respectively (unpublished
data).
/HCl 6
/
test). This non-linearity is characteristic of many b-
blockers at higher doses and has been particularly well
studied with propranolol [15].
/
/
/
/
/
/
/
/
5.2.2. Four week intravenous bolus injection study in
conscious rats
/
/
/
D140S×/HCl 6 plasma concentrations for rats 10 min
B
/
after receiving the final dose of 10.0, 20.0 and 30.0 mg
kgꢀ1 bd are listed in Table 3. At 10.0 and 20.0 mg kgꢀ1
bd dose levels there was no significant difference in
5.2. D140S×
/
HCl (6) pharmacokinetics
D140S×
females (P ꢀ
observed at 30 mg kgꢀ1 bd (Pꢁ
/
HCl 6 plasma concentrations between males and
0.1, Student’s t-test). A difference was
0.04, Student’s t-test)
/
/
5.2.1. Seven day continuous intravenous infusion in
conscious rats
but in contrast to the previous experiment the levels
were greater in males when compared with females (see
Section 5.2.1) suggesting there were no consistent gender
differences in these two studies. In contrast to the
previous study (Section 5.2.1), there appeared to be a
The plasma concentrations for rats at the end of a 7
day continuous infusion of 5.0, 10.0 and 15.0 mg kgꢀ1
hꢀ1 D140S×
/
HCl 6 are listed in Table 2. Samples taken
from the rats on the last day of the study showed a small
but significant increase in D140S×HCl 6 plasma con-
centrations at 5.0 and 15.0 mg kgꢀ1 hꢀ1 in females
compared with males (P B0.05; Student’s t-test) but no
significant difference at 10.0 mg kgꢀ1 hꢀ1 (Pꢁ
0.99,
Student’s t-test). There was evidence of non-linearity
with dose (P B0.01, two-way analysis of variance
dose linearity with plasma D140S×
/
HCl concentrations
1.00) at all doses
/
(for males r2ꢁ0.99, for females r2ꢁ
/
/
studied which may reflect the lower overall doses
studied. The maximum dose studied was 60.0 mg kgꢀ1
per day, as a bi-daily dosage, whereas the total daily
dose administered in the continuous infusion studies
were 120, 240 and 360 mg kgꢀ1 per day (Section 5.2.1).
/
/
/
(ANOVA) on steady state concentration/dose) and the
Table 5
Mean t1/2 of D140S×HCl 6 after intravenous bolus injection in conscious dogs in a 4 weeks study
a
a
a
D140S×HCl 6 dose (mg kgꢀ1 bd)
t1/29S.E.M. day 1 (min)
t1/29S.E.M. day 14 (min)
t1/29S.E.M. day 28 (min)
10.0
15.0
25.893.0
27.091.2
25.292.4
28.293.0
24.091.2
25.291.8
a
A two-way ANOVA found no difference in t1/2 between dogs (male and female), dose level or day of sampling, P ꢀ0.1. Nꢁ16 beagle dogs, two
of each sex at each dose level. Data for 0 and 5.0 mg kgꢀ1 bd dose have not been included in the table as the plasma levels of D140S×HCl 6 were
below the assay levels of detection (refer to Section 7.2.2.3), except for the 0.5 and 1 h levels at the 5.0 mg kgꢀ1 bd dose.

Table 6
The effect of D140S×HCl 6, esmolol and atenolol on heart rate responses before and after 0.1 mg kgꢀ1 isoprenaline intravenous bolus,% inhibition of isoprenaline-induced heart rate increase and the
pharmacodynamic t1/2 of D140S×HCl 6, esmolol and atenolol
Compound
Dose (mg
Control resting
heart rate (bpm)
Control heart rate increase in
response to isoprenaline (bpm)
Heart rate increase in response to Percent Inhibition of isoprenaline-induced Pharmacodynamic t1/2
e
a
b
c
kgꢀ1 hꢀ1
)
isoprenaline after 30 min infusion heart rate increase after 30 min infusion
d
(bpm)
(min)
D140S×HCl 6
Esmolol
5.0
10.0
20.0
5.0
389926
378931
403927
406924
397922
386921
382939
397923
392931
169919
150917
147921
134919
143935
136913
157914
136939
160939
143922
107912
89925
131923
119927
104916
96919
42919
57929
1696
2995
40912
2911
1698
24914
3998
7098
65910
B10
B10
10Á15
B10
B10
/
10.0
20.0
5.0
B10
f
Atenolol
ꢀ60
ꢀ60
ꢀ60
f
f
10.0
20.0
a
Infusion period was 30 min.
Control resting heart rate9S.E.M. bpm.
b
c
d
e
f
Increase in the control resting heart rate9S.E.M. bpm in response to a 0.1 mg kgꢀ1 bolus dose of isoprenaline.
Maximum increase in heart rate in response to the bolus dose of isoprenaline9S.E.M.. Isoprenaline was given after the 30 min drug infusion ceased.
Expressed as the percentage change from predrug values9S.E.M.
It was difficult to calculate t1/2 for atenolol because at the end of the follow up time (2 h) isoprenaline responses had not recovered to basal levels, in contrast to D140S×HCl and esmolol. Nꢁ4Á
/
6
individual experiments for each dose studied.

G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á
/741
737
Table 7
Summary of the toxicological studies carried out on D140S×HCl 6
a
Laboratory Result
Type of study
b
Acute toxicity, intravenous injection in albino CD-1 mice
HWI
HWI
LD50ꢀ60 mg kgꢀ1 NOEL 30 mg
kgꢀ1
b
Acute toxicity, intravenous injection In SpragueÁ
/
Dawley rats
LD50ꢀ40 mg kgꢀ1 NOEL 20 mg
kgꢀ1
c
Acute toxicity, oral (gavage) dose in CD-1 mice
IRI
IRI
LD50 ca. 1000 mg kgꢀ1
LD50 ca 516 mg kgꢀ1
NOEL 10 mg kgꢀ1 hꢀ1
NOEL 5 mg kgꢀ1 hꢀ1
NOEL 40 mg kgꢀ1 per day
NOEL 10 mg kgꢀ1 per day
NOELꢀ400 mg kgꢀ1 per day
NOELꢀ8 mg per day
NOELꢀ5 mg per day
negative
c
Acute toxicity, oral (gavage) dose in SpragueÁDawley rats
/
b
Seven days continuous intravenous infusion in conscious SpragueÁ
Seven days continuous intravenous infusion in conscious beagle dogs
Four weeks intravenous bolus injection study in conscious SpragueÁ
Four weeks intravenous bolus injection study in conscious beagle dogs
c
Two weeks oral (gavage) dose in male SpragueÁDawley rats
/
Dawley rats
HWI
HWI
HWI
HWI
IRI
b
b
/
Dawley rats
c
/
b
Five days ocular toxicity study in beagle dogs
Four weeks ocular toxicity study in New Zealand White rabbits
IRI
BDL
IRI
b
c
Buehler sensitisation test in albino DunkinÁHartley guinea pigs
/
Mutagenic activity with S. typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia IRI
c
negative
Coli WP2uvrA
Chromosomal aberrations assay with Chinese Hamster ovary cells in vitro
c
IRI
IRI
positive
negative
c
Micronucleus test in bone marrow of CD-1 mice
a
HWI, Hazelton laboratories, Madison, WI, USA. IRI, Inveresk Research International Limited, Tranent, Scotland. BDL, Biodevelopment
Laboratories, Cambridge, MA, USA.
b
D140S×HCl 6 was made up as a 4% parenteral formulation in phosphate buffered saline, pH 7.4.
D140S×HCl 6 was used as the unformulated crystalline solid.
c
5.2.3. Seven day continuous intravenous infusion in
conscious dogs
these very high doses ranged from 24 to 28 min. Two
way ANOVA found no difference in t1/2 between dogs
(male and female), dose level or day of sampling at each
The measured D140S×
/
HCl 6 plasma concentrations
on days 2 and 7 of the 7 day study are tabulated in Table
4. After day 4, the 10.0 mg kgꢀ1 hꢀ1 dose was reduced
to 7.5 mg kgꢀ1 hꢀ1 due to the four dogs developing
side-effects, which included hypoactivity, tremors, heart
rate reduction and decreased food consumption. Plasma
concentrations on days 2 and 7 increased disproportio-
dose (P ꢀ
centration appeared to be linearly related to dose on all
three study days (data not shown, r2ꢁ
0.88Á0.96, P B
/
0.1). Maximum D140S×
/
HCl 6 plasma con-
/
/
/
0.01). There was no evidence of saturation kinetics in the
doses studied.
nately with D140S×
P B0.02 for day 7, ANOVA on concentration/dose).
Above 2.5 mg kgꢀ1 hꢀ1 the relationship between dose
and plasma concentration is non-linear (P B0.03 for
both days 2 and 7, paired t-test on plasma concentra-
tion/dose). For both the 2.5 and 5.0 mg kgꢀ1 hꢀ1
/
HCl 6 dose (P B
/
0.01 for day 2 and
5.3. Pharmacodynamics
/
In conscious cannulated rats, the effects of D140S×
/
/
HCl 6, esmolol and atenolol on; (i) heart rate; (ii)
isoprenaline-induced increase in heart rate and (iii) their
pharmacodynamic t1/2 at the end of the b-blocker
infusion were determined and are listed in Table 6.
D140S×
/
HCl doses, which remained constant for the 7
days study, the plasma concentrations were higher on
D140S×
/
HCl and esmolol had little effect on resting heart
day 7 than on day 2 (P B0.03, paired t-test for both the
/
2.5 and 5.0 mg kgꢀ1 hꢀ1 doses).
rate. In contrast, atenolol caused a significant fall in
resting heart rate at all three doses (average of 24 bpm at
5 mg kgꢀ1 minꢀ1 increasing to 48 bpm at 20 mg kgꢀ1
5.2.4. Four week intravenous bolus injection study in
conscious dogs
minꢀ1, P B
0.05, 2 way ANOVA on control resting heart
/
rate and heart rate after 30 min infusion). In these
studies, the increase in control heart rate responses to
0.1 mg kgꢀ1 isoprenaline ranged from 1349
19 bpm. Inhibition of this heart rate increase at the
maximum dose studied (20.0 mg kgꢀ1 minꢀ1) ranged
from an average of 32 bpm for esmolol, 56 bpm for
The predose concentration of D140S×
measured 5 h after the first dose on day 28 when at
5.0 and 10.0 mg kgꢀ1 bd no D140S×
HCl was detected.
At the highest dose (15.0 mg kgꢀ1 bd) the predose
concentration of D140S×HCl 6 was close to the limit of
detection (0.1 mg mLꢀ1). The t1/2 post dose for D140S×
/
HCl 6 was
/
19 to 1699
/
/
/
/
D140S×
isoprenaline heart rate response was significant at all
doses for D140S×HCl and atenolol but not at the 5.0 mg
kgꢀ1 minꢀ1 infusion rate for esmolol (P B
0.01 for both
/
HCl and 103 bpm for atenolol. Inhibition of the
HCl 6 was estimated from plasma samples taken 0.5, 1,
2, 4, 8 and 12 h post dose for each day and the mean
results are given in Table 5, where the average t1/2 at
/
/

738
G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á741
/
atenolol and D140S×
/
HCl at all three doses and esmolol
beagle dog study (i.e. hypoactivity, tremors, excessive
for the 10.0 and 20.0 mg kgꢀ1 minꢀ1 doses, two-way
ANOVA on heart rate increase in response to isoprena-
line in control and infusion periods). Moreover there
was evidence of plateauing of the inhibition above 10.0
mg kgꢀ1 minꢀ1 for all three b-blockers. At the two
higher doses studied (10.0 and 20.0 mg kgꢀ1 minꢀ1),
salivation, decreased food consumption, heart rate
reduction and liquid, few or no faeces) are consistent
with those seen with high doses of other b-blockers [16].
D140S×
and there was no evidence of chromosomal damage in
these studies (Table 7). Ocular doses of 5Á8 mg per eye
/
HCl 6 was not mutagenic in a number of tests
/
D140S×
29 and 40% which was nearly double the effect of
racemic esmolol (16 and 24%). Once the D140S×HCl 6,
or esmolol, infusion ceased the effect on heart rate wore
off quickly (pharmacodynamic t1/2
15 min for D140S×
HCl and B10 min for esmolol). The pharmacodynamic
t1/2 10 min observed for esmolol was consistent with
/HCl 6 inhibited the isoprenaline response by ca.
per day were well tolerated in a 5 days study in dogs and
a 4 weeks study in rabbits (Table 7). In the latter study
eye observations were similar to the control group at all
doses tested, there were no microscopic changes in any
/
B
/
/
of the eye tissues and adnexa examined and D140S×
/
HCl
/
6 did not cause any systemic toxicity. The absence of
local irritancy in all parenteral studies, ocular studies
and in the Beuhler sensitisation test is an important
B
/
previously reported values [4,7]. Atenolol was the most
potent in reducing the heart rate and the duration of
action was much longer (pharmacodynamic t1/2
feature of D140S×
/
HCl 6, unlike esmolol formulations
ꢀ
/
60
which produce local tissue irritation [1].
min at all doses). The present study probably under-
estimates the t1/2 because a 30 min infusion does not
achieve steady state for atenolol (see Section 3.3).
6. Conclusions
Esmolol and D140S×
/
HCl 6 both behaved as short
acting b1-adrenoceptor blocking drugs when adminis-
tered intravenously. Atenolol, as expected, was less b1-
selective, more potent in reducing the heart rate
response to isoprenaline and had a much longer dura-
tion of action. The endogenous neurotransmitter nora-
drenaline is more b1-specific and is a weak b2-
adrenoceptor agonist compared with isoprenaline and
The results demonstrate that D140S×
acting, highly specific b1-adrenoceptor antagonist which
appears to lack the local tissue irritancy seen with
/
HCl 6 is a short
esmolol formulations. D140S×
/
HCl t1/2 was of the order
of 10Á15 min in the pharmacodynamic studies in the rat
/
and up to 28 min in the toxicology studies in the dog.
Pharmacokinetic non-linearity was seen in the toxicol-
ogy studies when given as a 7 days continuous intrave-
nous infusion above 10 mg kgꢀ1 hꢀ1 in the rat and
above 2.5 mg kgꢀ1 hꢀ1 in the dog. There was no
evidence of saturation kinetics in the other dose ranges
studied. The high degree of b1-specificity exhibited by
it is likely that D140S×
/
HCl 6, like esmolol, will be more
effective at inhibiting tachycardia resulting from endo-
genous sympathetic over-activity involving noradrena-
line.
5.4. Toxicology
D140S×
/
HCl 6 should help to avoid adverse effects on
the b2-adrenoceptors present in lung and other tissues.
The results of the toxicological tests (Table 7) indicate
that D140S×
no effect level (NOEL) for the intravenous dose in
rodents of 20Á
30 mg kgꢀ1 is about 5% of the NOEL for
the oral dose (516Á
1000 mg kgꢀ1) and is almost
identical to that reported for betaxolol and similar to
other b-blockers [16]. D140S×HCl 6 also shows a high
/
HCl 6 showed no unexpected toxicity. The
7. Experimental
/
/
7.1. Chemistry
/
level of safety in subacute intravenous, oral and ocular
studies of between 7 and 28 days duration in rats, dogs
and rabbits. For example in the 4 weeks intravenous
bolus injection study in conscious beagle dogs, the
7.1.1. General
Melting points (m.p.) were determined using a manual
Gallenkamp electrothermal apparatus (range 0Á
400 8C) in glass capillary tubes and are uncorrected.
IR spectra were recorded on a PerkinÁElmer FTIR 1600
/
NOEL for D140S×
/
HCl 6 was 10 mg kgꢀ1 per day
/
compared with a reported figure of 3 mg kgꢀ1 per day
for betaxolol in a similar study [16]. Similarly, the
spectrometer. H-NMR spectra were recorded on a 60
MHz Varian EM 360 spectrometer. Chemical ionisation
(CI, methane gas) mass spectra were recorded on either
a Finnigan GCQTM or a Finnigan 4000 series GCMS
mass spectrometer. All spectra were consistent with the
assigned structures. Analyses indicated by the symbols
1
NOEL for D140S×
/
HCl 6 in the 4 weeks intravenous
Dawley rats
bolus injection study in conscious SpragueÁ
/
was 40 mg kgꢀ1 per day compared with betaxolol 6 mg
kgꢀ1 per day [16]. It is well recognised that dogs are
more sensitive than rats to the toxic effects of b-blockers
and the toxic effects seen at the 10 mg kgꢀ1 minꢀ1 dose
in the 7 days continuous intravenous infusion conscious
of the elements or functions were within 90.4% of the
theoretical values and were carried out by The Campbell
Microanalytical Laboratory, Department of Chemistry,
/

G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á
/741
739
University of Otago, Dunedin, New Zealand. No
attempts were made to maximise yields.
7.1.4. S-(ꢀ)-1-(4-(2-Ethoxyethoxy)phenoxy)-2-
/
hydroxy-3-(2-(3,4-dimethoxyphenyl)ethyl-
amino)propane hydrochloride (6)
NaH, 60% suspension in mineral oil (32 g, 0.8 mol),
was washed with dry ether (3ꢃ100 mL) to remove the
/
7.1.2. Materials
3,4-Dimethoxyphenethylamine (homoveratrylamine),
oil and suspended in anhydrous DMF (400 mL) under
nitrogen. 4-Ethoxyethoxyphenol 4 (141 g, 0.8 mol) in
anhydrous DMF (200 mL) was added dropwise over
2S-(ꢂ)-glycidyl-3-nitrobenzenesulfonate, p-toluenesul-
/
fonyl chloride and dimethylformamide (DMF) were
purchased from Aldrich Chemicals. 10% Palladium on
carbon, 4-benzyloxyphenol (hydroquinone monobenzy-
lether) and sodium hydride were obtained from Fluka
Chemicals. 2-Ethoxyethanol and inorganic reagents
were supplied by Ajax Chemicals. Analytical grade
and HPLC grade solvents were purchased from Rhone
30Á60 min to the NaH suspension while stirring.
Stirring continued at r.t. for a further 30 min. The
resulting mixture was cooled to 5 8C with the aid of an
/
ice bath and treated dropwise over 45Á
/
90 min with a
solution of 2S-(ꢂ)-glycidyl-3-nitrobenzenesulfonate
/
(200 g, 0.8 mol) in DMF (200 mL) while ensuring that
the temperature did not rise above 10 8C. The solution
was brought to r.t. and stirred for 2 h under nitrogen.
Distilled water (50 mL) was added cautiously to destroy
any excess NaH and the mixture diluted with 3 L of
Poulenc Australia. Silica plates (5ꢃ10 cm, Silica F254)
/
were purchased from Merck. Silica for column chroma-
˚
tography (100 A, 50 mm) was supplied by Amicon Inc.,
MA 01923, USA.
water and extracted with ethyl acetate (3ꢃ
The extracts were combined, washed with brine (2ꢃ
/
750 mL).
250
/
mL) and dried over anhydrous MgSO4. The filtrate was
freed from solvent under reduced pressure giving the
epoxide 5 as an oil (homogenous by TLC).
7.1.3. 4-Ethoxyethoxyphenol (4)
2-Ethoxyethanol 1 (298 g, 3.3 mol) was placed in a 2 L
round bottomed flask and p-toluenesulfonyl chloride
(381 g, 2.0 mol) added with stirring. The mixture was
cooled to 15 8C and a 25% aqueous solution of NaOH
(320 mL) was added dropwise over 15 min, the mixture
was then stirred for 3 h at 15 8C. 4-Benzyloxyphenol
(320 g, 1.6 mol) was added to the mixture with stirring,
followed by NaOH (64 g, 1.6 mol in 120 mL water) and
the solution heated to reflux for 30 min. NaOH (22.5 g,
0.56 mol as a 25% aqueous solution) was again added
and the mixture refluxed for a further 30 min. After the
solution had cooled, crude 1-benzyloxy,4-(2-ethox-
yethoxy)benzene (3) was filtered, washed with 5%
aqueous NaOH and three times with distilled water
and then dried.
The crude 1-benzyloxy,4-(2-ethoxyethoxy)benzene (3)
was suspended in 4 l of EtOH and hydrogenated over
10% Pd/C (2.5 g) at room temperature (r.t.) and atmo-
spheric pressure. When hydrogen absorption ceased, the
reaction mixture was filtered through celite and evapo-
rated to dryness. The solid residue was then dissolved in
toluene (1 L) and filtered. The solution was transferred
to a separating funnel and extracted with 5% aqueous
The epoxide 5 and homoveratrylamine (3,4-dimethox-
yphenylethylamine, 141 g, 0.8 mol) in dioxane (400 mL)
were heated to reflux for 2 h while stirring. The reaction
mixture was then evaporated to dryness under reduced
pressure. The residue was dissolved in anhydrous diethyl
ether (300 mL) and excess saturated ethereal HCl was
added with stirring. The mixture was then stirred
overnight and the precipitate subsequently isolated by
filtration. The precipitated hydrochloride 6 was washed
with ether, excess solvent removed by suction and then
recrystallised from hot isopropanol (800 mL). Yield of
fine white crystalsꢁ
/
206Á
/
212 g (63Á
/
65%); m. p.ꢁ
/
147Á
/
149 8C; MS m/e 151 ((CH3O)2PhCH2ꢂ), 165 ((CH3O)2-
PhCH2 CH2ꢂ) and 268 (CH3CH2OCH2CH2OPhO-
CH2CH(OH)ꢀ
/
CH2NHCH2ꢂ) from loss of (CH3O)2-
PhCH2. On treatment with D2O the m/e 268 ion shifts
1
to m/e 269 and 270 from mono and dideuteration; H-
NMR (D2O) d 7.1 (7H, s), d 3.0Á
(3H, s), d 3.93 (3H, s), d 3.7 (2H, q, Jꢁ
(3H, t, Jꢁ 31.5 to 32.58 (4.0% aqueous
7 Hz); [a]2D3ꢁ
solution); Anal.: C23H34ClNO6(C, H, N, Cl).
/4.3 (13H, m), d 3.96
/7 Hz), d 1.27
/
/
ꢀ
/
7.1.5. Enantiomeric purity of D140S×
Solutions of D140S×HCl 6 were injected onto a
column of Chiracel OD (4ꢃ250 mm, Daicel Chemical
Industries) using a mobile phase of isopropanolÁhexane
/
HCl (6)
NaOH (3ꢃ
acidified using dilute HCl. The precipitate was extracted
with diethyl ether (2ꢃ500 mL), the extracts were
/
400 mL). The extracts were combined and
/
/
/
/
combined and dried over anhydrous MgSO4. The
diethyl ether was removed under reduced pressure to
leave a brown oil, which was then distilled under
reduced pressure.
(9:1) with 0.02% triethylamine at a flow rate of 0.8 mL
minꢀ1. Detection was by UV absorbance at 280 nm.
Samples were dissolved in EtOHÁ
/
hexaneÁisopropanol
/
(2:1:1), at a concentration of ca. 0.5 mg mLꢀ1, and 10
mL was injected onto the column. The R-isomer eluted
at 15 min and the S-isomer 6 at 24 min. The isomers
were well resolved and the ratio of the peak areas was
Yieldꢁ
/
190Á
/
235 g (65Á
/
81%); b.p.ꢁ
/
125Á130 8C at
/
1
1.5 mmHg; H-NMR (CDCl3) d 6.8 (4H, s) d 3.9(4H,
m) d 3.6(2H, q), d 1.2 (3H, t).

740
G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á741
/
used to estimate enantiomeric purity. D140S×
consistently assayed at ꢀ99% enantiomeric purity.
Neither acid nor alkaline conditions have been found
to promote racemisation of D140S×HCl 6.
/
HCl 6
CO2 in O2. The atria were dissected free of the ventricles
and overlying tissue and placed in a 20 mL bath
maintained at 37 8C and connected to an isometric
transducer. A tension of 1 g was applied and chrono-
tropic activity was amplified and recorded on a Grass
Polygraph.
/
/
7.2. Pharmacology
All studies were performed in agreement with and
according to the Prevention of Cruelty to Animals Act
(1986), the NH and MRC/CSIRO/AAC Australian
Code of Practice for the Care and Use of Animals for
Scientific Purposes (1990) and with the approval of the
Animal Welfare Committee at the Austin and Repatria-
tion Medical Centre.
7.2.2.2. Rat isolated tracheal chains. Tracheas were
excised from adult rats, dissected free of overlying tissue
and cut transversely into segments about 2 mm wide. Five
segments were mounted in a 20 mL bath maintained at
37 8C at a tension of 1 g. Relaxation of the segments by
(ꢀ)-isoprenaline were recorded by an isotonic transducer
/
connected to a Grass Polygraph after tone had been
established by administration of 1 mM carbachol (45 min
7.2.1. Drugs and chemicals
prior to concentrationÁresponse curves).
/
(ꢀ
chased from Sigma Chemical Co. (St. Louis, MO,
USA). (9
)-Esmolol (Brevibloc† injection 100 mg per
10 mL esmolol hydrochloride) was purchased from Du
Pont Pharmaceuticals (Aguadilla, PR, USA). (9)-Ate-
nolol, (9)-metoprolol, (9)-betaxolol and (9)-LK204-
545 ((9)-1-(2-(3-(2-cyano-4-(2-cyclopropylmethox-
/
)-Isoprenaline and (9
/
)-propranolol were pur-
7.2.2.3. Plasma assay for pharmacokinetic studies.
D140S×HCl 6 was extracted from plasma, after addition
of 1 mg of an internal standard (the methoxyethoxy
analogue of D140S×HCl), by passage through BondElut
/
/
/
/
/
/
/
Certify cartridges (Varian Pty. Ltd.). The cartridges
/
were then washed with 2 mL distilled water, 1 mL of 0.1
yethoxy)phenoxy)-2-hydroxypropylamino)-ethyl)-3-(4-
hydroxyphenyl)urea) [14] were synthesised as the hy-
drochloride salts within our department by Dr. D.
Iakovidis. All other chemicals were of reagent grade
from BDH Chemicals (Kilsyth, Australia).
M sodium acetate pH 4 and 2 mL MeOHÁ
/
water (4:2).
HCl
The drug was eluted with 2 mL of CH2Cl2ÁMeOHÁ
/
/
(80:20:1). The eluates were reduced to dryness at 60 8C
before being reconstituted in 150 mL of the mobile
phase. Recoveries of D140S×
standard were ca. 90%. The extracts were analysed by
HPLC with a Novapak C18, 5 mm, 3.9ꢃ150 mm
column (Waters Assoc.) and a mobile phase of waterÁ
acetonitrileÁ70% perchloric acidÁtriethylamine
(70:30:0.1:0.02) and a flow rate of 1 mL minꢀ1
Detection was by UV absorbance at 224 nm. D140S×
HCl 6 elutes at ca. 7 min. Plasma blanks in rats and dogs
/HCl 6 and the internal
7.2.2. Isolated tissue preparations
/
Male and female SpragueÁ
/
Dawley rats, 200Á/300 g,
/
were used and killed by a blow to the neck followed by
cervical dislocation and/or decapitation. Studies were
carried out on rat isolated atria and tracheal rings as we
described previously [14]. All tissues were allowed to
equilibrate for 45 min with Krebs Ringer physiological
salt solution; the composition of which in mmol Lꢀ1
was NaCl, 120; KCl 5.6; MgSO4 1.2; CaCl2, 2.5;
KH2PO4, 1.4; NaHCO3, 25; glucose 11.2 and EGTA,
/
/
.
/
gave readings of 0.089
/
0.04 mg mLꢀ1 and 0.109
0.07 mg
/
mLꢀ1, respectively, and only values exceeding twice
these readings were used in the calculations.
0.0025. Cumulative concentrationÁ
obtained from the non-selective b-adrenoceptor agonist
)-Isoprena-
line was dissolved and diluted in 1 mg mLꢀ1 ascorbic
acid to prevent oxidation. For the measurement of
antagonist activity the appropriate agent was added to
the organ bath at least 30 min after the first control
/
response curves were
7.2.3. Pharmacodynamic studies
The pharmacodynamic t1/2 of D140S×
/
HCl 6 was
(ꢀ
/
)-isoprenaline in each preparation [14]. (ꢀ
/
compared with esmolol (a short acting b-blocker) and
atenolol (a conventional cardioselective b-blocker) by
the inhibition of cardiovascular responses to a standard
dose of (ꢀ
/
)-isoprenaline in a conscious rat model.
Healthy male SpragueÁ
/
Dawley rats (200Á300 g) were
/
concentrationÁ
lowed to equilibrate for 10 min before the next
concentrationÁresponse curve established. The shift in
this curve to the right was calculated as a pA2 value [17].
At least three concentrations of each antagonist were
examined to verify the pA2.
/
response curve was completed and al-
prepared under anesthesia with indwelling catheters in
the femoral artery and femoral vein and allowed to
recover for at least 24 h. On the day of the experiment,
the arterial catheter was attached to a flexible swivel and
thence to a pressure transducer for the continuous
recording of heart rate and blood pressure. The venous
catheters were attached to an infusion pump for drug
administration or to a syringe for bolus injections of
/
7.2.2.1. Rat isolated spontaneously beating atria. Rat
hearts were removed from adult animals and placed in
Krebs Ringer salt solution (pH 7.4) aerated with 5%
(ꢀ
/
)-isoprenaline (0.1 mg kgꢀ1). The catheters were
suspended out of the animal’s way and the rat was

G.P. Jackman et al. / European Journal of Medicinal Chemistry 37 (2002) 731Á
/741
741
[2] S.T. Kam, W.L. Matier, K.X. Mai, C. Barcelon-Yang, R.J.
Borgman, J.P. O’Donnell, H.F. Stampfli, C.Y. Sum, W.G.
Anderson, R.J. Gorczynski, R.J. Lee, J. Med. Chem. 27 (1984)
allowed to move freely in a small covered enclosure
isolated from outside disturbance. When the blood
pressure and heart rate were stable, isoprenaline boluses
(0.1 mg kgꢀ1) were given at intervals of 10 min until
maximum responses in heart rate were reproducible
1007Á
[3] P.W. Erhardt, C.M. Woo, R.J. Gorczynski, W.G. Anderson, J.
Med. Chem. 25 (1982) 1402Á1407.
[4] C. Volz-Zang, B. Eckrich, P. Jahn, B. Schneidrowski, B. Schulte,
D. Palm, Eur. J. Clin. Pharmacol. 46 (1994) 399Á404.
/1016.
/
(910%). The doses were repeated for at least 40 min and
/
/
the last five readings were considered to constitute the
baseline response. The doses were continued at the same
interval throughout the test period of a further 150 min.
Ca. 3.5 min before the next dose the drug infusion was
started, to allow for dead volume in the infusion line,
and continued for a further 33.5 min. Rats were used for
more than one experiment if their catheters remained
patent with daily flushing with heparinised saline, but at
[5] I. Ahmet, N. Fukushima, Y. Sawa, T. Masai, K. Kadoba, K.
Kagisaki, J.C. Chang, T. Yamaguchi, H. Matsuda, Surg. Today
29 (1999) 248Á
[6] S. Motomura, A. Hagihara, Y. Narumi, K. Hashimoto, J.
Cardiovasc. Pharmacol. 31 (1998) 431Á440.
[7] P.W. Erhardt, C.M. Woo, W.G. Anderson, R.J. Gorczynski, J.
Med. Chem. 25 (1982) 1408Á1412.
[8] N. Freemantle, J. Cleland, P. Young, Br. Med. J. 318 (1999)
1730Á1737.
/
254.
/
/
/
least 24 h separated each experiment. D140S×
/HCl 6,
[9] E. Braunwald, E.M. Antman, J.W. Beasley, R.M. Califf, M.D.
Chietlin, J.S. Hochman, R.H. Jones, D. Kereiakes, J.
Kupersmith, T.N. Levin, C.J. Pepine, J.W. Schaeffer, E.E. Smith,
III, D.E. Steward, P. Theroux, R.J. Gibbons, J.S. Alpert,
K.A. Eagle, D.P. Faxon, V. Fuster, T.J. Gardner, G. Gregoratos,
R.O. Russell, S.C. Smith, Jr., Circulation 102 (2000)
esmolol and atenolol were prepared in normal saline and
infused at 5.0, 10.0 or 20.0 mg kgꢀ1 minꢀ1
.
Acknowledgements
1193Á/1209.
[10] W. Frisman, A. Chen, Am. Heart J. 137 (1999) 525Á
/
533.
[11] W.J. Louis, G.P. Jackman, D. Iakovidis, S.N.S. Louis, O.H.
Drummer, University of Melbourne, Australia, World Patent
Application 9713744, April 17, 1997.
The authors would like to thank Ms. Felicia M.
Colagrande, Ms. Leanne Styan and Mr. Peter Rowe for
their technical assistance. This work was supported by
grants from the National Health and Medical Research
Council of Australia, the Austin Hospital Medical
Research Foundation and the Sir Edward Dunlop
Medical Research Foundation.
[12] J.M. van Rossum, J.A.T.M. Hurkmans, C.J.J. Wolters, Arch. Int.
Pharmacodyn. Ther. 143 (1963) 299Á
[13] B.R. Zaborowsky, W.C. McMahan, W.A. Griffin, F.H.
Norris, R.R. Ruffolo, Jr., J. Pharmacol. Methods 4 (1980) 165Á
/
330.
/
178.
[14] S.N.S. Louis, T.L. Nero, D. Iakovidis, G.P. Jackman, W.J. Louis,
Eur. J. Pharmacol. 367 (1999) 431Á435.
/
[15] J.M. Cruickshank, B.N.C. Prichard, Beta-Blockers in Clinical
References
Practice, Churchill Livingstone, London, 1987, p. 214.
[16] P.L. Morselli, J.R. Kilborn, I. Cavero, D.C. Harrison, S.Z.
Langer (Eds.), Betaxolol and Other Beta1-Adrenoceptor Antago-
nists, vol. 1, Raven Press, New York, 1983, p. 43.
[1] J.E.F. Reynolds (Ed.), Beta-Adrenoceptor Blocking Agents.
Martindale the Extra Pharmacopoeia, 30th ed., The Pharmaceu-
tical Press, London, 1993, pp. 624Á
/641.
[17] D. Mackay, J. Pharm. Pharmacol. 30 (1978) 312Á313.
/