A new aspect of view in synthesizing new type β-adrenoceptor blockers with ancillary antioxidant activities

Article abstract of DOI:10.1016/S0968-0896(01)00067-0

A series of vanilloid-type β-adrenoceptor blockers derived from traditional Chinese herbal medicines were synthesized and tested for their antioxidant and adrenoceptor antagonistic activities. They all possessed significant β-adrenoceptor blocking activities under in vitro experiments and radioligand binding assays. In addition, some compounds were further examined in in vito tests and produced antagonist effects matching that of propranolol and labetalol by measurements of antagonism toward (-)isoproterenol-induced tachycardia and (-)phenylephrine-induced pressor responses in anesthetized rats. Furthermore, all of the compounds had antioxidant effects inherited from their original structures. In conclusion, compound 11 had the most potent β-adrenoceptors blocking activity, 12 and 13 possessed high cardioselectivity, whereas 14, 15 and 16 possessed additional α-adrenoceptor blocking activity and 15 is the most effective antioxidant of all. the antioxidant activity may be due to their @α and β unsaturated side chain at position 1 and ortho-substituted methoxy moiety on 4-phenoxyethylamine. Copyright

Full text of DOI:10.1016/S0968-0896(01)00067-0

Bioorganic & Medicinal Chemistry 9 (2001) 1739–1746
A New Aspect of View in Synthesizing New Type ꢀ-adrenoceptor
Blockers with Ancillary Antioxidant Activities
Yeun-Chih Huang,a Bin-Nan Wu,b Jwu-Lai Yeh,b Sheue-Jiun Chen,c
Jyh-Chong Liang,b Yi-Ching Lob and Ing-Jun Chenb,*
aDevelopment Center for Biotechnology, 81 Chang-Hsing Street, Taipei 106, Taiwan
bDepartment of Pharmacology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaoshing 807, Taiwan
cDepartment of Nursing, Foo-Yin Institute of Technology, 151 Chin-Shueh Road, Taliou, Kaohsiung 831, Taiwan
Received 31 May 2000; accepted 3 March 2001
Abstract—A series of vanilloid-type b-adrenoceptor blockers derived from antioxidant traditional Chinese herbal medicines were
synthesized and tested for their antioxidant and adrenoceptor antagonistic activities. They all possessed significant b-adrenoceptor
blocking activities under in vitro experiments and radioligand binding assays. In addition, some compounds were further examined
in in vivo tests and produced antagonist effects matching that of propranolol and labetalol by measurements of antagonism toward
(ꢀ)isoproterenol-induced tachycardia and (ꢀ)phenylephrine-induced pressor responses in anesthetized rats. Furthermore, all of the
compounds had antioxidant effects inherited from their original structures. In conclusion, compound 11 had the most potent b-
adrenoceptors blocking activity, 12 and 13 possessed high cardioselectivity, whereas 14, 15 and 16 possessed additional a-adreno-
ceptor blocking activity and 15 is the most effective antioxidant of all. The antioxidant activity may be due to their a and b unsa-
turated side chain at position 1 and ortho-substituted methoxy moiety on 4-phenoxyethylamine. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
blockade at peripheral vessels, which decreases preload,
afterload and a1-adrenoceptor mediated cardiac remo-
deling associated hypertrophy.13,14
The use of b-adrenoceptor blockers is well established in
the treatment of various cardiovascular disorders.1 Their
benefits have been demonstrated in many clinical investi-
gations.2,3 Recently, some b-adrenoceptor blockers have
been found to inhibit lipid peroxidation in canine and
swine models,4,5 and it has been suggested that the inhi-
bition of lipid peroxidation may provide additional
cardioprotective effects for b-adrenoceptor blockers.6,7
More recently, the third-generation b-adrenoceptor
blockers, which possess ancillary cardiovascular actions
other than b-adrenoceptor blockade, or cardioselective
b-adrenoceptor blockers, have been shown to improve
left ventricular function and decrease the risk of chronic
heart failure.8ꢀ10 The mechanism underlying the dra-
matic cardioprotective effects has been suggested to be
reduction in myocardial workload and oxygen con-
sumption.11ꢀ12 Additional benefit is provided by vaso-
dilation resulted from particular a1-adrenoceptor
From the history of developing b-adrenoceptor block-
ers, it has been generally accepted that the structure of
aryloxypropanolamines led to b-adrenoceptor blocking
activity15,16 and the effect was also confirmed in our
previous report.17 Numerous natural products, includ-
ing dehydrozingerone (1), zingerone (2), vanillin (3),
eugenol (4), isoeugenol (5), and ferulic acid (6) (Scheme
1), used in traditional Chinese medicines have been
shown to produce novel antioxidant activities.18,19 All
of them contain vanilloid (3-methoxy-4-hydroxy ben-
zene) moiety and the phenolic hydroxyl groups provide
the possibility to introduce a 4-ether-linked propanola-
mine side chain. Additionally, it is also well known that
most of the initial b-adrenoceptor blockers contained an
isopropyl- or tert-butylamine structure,20 however, the
substitution with aiacoxyethylamine was found to pro-
duce the a-adrenoceptor blocking activity.21 In this
article, we describe the synthesis of a series of 10 new b-
adrenoceptor blockers derived from vanilloid nucleus
(Table 1) and discuss the results from the examination
*Corresponding author. Tel.: +886-7-323-4686; fax: +886-7-323-
4686; e-mail: ingjun@kmu.edu.tw
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00067-0

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Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
of them under in vivo, in vitro and receptor binding
assays.
Pharmacology
The adrenoceptor blocking activities of compounds 7–
16 were evaluated by in vitro experiments and receptor
binding assays. From the in vitro studies, all of the
newly synthesized vanilloid derivatives concentration-
dependently inhibited (ꢀ)isoproterenol-induced positive
inotropic and chronotropic effects of the atria and tra-
cheal relaxant responses in isolated guinea pig tissues.
In addition, compounds 14, 15 and 16 also produced
significantly competitive antagonism of the nor-
epinephrine-induced contraction in isolated rat thoracic
aorta. The antagonistic potency of compounds 7–16 and
the reference compounds were evaluated from the
measurement of pA2 values shown in Table 2. Further-
more, all of the compounds were then investigated in
receptor binding assays using [3H]CGP12177 as a ligand
for b-adrenoceptors, and compounds 14, 15 and 16 were
further tested using [3H]prazosin for a-adrenoceptors.
Scatchard analysis22 of the data was used to estimate
the affinity and number of binding sites. The
[3H]CGP12177 was bound to b1- and b2-adrenoceptors
in rat ventricle and lung membranes both in concentra-
tion-dependent and saturable manners with Kd values of
0.18ꢁ0.04 and 1.25ꢁ0.09 nM, respectively, and the
Bmax values were 45.2ꢁ2.1 and 296.1ꢁ18.4 fmol per
mg protein at 25 ꢂC, respectively. Compounds 7–16
competitively antagonized [3H]CGP12177 bound to rat
ventricle and lung membranes with Ki values shown in
Table 3. [3H]prazosin also concentration-dependently
bound to a-adrenoceptors in rat brain membranes. The
Kd value was 0.25ꢁ0.01 nM and the Bmax value was
71.3ꢁ1.7 fmol per mg protein at 25 ꢂC. The inhibi-
tory constant Ki value of labetalol, an a/b-adreno-
ceptor blocker was 51.9 nM, and those of
compounds 14, 15 and 16 were 38.7, 38.9 and 32.5
nM, respectively.
Results
Chemistry
The synthesis of compounds 7–16, as shown in Scheme
2, represents a typical example of the general synthesis
of vanilloid derivatives of 1–6. Propanolamine deriva-
tives were obtained by reacting compounds 1–6 with
epichlorohydrin, and the obtained epoxide compounds
were then reacted with isopropylamine, tert-butylamine
or guaiacoxyethylamine, respectively, to yield 7–16.17
Scheme 1.
Table 1. Physicochemical data of newly synthesized b-adrenoceptor blockers
Compounds
R1
R2
mp, ꢂ
C
Recrystn solv
Yield (%)
Formulaa
7
8
9
10
11
12
13
14
15
16
CH¼CHCOCH3
CH¼CHCOCH3
CH2CH2COCH3
CHO
CH2CH¼CH2
CH¼CHCH3
CH¼CHCOOEt
CH2CH¼CH2
CH¼CHCH3
CH¼CHCOOEt
CH(CH3)2
C(CH3)3
C(CH3)3
C(CH3)3
CH(CH3)2
109–111
96–98
75–77
135–137
42–44
120–122
149–151
46–48
125–127
167–169
CH2Cl2
CH2Cl2
EtOH
55
35
30
50
68
31
45
25
52C
37
C17H25NO4
C18H27NO4
C17H27NO4
C15H23NO4
C16H25NO3
C16H25NO3
C19H29NO5
C22H29NO5
22H29NO5
EtOH
n-Hexane
n-Hexane
MeOH/Et2O
n-Hexane
n-Hexane
n-Hexane
CH(CH3)2
C(CH3)3
CH2CH2O(C6H4)-2-OCH3
CH2CH2O(C6H4)-2-OCH3
CH2CH2O(C6H4)-2-OCH3
C24H31NO7
aC, H, N were analyzed: the values are at ꢁ0.4% of the theoretical values.

Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
1741
Scheme 2.
Table 2. pA2 and b1/b2-selectivity values from in vitro experiments
b1
b2
a1
pA2 valuea
pA2 valuea
Trachea
pA2 valuea
Aorta
b1/b2 ratiob
Compounds
Right atrium
Left atrium
ratio
7
8
9
10
11
12
13
14
15
16
7.57ꢁ0.09
7.68ꢁ0.06
7.50ꢁ0.07
7.67ꢁ0.03
8.23ꢁ0.04
7.63ꢁ0.08
7.62ꢁ0.05
7.88ꢁ0.127.52
7.83ꢁ0.127.80
8.04ꢁ0.09
8.24ꢁ0.06
7.91ꢁ0.09
7.34ꢁ0.03
7.42ꢁ0.08
7.53ꢁ0.026.76
7.62ꢁ0.09
7.89ꢁ0.11
8.36ꢁ0.13
7.89ꢁ0.126.12
7.54ꢁ0.07
ꢁ0.05
6.93ꢁ0.04
ꢁ0.05
< 5.00
< 5.00
< 5.00
< 5.00
< 5.00
< 5.00
< 5.00
7.05ꢁ0.03
7.47ꢁ0.45
7.05ꢁ0.03
< 5.00
4.4
8.3
5.4
1.0
6.77ꢁ0.05
7.66ꢁ0.15
8.18ꢁ0.12
ꢁ0.05
1.1
32.4
21.9
3.6
1.2
3.4
1.5
2.3
34.7
6.28ꢁ0.11
7.33ꢁ0.15
7.76ꢁ0.11
7.51ꢁ0.06
ꢁ0.12
ꢁ0.09
8.03ꢁ0.15
8.07ꢁ0.128.07
7.54ꢁ0.16
5.70ꢁ0.06
Propranolol
Labetalol
Atenolol
7.54ꢁ0.16
6.87ꢁ0.08
5.81ꢁ0.06
< 5.00
apA2 values were obtained from the formula pA2=[log(DRꢀ1)-log molar concentration antagonist] and calculated from individual Schild plot by
regression. Each value was the meanꢁSEM of six to eight experimental results.
bThe b1/b2-selectivity ratios were obtained from the antilogarithm of the differences between the mean pA2 values obtained from right atrium and
trachea.

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Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
Furthermore, compounds 14, 15 and 16 were investi-
gated under in vivo experiments. The results indicated
that in ganglion-blocked anesthetized rats, injection of
compounds 14, 15, 16 and propranolol (0.5 mg/kg, iv)
all significantly decreased the (ꢀ)isoproterenol-
induced tachycardia responses (Fig. 1A). Likewise, in
reserpine-pretreated rats, intravenous administration
of (ꢀ)phenylephrine (1.0 mg/kg) increased the blood
pressure 63.1ꢁ3.5 mmHg (meanꢁSEM; n=8), which
was markedly inhibited by the iv injection of labetalol
or compounds 14, 15 and 16 (1.0 mg/kg, iv) but was not
significantly affected by propranolol (1.0 mg/kg) (Fig. 1B).
Table 3. Ki values from receptor binding assays
In the antioxidant experiments, in order to eliminate the
possibility of test compounds interfering with the assay,
the test agents added directly to malondialdehyde (MDA)
standards before the TBA agents were added. The
results indicated that the test compounds nearly had no
direct effects on the TBARS assay (data not shown).
The abilities of test compounds and other reference
agents on inhibiting lipid peroxidation in rat brain
homogenates are compared in Table 4. Furthermore,
compounds 14, 15 and 16 were further investigated the
scavenging activity of hydroxyl free radicals by EPR
experiments. The formation of the spin adducts DMPO–
OH, after the addition of DHF/Fe2+–ADP to DMPO
(90 mM in 0.9% saline), was evidenced by the appear-
ance of the characteristic 1:2:2:1 EPR hyperfine splitting
pattern (Fig. 2). Compounds 14, 15 and 16 (10 mM) all
significantly inhibited the height of DMPO–OH signal.
b1 (Ventricle)
Kia (nM)
b2 (Lung)
Ki (nM)
a1 (Brain)
Ki (nM)
b1/b2
ratio
Compounds
7
8
9
10
11
122
13
14
15
16
63.1ꢁ2.4
93.2ꢁ3.1
56.8ꢁ1.9
8.0ꢁ0.29.8
0.2ꢁ0.01
ꢁ0.7
498.5ꢁ22.4
1248.6ꢁ55.3
511.1ꢁ19.9
ꢁ0.3
NDb
ND
ND
ND
7.9
13.4
9.0
1.1
0.7ꢁ0.02ND
3.5
09.0
6859.0ꢁ231.5
2412.0ꢁ87.9
48.3ꢁ0.9
ND
ND
32.2
23.4
5.0
1.2
4.1
103.0ꢁ4.6
9.7ꢁ0.5
38.7ꢁ0.9
38.9ꢁ1.8
32.5ꢁ1.3
43.4ꢁ2.0
3.4ꢁ0.1
53.5ꢁ2.5
13.8ꢁ0.4
Propranolol
Labetalol
Atenolol
0.2ꢁ0.01
4.1ꢁ0.214.9
262.8ꢁ9.8
0.6ꢁ0.02ND .4
2
ꢁ0.5
51.9ꢁ2.1
3.7
8478.6ꢁ387.2ND .3 32
aKi values were calculated from the equation Ki=IC50/(1+[3H]ligand/
Kd). Kd and [3H]ligand denote the apparent dissociation constant and
the free concentration of the radiolabel, respectively.
bND, not determined.
Discussion
In the present studies, compounds 7–16 were evaluated
for their adrenoceptor blocking activities in vitro.
Pharmacological and radioligand binding techniques
were used to investigate the blocking activities at a- and
b-adrenoceptors. In isolated guinea pig tissues, com-
pounds 7–16 antagonized the (ꢀ)isoproterenol-induced
positive inotropic and chronotropic responses of the
atria and relaxant effects of the trachea, indicating that
these newly synthesized compounds all possessed b-
adrenoceptor blocking activities. The dramatic blocking
activities of compounds 7–16 may be contributed to the
basic propanolamine configuration. Most compounds
Table 4. Fifty concentration (IC50) required inhibiting lipid perox-
idation initiated by Fe2+-ascorbic acid in rat brain homogenatesa
Compounds
IC50 (mM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
35.7ꢁ1.5ꢃ
28.2ꢁ0.8ꢃ
6.9ꢁ0.3ꢃꢃ
12.8ꢁ0.4ꢃꢃ
1.5ꢁ0.1ꢃꢃ
6.3ꢁ0.3ꢃꢃ
83.4ꢁ4.0ꢃ
31.1ꢁ1.1ꢃ
103.0ꢁ4.9
15.7ꢁ0.9ꢃꢃ
67.4ꢁ3.5ꢃ
35.8ꢁ1.4ꢃ
31.8ꢁ1.2ꢃ
55.1ꢁ2.7ꢃ
0.7ꢁ0.1ꢃꢃ
7.1ꢁ0.4ꢃꢃ
1.8ꢁ0.1ꢃꢃ
3.0ꢁ0.2ꢃꢃ
4.1ꢁ0.2ꢃꢃ
Figure 1. Effects of intravenous injection of (ꢀ)isoproterenol (0.5 mg/
kg, panel A) in causing a tachycardia and (ꢀ)phenylephrine (10 mg/kg,
panel B) in causing a pressor responses before , CTL) and after (
propranolol;
,
a-Tocopherol
Trolox
Ascorbic acid
labetalol;
14;
15;
16, 0.5 mg/kg for b-adreno-
ceptor blockade and 1.0 mg/kg for a-adrenoceptor blockade) in gang-
lion-blocked anesthetized rats. Vertical bars, SEM changes from the
baseline value, which was 269ꢁ18 beats/min for heart rate and
108ꢁ12mmHg for blood pressure. Each value represents the average
of six to eight rats. *p<0.05 (paired Student’s t-test).
aEach value represents the mean. SEM of six to eight results
(ꢃ p<0.05, ꢃꢃ p<0.01).

Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
1743
were less potent than propranolol, but more potent than
atenolol in comparison with the estimated pA2 values at
b-adrenoceptors (Table 2). Particularly, compound 11
possesses the highest potency of all and compounds 12
and 13 are cardioselective b-adrenoceptor blockers with
b1/b2 ratios of 32.4 and 21.9, respectively. These new
findings encouraged us to modify vanilloid compounds
4, 5 and 6 to obtain compounds 11, 12 and 13 (Scheme
1), respectively, and to synthesize third generation b-
adrenoceptor blockers 14, 15 and 16 by substituting the
epoxide compounds with guaiacoxyethylamine. Further
examinations of a-adrenoceptor blocking activities were
performed in rat aorta, precontracted with nor-
epinephrine. The resulting pA2 values indicated that 14,
15 and 16 possess significant a-adrenoceptor blocking
activities, however, the other compounds had only
minor antagonistic actions and all their values were less
than 5. These results further confirmed the previous
report of Augstein et al.,21 which suggested that a high
than that of atenolol, which is a highly selective b1-
adrenoceptor blocker with the selective ratio of 34.7.
The characterizations of the adrenoceptor blocking
activities of 7–16 were determined from the receptor
binding assays and compared with those of propranolol,
labetalol and atenolol (Table 3). In the b-adrenoceptor
binding study, all of the compounds showed various
affinities at both b1- and b2-adrenoceptors. Above all,
the binding affinity of compound 11 was similar to that
of propranolol. All of the compounds showed the
higher binding affinity than that of atenolol. In addi-
tion, 14, 15 and 16 were further tested for their binding
affinities on a-adrenoceptors and the obtained Ki values
indicated that these agents displayed the higher binding
affinity than that of labetalol. These results were then
confirmed by in vivo experiments (Fig. 1). Compounds
14, 15 and 16, as well as propranolol and labetalol, all
blocked (ꢀ)isoproterenol-induced tachycardia effects
and indicated that they had b-adrenoceptor blocking
activities. Additionally, 14, 15, 16 and labetalol (a dual
a- and b-adrenoceptor antagonist) showed significant
inhibition of pressor responses to (ꢀ)phenylephrine,
suggesting that these agents also possess a-adrenoceptor
blocking activities. However, propranolol did not show
any blockade on a-adrenoceptors.
degree
molecule,
requires
2-(2-methoxyphenox-
y)ethylamine for optimal a-adrenoceptor blocking
activity. In the a-adrenoceptor blocking experiments,
the pA2 values of 14, 15 and 16 were greater than those
of labetalol, a a/b-adrenoceptor blocker. However, 15
and 16 did not display the cardioselectivity that found
in 12 and 13. The b1/b2 ratios of 15 and 16 are 1.2and
3.4, respectively, denoting the lower cardioselectivity
The brain tissue, rich in lipids, was a commonly used
model23 for the study of lipid peroxidation and there-
fore in the present study it was chosen to evaluate
the inhibition of lipid peroxidation of compounds 1–16
(Table 4). MDA formation assayed by the TBA
method was used as an index of membrane lipid
peroxidation because of its sensitivity and simplicity. In
this test, all of the compounds, including the vanilloid
type natural products 1–6 and the newly synthesized
b-adrenoceptor blockers 7–16, showed remarkable
inhibitory actions on Fe2+-induced lipid peroxidation.
The antioxidant effect of these compounds may be due
to their a and b unsaturated side chain at the 1-position
and ortho-substituted24 methoxy group at 3-position
of 4-phenoxyethylamine derived from vanilloid base
(7–16). To ensure the scavenging activity, compounds
14, 15 and 16 were further investigated by using EPR
spectroscopy to determine whether or not these com-
pounds inhibit OH radical generation in a Fenton-type
reaction. Because EPR signal height is proportional
to the amount of DMPO-OH adduct produced, the
decrease of signal in the presence of compounds 14, 15
and 16 indicates that these compounds effectively
suppress the formation of DMPO-OH (Fig. 2). In addi-
tion, there are significantly positive correlations between
the newly synthesized vanilloid compounds and their
original structures, indicating that the antioxidant
activities are inherited from their basic vanilloid moiety.
For example, the rank of antioxidant potency was
5>6>4, whereas the potency of compounds 15, 16 and
14, which were derived from 5, 6 and 4, respectively,
was also 15ꢄ16ꢄ14. The measured IC50 values of 14,
15 and 16 were less than those of 11, 12 and 13, and
indicated that the guaiacoxyethylamine series is more
potent than the tert-butylamine series in their anti-
oxidant activities.
Figure 2. EPR spectra of DMPO–OH adduct formation in the
absence (control) or presence of test compounds. The panel shows the
representative tracings. The EPR signals were initiated by adding
DHF/Fe2+–ADP into 0.9% saline solutions containing 90 mM
DMPO, with or without test compounds. These tracings were recor-
ded at 10 min after the addition of DHF/Fe2+–ADP.

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Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
In summary, all of the new vanilloid derivatives descri-
bed in this article have potent b-adrenoceptor blocking
activities, which match those of reference compounds in
in vitro and receptor binding studies. Compound 12 and
13 have the high cardioselectivity equal to that of ate-
nolol. Compounds 14, 15 and 16, which are substituted
with guaiacoxyethylamine, all possess a-adrenoceptor
blocking activities. Additionally, we have demonstrated
that all of the compounds possessed potent antioxidant
activities inherited from their original structures. These
findings suggested that it is valuable to modify the nat-
ural products from traditional Chinese herbal medicines
to synthesize new b-adrenoceptor blockers with the
vasodilatoty a-adrenoceptor blocking activity by sub-
stituting the 4-hydroxyl group.
Ar–CH=CH); IR (KBr) 3300, 1690, 1600 cmꢀ1; MS m/
z 322 (M+H)+.
1-{[4-(3-Butanone)-2-methoxy]phenoxy}-3-(isopropyl-
1
amino)propanol (9). H NMR (CDCl3) d 1.10–1.13 (s,
6H, CH3ꢅ2), 2.15 (s, 3H, COCH3), 2.51 (br s, 1H,
exchangeable OH), 2.74–2.79 (s, 4H, Ar–CH2CH2),
2.80–2.88 (s, 3H, CH2NHCH-), 3.85 (s, 3H, Ar–OCH3),
4.05–4.19 (m, 3H, Ar–OCH2CH(OH)), 5.81 (s, 1H, -
NH-), 6.68–6.86 (m, 3H, Ar–H); IR (KBr) 3300, 1710,
1590 cmꢀ1; MS m/z 310 (M+H)+.
1-[(4-Aldehyde-2-methoxy)phenoxy]-3-(tert-butylamino)-
1
propanol (10). H NMR (CDCl3) d 1.25 (s, 9H, CH3 ꢅ
3), 2.45 (br s, 1H, exchangeable OH), 3.17–3.39 (m, 2H,
-CH2NHC-), 3.90 (s, 3H, Ar–OCH3), 4.16–4.24 (m, 3H,
Ar–OCH2CH(OH)), 5.71 (s, 1H, -NH-), 6.99–7.40 (m,
3H, Ar–H), 9.85 (s, 1H, CHO); IR (KBr) 3250, 2900,
1670 cmꢀ1; MS m/z 282 (M+H)+.
Experimental
General information
1-[(4-Allyl-2-methoxy)phenoxy]-3-(isopropylamino)-pro-
1
All melting points were measured with a Yanaco MP-J3
micromelting point apparatus and are uncorrected.
Infrared spectra were recorded through a KBr disk (n in
panol (11). H NMR (CDCl3) d 1.06–1.09 (d, 6H, CH3
ꢅ 2), 1.95 (d, 1H, exchangeable OH), 2.69–2.89 (m, 3H,
-CH2NHCH-), 3.32–3.35 (dd, 2H, Ar–CH2), 3.85 (s,
3H, Ar–OCH3), 3.91–4.07 (m, 3H, Ar–OCH2CH(OH)),
5.04–5.10 (m, 2H, -CH=CH2), 5.86–6.06 (m, 1H, -
CH=CH2), 6.69–6.88 (m, 3H, Ar–H); IR (KBr) 3300,
3150, 1600 cmꢀ1; MS m/z 280 (M+H)+.
1
cmꢀ1) on a Hitachi 270-30 IR spectrophotometer. H
nuclear magnetic resonance spectra were recorded on a
Varian Gemini 400 FT-NMR spectrophotometer, using
CDCl3 as solvent and TMS as internal standard (che-
mical shift in d, ppm). Mass spectra were recorded with
a JEOL-D100 GC-mass spectrophotometer. Elemental
analyses were performed on a Heraeus CHN-O-Rapid
analyzer and were within 0.4% of the theoretical values
unless otherwise indicated.
1-[(4-Propenyl-2-methoxy)phenoxy]-3-(isopropylamino)-
1
propanol (12). H NMR (CDCl3) d 1.18–1.22 (d, 6H,
CH3ꢅ2), 1.85–1.89 (d, 3H, Ar–CH¼CHCH3), 2.13 (br
s, 1H, exchangeable OH), 2.83–3.08 (m, -CH2NHCH-),
3.86 (s, 3H, OCH3), 4.03–4.06 (m, 2H, Ar–OCH2), 4.14–
4.21 (m, 1H, -CHOH), 6.02–6.19 (m, 1H, Ar–
CH¼CHCH3), 6.29–6.38 (d, 1H, Ar–CH¼CHCH3),
6.84–6.88 (t, 3H, Ar–H); IR (KBr) 3600, 3300, 1600
cmꢀ1; MS m/z 280 (M+H)+.
Vanillin, eugenol, isoeugenol, ferulic acid and guaiacol
were purchased from Tokyo Chemical Industry Co.
(TCI). Epichlorohydrin and CDCl3 were obtained from
Janssen. Guaiacoxyethylamine was synthesized via
Mannich reactions19 from guaiacol. All the other
reagents used in this study were EP-grade products of E.
Merck. Animals were obtained from the Experimental
Animal Center, Cheng-Kung National University Med-
ical College, Tainan, Taiwan.
1-[(4-Propenoic acid ethyl easter-2-methoxy)phenoxy]-3-
(tert-butyl amino)propanol (13). 1H NMR (CDCl3) d
1.12(s, 9H, C H3ꢅ3), 1.34 (q, 3H, -COOCH2CH3),
2.60–2.91 (m, 2H, -CH2NH-), 3.88 (s, 3H, ArOCH3),
3.90-4.l0 (m, 2H, ArOCH2-), 4.21–4.32 (m, 2H, -
COOCH2CH3), 4.60–4.62(m, 1H, -C HOH), 6.27–6.35
(d, 1H, Ar–CH¼CH-), 6.88–7.10 (m, 3H, Ar–H), 7.57–
Synthesis
1-{[4-(1-Buten-3-one)-2-methoxy]phenoxy}-3-(isopropyl-
1
7.67 (d, 1H, Ar–CH=CH-), IR (KBr) 3300, 1700 cmꢀ1
,
+
amino)-propanol (7). H NMR (CDCl3) d 1.08–1.11 (d,
MS m/z 352(M+H)
.
6H, CH3ꢅ2), 2.37 (s, 3H, COCH3), 2.57 (br s, 1H,
exchangeable OH), 2.80–2.86 (s, 3H, -CH2NHCH-),
3.89 (s, 3H, Ar–OCH3), 4.07–4.20 (m, 3H, Ar–OCH2-
CH(OH)), 5.71 (s, 1H, -NH-), 6.56–6.64 (d, 1H, Ar–
CH¼CH), 6.89–7.12(m, 3H, Ar– H), 7.42–7.50 (d, 1H,
Ar–CHCH); IR (KBr) 3300, 1680, 1600 cmꢀ1; MS m/z
308 (M+H)+.
1-[(4-Allyl-2-methoxy)phenoxy]-3-[(2-methoxyphenoxye-
thyl)amino]-propanol (14). 1H NMR (CDCl3) d 2.41–
2.79 (br s, 1H, -NH-), 2.93–3.16 (m, 4H, -CH2NHCH2-
), 3.31–3.34 (t, 2H, Ar–CH2), 3.80–3.83 (s, 6H, 2ꢅAr–
OCH3), 4.00–4.03 (m, 4H, 2ꢅAr–OCH2-), 4.14–4.16 (m,
1H, -CH(OH)), 5.03–5.12(m, H2 , -CH ¼CH2), 5.88–
6.01 (m, 1H, -CH¼CH2), 6.67–6.92(m, 7H, Ar– H); IR
(KBr) 3300, 3150, 1600 cmꢀ1; MS m/z 388 (M+H)+.
1-{[4-(1-Buten-3-one)-2-methoxy]phenoxy}-3-(tert-butyla-
mino)propanol (8). 1H NMR (CDCl3) d 1.03 (s, 9H,
CH3ꢅ3), 2.31 (s, 3H, COCH3), 2.45 (br s, 1H,
exchangeable OH), 2.54–2.69 (s, 3H, -CH2NHC-), 3.83
(s, 3H, Ar–OCH3), 3.90–4.05 (m, 3H, Ar–OCH2-
CH(OH)), 5.78 (s, 1H, -NH-), 6.70–6.80 (d, 1H, Ar–
CH¼CH), 7.01–7.45 (m, 3H, Ar–H), 7.52–7.60 (d, 1H,
1-[(4-Propenyl-2-methoxy)phenoxy]-3-[(2-methoxyphe-
noxyethyl)amino]-propanol (15). 1H NMR (CDCl3) d
1.85–1.88 (d, 3H, Ar–CH=CH-CH3), 2.15–2.22 (br s,
1H, -NH-), 2.83–3.10 (m, 4H, -CH2NHCH2-), 3.84–3.85
(s, 6H, 2ꢅAr–OCH3), 4.03–4.04 (m, 2ꢅ4H, Ar–OCH2-),

Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
1745
4.12–4.15 (m, 1H, -CH(OH)), 6.05–6.17 (m, 1H, Ar–
CH=CH-CH3), 6.30–6.36 (m, 1H, Ar–CH=CH-CH3),
6.84–6.92(m, 6H, Ar– H); IR (KBr) 3600, 3300, 1600
cmꢀ1; MS m/z: 388 (M+H)+.
lung preparations were homogenized with a polytron
(IKA-Labortechnik, Model T25-S1, Germany) in 20
volumes of buffer containing 10 mM Tris–HCl, 1 mM
EDTA and 0.1 mM ascorbic acid (pH 7.4) under ice-
cold conditions. The homogenate was centrifuged fur-
ther at 1000 g for 10 min at 4 ꢂC and then the super-
natant was centrifuged again at 10,000 g for 12min. The
second supernatant was further centrifuged at 30,000 g
for 15 min at 4 ꢂC and the final pellet was resuspended
in an assay buffer (75 mM Tris–HCl, 25 mM MgCl2, pH
7.4) and stored at ꢀ80 ꢂC. The membrane preparation
for a1-adrenoceptor binding assay was prepared
according to the method of Miach et al.29 The cortex
and the whole brain minus the cerebellum were homo-
genized in 20 volumes of ice-cold buffer (0.25 mM
sucrose, 1 mM MgCl2, 5 mM Tris–HCl, 0.05% ascorbic
acid, pH 7.4). The homogenate was centrifuged at 900 g
for 10 min at 4 ꢂC and then the supernatant was cen-
trifuged at 12,000 g for 20 min at 4 ꢂC. The resulting
pellet was resuspended in the incubation buffer (mM:
MgCl2 12.5, Tris–HCl 62.5, pH 7.5) and stored at
ꢀ80 ꢂC. Protein concentration was determined using the
method of Lowry et al.,30 using bovine serum albumin
as standards.
1-[(4-Propenoic acid ethyl easter-2-methoxy)phenoxy]-3-
1
[(2-methoxyphenoxyethyl)amino]-propanol (16). H NMR
(CDCl3) d 1.34 (t, 3H, -COOCH2CH3), 2.93–3.18 (m,
4H, -CH2NHCH2-), 3.77–3.84 (s, 6H, 2ꢅArOCH3),
4.06–4.14 (m, 4H, 2ꢅArOCH2-), 4.17–4.20 (m, 3H, -
COOCH2CH3), 4.24–4.31 (m, 1H, CH(OH)), 6.26–6.34
(d, 1H, Ar–CH=CH), 6.86–7.04 (m, 7H, Ar–H), 7.57–
7.65 (d, 1H, Ar–CH¼CH-), IR (KBr) 3300, 1700 cmꢀ1
,
MS m/z 446 (M+H)+.
Pharmacology
Measurements of isolated atria and trachea of the guinea
pigs. Experiments were performed following the
method described in a previous report from our labora-
tory.25 Right atria preparations, which retained a spon-
taneous rhythm, were used for assessment of
chronotropic effects, while inotropic effects were exam-
ined with left atrial preparations. The experiments were
carried out at 37 ꢂC containing Kreb’s solution of the
following composition (mM) NaCl 113, KCl 4.8, CaC12
2.2, KH2PO4 1.2, MgCl2 1.2, NaHCO3 25, dextrose
11.0, bubbled with 95% O2 and 5% CO2. As b-agonist,
(ꢀ)isoproterenol was administrated to the preparations
in cumulative fashion after an equilibration period of 90
min in Kreb’s solution and the concentration-response
curve was established. The atria were then allowed a
30–60 min washout period to restablize, after which
time various concentrations of the test compounds
were incubated with the atrium 30 min before
(ꢀ)isoproterenol. The activity of the test compound was
expressed as pA2 value, which was calculated from the
parallel shifts of the cumulative concentration-response
curves of (ꢀ)isoproterenol.26
Adrenoceptor binding assay, including [3H]CGP-12177
binding to rat ventricular and lung membranes and
[3H]prazosin to rat brain membrane, were determined
by the methods of Huang et al.25 [3H]CGP-12177 and
ventricular or lung membranes (200–300 mg) were incu-
bated for 60 min at 25 ꢂC in 75 mM Tris–HCl buffer
composed of MgCl2 25 mM, with or without the addi-
tion of 10 mM propranolol, to make a final volume of
250 mL. [3H]prazosin and brain membrane (250–300 mg)
were incubated for 60 min at 37 ꢂC with or without 10
mM phentolamine, in 50 mM Tris–HCl buffer including
MgCl2 10 mM, to make a final volume of 500 mL.
After incubation, the mixture was filtered rapidly
through Whatman GF/C glass fiber filters supported on
a 12-port filter manifold (Millipore). The filters were
immediately washed three times with 5 mL of each of
the ice-cold buffer (75 mM Tris–HCl and 25 mM MgCl2
buffer for b-adrenoceptor binding and 50 mM Tris–HCl
and 10 mM MgCl2 buffer for a-adrenoceptor binding),
and dried in an oven at 80 ꢂC for 2h. The radioactivity
was counted in 4 mL of Triton-toluene based scintilla-
tion fluid with about 45% efficiency in a Beckman
LS6500 scintillation system (Fullerton, CA, USA). Spe-
cific binding was defined as the excess over blank in the
presence of 10 mM propranolol for b-adrenoceptors, 10
mM phentolamine for a-adrenoceptors.
Measurement of isolated aorta of the rats. The prepara-
tion of Wistar thoracic aorta rings was similar to that
originally described by Honda et al.27 In brief, the rat
thoracic aorta preparations were set up in 10 mL organ
baths containing Kreb’s solution at 37 ꢂC under a rest-
ing tension of 1.5 g. Cumulative concentration-response
curves for norepinephrine were constructed by increas-
ing the concentration of the agonist bath approximately
3-fold. Test compounds were added to the bath medium
after a control concentration-response curve to nor-
epinephrine had been obtained. The tissues were
exposed to the test compounds for 30 min before
rechallenging with norepinephrine. Contractions were
expressed as a percentage of the maximum contraction
obtained by the first challenge to the tissues.
Measurement of lipid peroxidation in rat brain homo-
genates. The rat brain homogenate was made in 0.9%
saline containing 10 mg tissue/mL. The rates of mem-
brane lipid peroxidation were measured by the forma-
tion of TBARS. Rat brain homogenates (1 mL) were
incubated at 37 ꢂC for 5 min with 10 mL of test com-
pound or vehicle. Lipid peroxidation was initiated by the
addition of 0.1 mL of 0.25 mM FeCl2 and 1 mM ascorbic
acid.23 After 30 min of incubation, the reaction was
arrested by adding 0.1 mL of 0.2% BHT. Thiobarbituric
Receptor binding assay procedures. Wistar rats (either
sex, 250–300 g) were sacrificed and their ventricle and
lung were removed quickly and chilled in ice-cold TE
buffers (10 mM Tris–HCl, 1mM EDTA, 0.1 mM ascor-
bic acid, pH 7.4). The membrane preparations for b1-
and b2-adrenoceptor binding assays were prepared
according to the methods of Wu et al.28 The ventricle or

1746
Y.-C. Huang et al. / Bioorg. Med. Chem. 9 (2001) 1739–1746
acid reagent was then added and the mixture was heated
for 30 min in a boiling water bath. The TBARS was
extracted with n-butanol and measured at 532nm. The
amount of TBARS was quantified using the linear
regression obtained from malondialdehyde standards.
6. Weglicki, W. B.; Mak, I. T.; Simic, M. G. J. Mol. Cell.
Cardiol. 1990, 22, 1190.
7. Feuerstein, G. Z.; Yue, T. L.; Ma, X. L.; Ruffolo, R. R.
Prog. Cardiovasc. Dis. 1998, 41 (Suppl. 1), 17.
8. Heesch, C. M.; Marcoux, L.; Hatfield, B.; Eichhorn, E. J.
Am. J. Cardiol 1995, 75, 360.
9. Packer, M.; Bristow, M. R.; Cohn, J. N.; Colucci, W. S.;
Fowler, M. B.; Gilbert, E. M.; Shusterman, N. H. N. Engl. J.
Med. 1996, 334, 1349.
10. Packer, M. Prog. Cardiovasc. Dis. 1998, 41 (Suppl. 1), 39.
11. Bril, A.; Slivjak, M.; DiMartino, M. J.; Feuerstein, G. Z.;
Linee, P.; Poyser, R. H.; Ruffolo, R. R.; Smith, E. F. Cardio-
vasc. Res. 1992, 26, 518.
12. Feuerstein, G. Z. J. Cardiovasc. Pharmacol. 1992,
19 (Suppl. 1), S138.
13. Bristow, M. R. Am. J. Cardiol. 1993, 71, 12 C.
14. Hamburger, S. A.; Barone, F. C.; Feuerstein, G. Z.; Ruf-
folo, R. R. Pharmacol. 1991, 43, 113.
15. Wu, E. S.; Cole, T. E.; Davidson, T. A.; Blosser, J. C.;
Borrelli, A. R.; Kinsolving, C. R.; Milgate, T. E.; Parker, R. B.
J. Med. Chem. 1987, 30, 788.
16. Hoffman, B. B.; Lefkowitz, R. J. In The Pharmacological
basis of Therapeutics, 8th ed; Gilman, A. G., Rall, T. W., Nies,
A. S., Taylor, P., Eds. New York: Pergamon; 1991; pp 221–
243.
17. Chen, I. J.; Yeh, J. L.; Liou, S. J.; Shen, A. Y. J. Med.
Chem. 1994, 37, 938.
18. Rajakumar, D. A.; Rao, M. N. A. Biochem. Pharmacol.
1993, 46, 2067.
19. Uchida, M.; Nakajin, S.; Toyoshima, S.; Shinoda, M.
Biol. Pharm. Bull. 1996, 19, 623.
20. Blanc, M.; Tamir, A.; Aubriot, S.; Michel, M. C.; Bou-
zoubaa, M.; Leclerc, G.; Demenge, P. J. Med. Chem. 1998, 41,
1613.
21. Augstein, J.; Austin, W. C.; Boscott, R. J.; Green, S. M.;
Worthing, C. R. J. Med. Chem. 1965, 8, 356.
22. Scatchard, G. Ann. N. Y. Acad. Sci. 1949, 51, 660.
23. Braughler, J. M.; Pregenzer, J. F.; Chase, R. L.; Duncan,
L. A.; Jacobsen, E. J.; Mccall, J. M. J. Biol. Chem. 1987, 262,
10438.
24. Yue, T. L.; Cheng, H. Y.; Lysko, P. G.; Mckenna, P. J.;
Feuerstein, R.; Gu, J. L.; Lysko, K. A.; Davis, L. L.; Feruer-
stein, G. J. Pharmacol. Exp. Ther. 1992, 263, 92 .
25. Huang, Y. C.; Yeh, J. L.; Wu, B. N.; Lo, Y. C.; Liang,
J. C.; Lin, Y. T.; Sheu, S. H.; Chen, I. J. Drug Dev. Res. 1999,
47, 77.
Spin-trapping experiments. The procedure for spin-trap-
ping experiments was described previously by Yue et
al.24 (1989). Briefly, oxygen-derived free radicals were
generated from auto-oxidation of DHF (830 mM) in the
presence of FeCl3 (25 mM) chelated by ADP (250 mM).
DMPO (90 mM) dissolved in 0.9% saline was exposed
to the free radical generation system in the presence or
absence of test compounds. The formation of the radi-
cal spin adduct, DMPO–OH, was monitored using a
Bruker EMX-10 EPR spectrometer interfaced to an
IBM PC/ATX computer.
Measurement of ꢀ- and ꢁ-adrenergic responses. Rats
were pretreated with mecamylamine (5 mg/kg, iv), a
ganglion-blocking agent, to ensure uniform initial heart
rate. (ꢀ)Isoproterenol (0.5 mg/kg) was administered via
a femoral vein and the resultant tachycardia recorded as
the control. A single dose of test compound was then
administered intravenously. After 10 min, a further
injection of (ꢀ)isoproterenol was given. Additionally,
rats were pretreated with reserpine (5 mg/kg, ip) 24 hr
prior to the injection of (ꢀ)phenylephrine (10 mg/kg, iv),
followed 15 min later by the intravenous injection of a
single dose of test compound. After 10 min, a further
injection of (ꢀ)phenylephrine was given.
Acknowledgements
This work was supported by research grants from
National Science Council of ROC (NSC 87-2314-B-037-
095)
References and Notes
26. Arunlakshana, O.; Schild, H. O. Br. J. Pharmacol. 1959,
14, 48.
27. Honda, K.; Takenaka, T.; Miyata-osawa, A.; Teral, M. J.
Pharmacol. Exp. Ther. 1986, 236, 776.
28. Wu, B. N.; Huang, Y. C.; Wu, H. M.; Hong, S. J.;
Chiang, L. C.; Chen, I. J. J. Cardiovasc. Pharmacol. 1998, 31,
750.
29. Miach, P. J.; Dausse, J. P.; Cardot, A.; Meyer, P. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1980, 312, 2 3.
30. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall,
R. J. J. Biol. Chem. 1951, 193, 265.
1. Frishman, W. H. Med. Clin. North Am. 1988, 72, 37.
2. Sponer, G.; Bartsch, W.; Strein, K.; Mulle-Beckmann, B.;
Bohm, E. J. Cardiovasc. Pharmacol. 1987, 9, 317.
3. Ruffolo, R. R.; Boyle, D.; Venuit, R. P.; Lukas, M. Drug of
Today 1991, 27, 465.
4. Mak, I. T.; Arroyo, C. M.; Weglicki, W. B. Circ. Res. 1989,
65, 1151.
5. Yue, T. L.; Cheng, H. Y.; Lysko, P. G.; McKenna, P. J.;
Feuerstein, R.; Gu, J. L.; Lysko, K. A.; Davis, L. L.; Feuer-
stein, G. Z. J. Pharmacol. Exp. Ther. 1992, 263, 92 .