Inhibitory effects of benzyl benzoate and its derivatives on angiotensin II-induced hypertension

Article abstract of DOI:10.1016/j.bmc.2008.03.056

Hypertension is a lifestyle-related disease which often leads to serious conditions such as heart disease and cerebral hemorrhage. Angiotensin II (Ang II) plays an important role in regulating cardiovascular homeostasis. Consequently, antagonists that block the interaction of Ang II with its receptors are thought to be effective in the suppression of hypertension. In this study, we searched for plant compounds that had antagonist-like activity toward Ang II receptors. From among 435 plant samples, we found that EtOH extract from the resin of sweet gum Liquidambar styraciflua strongly inhibited Ang II signaling. We isolated benzyl benzoate and benzyl cinnamate from this extract and found that those compounds inhibited the function of Ang II in a dose-dependent manner without cytotoxicity. An in vivo study showed that benzyl benzoate significantly suppressed Ang II-induced hypertension in mice. In addition, we synthesized more than 40 derivatives of benzyl benzoate and found that the meta-methyl and 3-methylbenzyl 2′-nitrobenzoate derivatives showed about 10-fold higher activity than benzyl benzoate itself. Thus, benzyl benzoate, its derivatives, and benzyl cinnamate may be useful for reducing hypertension.

Full text of DOI:10.1016/j.bmc.2008.03.056

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 7843–7852
Inhibitory effects of benzyl benzoate and its derivatives
on angiotensin II-induced hypertension
Osamu Ohno,a Mao Ye,b Tomoyuki Koyama,b Kazunaga Yazawa,b Emi Mura,a
Hiroshi Matsumoto,a Takao Ichino,a Kaoru Yamada,a Kazuhiko Nakamura,a
Tomohiro Ohno,c Kohji Yamaguchi,c Junji Ishida,d
Akiyoshi Fukamizud and Daisuke Uemuraa,e,
*
aDepartment of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
bGraduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan,
Minato-ku, Tokyo 108-8477, Japan
cFANCL Research Institute, FANCL Corporation, 12-13 Kamishinano, Yokohama 244-0806, Japan
dCenter for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
eInstitute of Advanced Research, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, Japan
Received 14 February 2008; revised 21 March 2008; accepted 22 March 2008
Available online 27 March 2008
Abstract—Hypertension is a lifestyle-related disease which often leads to serious conditions such as heart disease and cerebral hem-
orrhage. Angiotensin II (Ang II) plays an important role in regulating cardiovascular homeostasis. Consequently, antagonists that
block the interaction of Ang II with its receptors are thought to be effective in the suppression of hypertension. In this study, we
searched for plant compounds that had antagonist-like activity toward Ang II receptors. From among 435 plant samples, we found
that EtOH extract from the resin of sweet gum Liquidambar styraciflua strongly inhibited Ang II signaling. We isolated benzyl ben-
zoate and benzyl cinnamate from this extract and found that those compounds inhibited the function of Ang II in a dose-dependent
manner without cytotoxicity. An in vivo study showed that benzyl benzoate significantly suppressed Ang II-induced hypertension in
mice. In addition, we synthesized more than 40 derivatives of benzyl benzoate and found that the meta-methyl and 3-methylbenzyl
20-nitrobenzoate derivatives showed about 10-fold higher activity than benzyl benzoate itself. Thus, benzyl benzoate, its derivatives,
and benzyl cinnamate may be useful for reducing hypertension.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
peptide hormone angiotensin II (Ang II) produced by
RAS is a potent vasoconstrictor, and thus plays an inte-
gral role in the pathophysiology of hypertension.1–5 Ang
II causes vasoconstriction when it binds to its receptors.
Therefore, research efforts to control hypertension have
recently focused on competition for Ang II binding to its
receptors.
Hypertension is a lifestyle-related disease that is strongly
influenced by excessive intake of salt, smoking, alcohol
consumption, obesity, and stress. If the symptoms of
hypertension continue long term, serious conditions
such as heart disease and cerebral hemorrhage can oc-
cur. Thus, compounds that can suppress hypertension
are important for treating hypertension itself and for
preventing even more serious conditions. The renin–
angiotensin system (RAS) plays a determinant role in
the regulation of cardiovascular homeostasis. The octa-
There are two types of Ang II receptors: Ang II type 1
(AT1) receptor and Ang II type 2 (AT2) receptor.
AT1 receptor is closely associated with the regulation
of blood pressure, fluid, and electrolyte balance in
healthy adults, whereas AT2 receptor is expressed in fe-
tal tissues, including brain, and plays a significant role in
fetal development.6,7 When Ang II binds to AT1 recep-
tors that appear on the surface of smooth muscle cells in
blood vessels, the vessel walls constrict and hypertensive
effects occur.8 Thus, antagonists that can selectively
Keywords: Hypertension; Angiotensin II; Liquidambar styraciflua; Benzyl
benzoate; Benzyl cinnamate; Losartan.
*
Corresponding author. Tel./fax: +81 52 789 3654; e-mail:
uemura@chem3.chem.nagoya-u.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.056

7844
O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
block the interaction between Ang II and AT1 receptors
are thought to be effective for treating hypertension.
O
O
O
O
AT1 receptor antagonists have been shown to lower
blood pressure effectively, and they are better-tolerated
than other classes of drugs.9,10 The search for such com-
pounds led to the discovery of the first potent and orally
active non-peptide Ang II antagonist: losartan.11–15
Currently, several kinds of AT1 receptor antagonists,
such as candesartan, eprosartan, and irbesartan, are
used in the treatment of hypertension.16–19 The anti-
hypertensive efficacy of AT1 receptor antagonists has
been demonstrated in mild to moderate hypertension
as well as in severe essential hypertension and isolated
systolic hypertension.
Benzyl benzoate (1)
Benzyl cinnamate (2)
O
O
R
R
1
2
3-43
Considering that hypertension is a lifestyle-related dis-
ease, it is important to find ways to prevent hypertension
by improving lifestyle factors, particularly diet. There-
fore, we focused on plant components, since many of
them can be used in foods. In this study, we constructed
a new screening system and searched for plant com-
pounds that show antagonistic activity toward Ang II
receptors.
Figure 1. Structures of benzyl benzoate, benzyl cinnamate, and
the derivatives of benzyl benzoate. Substituents of 3–43 are listed in
Table 1.
values were determined to be 107.2 lg/ml for benzyl
benzoate and 47.2 lg/ml for benzyl cinnamate. As a po-
sitive control, losartan was also evaluated with this as-
say system. Losartan significantly suppressed Ang II-
induced Ca2+ uptake in mAT1a(HA)/293T cells (IC50
value: 2.4 lg/ml) (Figs. 2A and 3A).
2. Results
2.1. Inhibition of the function of Ang II by benzyl
benzoate in Ang II receptor-overexpressing cells
2.2. Benzyl benzoate is not cytotoxic toward mAT1a
(HA)/293T cells
To examine antagonistic activity toward Ang II recep-
tor, we used human embryo kidney epithelial 293T cells
(mAT1a(HA)/293T cells) that stably express AT1 recep-
tors. By interacting with its receptors, Ang II induces the
cellular uptake of calcium ion (Ca2+), which acts as a
second messenger. mAT1a(HA)/293T cells were pre-
treated with fura 2-AM, which is a specific fluorescent
indicator of Ca2+. The Ang II-induced increase in the
Ca2+ concentration in cells was evaluated in the presence
or absence of plant samples by spectrometric analysis.
To exclude the possibility that the isolated compounds
inhibited the function of Ang II by cytotoxicity toward
mAT1a(HA)/293T cells, we examined cytotoxicity by
the trypan blue dye exclusion assay. As shown in Fig-
ures 3B and C, benzyl benzoate and benzyl cinnamate
did not show any cytotoxicity toward mAT1a(HA)/
293T cells at up to 100 lg/ml after 24 h. Thus, they
inhibited the function of Ang II without showing
cytotoxicity.
We screened 435 plant extracts for compounds which
have antagonistic activity toward Ang II receptors. We
only considered samples that were not cytotoxic. In
the course of our screening, we found that the EtOH ex-
tract of the resin of sweet gum Liquidambar styraciflua
showed marked inhibitory activity toward the function
of Ang II. From this resin extract, we isolated two active
principles through several chromatographic separations.
Spectroscopic analysis of the purified compounds re-
vealed that they were identical to known aromatic com-
pounds, benzyl benzoate (1) and benzyl cinnamate (2)
(Fig. 1). In the present study, we used commercially
available benzyl benzoate (Nacalai Tesque, Inc.) and
benzyl cinnamate (Tokyo Chemical Industry Co.).
2.3. Biological evaluation of derivatives of benzyl
benzoate
To improve the activity of benzyl benzoate, we synthe-
sized derivatives of benzyl benzoate that had various
substituents on the benzyl alcohol moiety. The newly
synthesized analogs 3–43 were assayed for inhibitory
activity toward the function of Ang II in mAT1a(HA)/
293T cells and for cytotoxicity. As shown in Table 1,
many of the synthesized compounds inhibited the Ang
II-induced uptake of Ca2+ in mAT1a(HA)/293T cells.
In particular, derivatives with a methyl group at the 2-,
3-, or 4-position of the benzyl alcohol moiety (18, 19,
and 20) showed about 5–10 times stronger activity than
benzyl benzoate (for 19, Figs. 2D and 3D). The 3-meth-
ylbenzyl 20-nitrobenzoate derivative (37) also showed
high activity (Figs. 2E and 3E). All of the synthesized
compounds were confirmed not to show any cytotoxicity
toward mAT1a(HA)/293T cells at up to 100 lg/ml after
24 h (data not shown).
Treatment of mAT1a(HA)/293T cells with 1 lM Ang II
increased the intracellular Ca2+ concentration. The
addition of benzyl benzoate or benzyl cinnamate prior
to Ang II markedly decreased the intracellular Ca2+ con-
centration in mAT1a(HA)/293T cells in a dose-depen-
dent manner (Figs. 2B and C and 3B and C). The IC50

O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
7845
C
A 120
B 120
120
100
80
Vehicle
10 μg/ml
100 μg/ml
Vehicle
10 μg/ml
100 μg/ml
Vehicle
10 μg/ml
100
80
60
40
20
0
100
80
60
40
20
0
60
40
20
0
-20
-20
-20
0
50
100
150
200
0
50
100
150
200
0
50
100
150
200
Time (second)
Time (second)
Time (second)
D
E
120
100
120
Vehicle
10 μg/ml
100 μg/ml
Vehicle
10 μg/ml
100 μg/ml
100
80
60
40
20
0
80
60
40
20
0
-20
-20
0
50
100
150
200
0
50
100
150
200
Time (second)
Time (second)
Figure 2. Inhibition of Ang II-stimulated Ca2+ uptake in Ang II receptor-overexpressing cells by benzyl benzoate, benzyl cinnamate, and the
derivatives of benzyl benzoate. Fura 2-AM-loaded mAT1a(HA)/293T cells were treated with 1 lM Ang II for 200 s in the absence or presence of the
indicated concentrations of losartan (A), benzyl benzoate (B), benzyl cinnamate (C), derivative 19 (D), or derivative 37 (E). Ang II was added at time
0, and the increase in the F340/F380 ratio (% of control) was monitored as the intracellular Ca2+ uptake rate. Results show a representative tracing of
three separate experiments.
2.4. Selective inhibition of radio-labeled Ang II binding to
AT1 receptors by benzyl benzoate
Ang II. As shown in Figure 4, oral administration of
benzyl benzoate (2 and 10 mg/kg) significantly inhibited
the elevation of systolic arterial pressure induced by Ang
II treatment. This result suggested that benzyl benzoate
may have a suppressive effect on Ang II-associated
hypertension by blocking AT1 receptors in vivo. Benzyl
cinnamate was also evaluated for its in vivo anti-hyper-
tensive effect and it showed inhibitory activity (supple-
mentary data).
Next, we examined the effect of benzyl benzoate on the
binding of [125I] (Sar1, Ile8) Ang II to cells that overex-
pressed AT1 receptors. We used CHO cells that stably
expressed AT1 receptors. A c-ray counter analysis re-
vealed the binding of [125I] Ang II to the CHO transfor-
mants, and the addition of benzyl benzoate markedly
decreased the binding of [125I] Ang II at 100 lM and
300 lM (Table 2). One of the derivatives of benzyl ben-
zoate (9) also inhibited this binding. On the other hand,
benzyl benzoate did not inhibit the binding of [125I]
CGP-42112A, a specific radioligand of AT2 receptors,
to HeLa cells that stably expressed AT2 receptors.
Derivative 9 also did not inhibit this binding. An excess
amount of non-labeled Ang II (cold Ang II) decreased
fluorescence in both of the experiments (data not
shown). Thus, benzyl benzoate was shown to block the
binding of Ang II to AT1 receptors selectively.
3. Discussion
We constructed a screening system and searched for
plant compounds that inhibit the function of Ang II.
In the screening of 435 plant samples, we isolated benzyl
benzoate and benzyl cinnamate (Fig. 1) from the resin of
sweet gum L. styraciflua as antagonist-like compounds
of Ang II receptors. Benzyl benzoate and benzyl cinna-
mate were shown to inhibit the function of Ang II in a
dose-dependent manner without showing cytotoxicity
(Figs. 2B and C and 3B and C).
2.5. Anti-hypertensive effect of benzyl benzoate in Ang II-
induced hypertensive mice
For the analysis of structure–activity relationships and to
improve upon the activity of benzyl benzoate, we synthe-
sized more than 40 derivatives of the mother molecule.
Synthesized analogs with various substituents in the ben-
zyl alcohol moiety were tested to determine their biologi-
cal activities. Among them, the meta-methyl derivative
Since benzyl benzoate was expected to suppress Ang II-
induced hypertension, we subjected benzyl benzoate to
an in vivo evaluation to determine its effect on Ang II-
induced hypertension in mice. The systolic blood pres-
sure was recorded before and 4 min after treatment with

7846
O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
B
C
A
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
0
1
3
10
0
10 30 100
0
10
30 100
Losartan ( g/ml)
Benzyl benzoate ( g/ml)
Benzyl cinnamate ( g/ml)
μ
μ
μ
D
E
120
120
100
80
60
40
20
0
100
100
80
60
40
20
0
100
80
60
40
80
60
40
20
0
20
0
0
10
30 100
0
10
30 100
Derivative 19 ( g/ml)
Derivative 37 ( g/ml)
μ
μ
Figure 3. Effects of benzyl benzoate, benzyl cinnamate, and the derivatives of benzyl benzoate on Ang II-stimulated Ca2+ uptake and viability of
mAT1a(HA)/293T cells. Columns: Fura 2-AM-loaded mAT1a(HA)/293T cells were pretreated with the indicated concentrations of losartan (A),
benzyl benzoate (B), benzyl cinnamate (C), derivative 19 (D), or derivative 37 (E) for more than 50 s, and then treated with 1 lM Ang II for 200 s.
The uptake rate of intracellular Ca2+ was then determined as described in Section 5. Values are means SD of triplicate determinations. Circles;
mAT1a(HA)/293T cells were treated with the indicated concentrations of the above-mentioned compounds for 24 h, and cell viability was determined
by trypan blue dye exclusion. Values are means SD of quadruplicate determinations.
(19) showed about 10-fold higher activity than benzyl
benzoate itself (Figs. 2D and 3D). According to this
observation, various derivatives at the benzoate moiety,
including 3-methyl benzyl ester, were synthesized for bio-
logical trials. 3-Methylbenzyl 20-nitrobenzoate (37)
showed the best activity among them (Figs. 2E and 3E),
but the results were not satisfactory compared to the po-
sitive control, losartan (Figs. 2A and 3A). The results are
summarized in Table 1. From those results, it was esti-
mated that substituents with a certain size, such as methyl
and chloro groups, on benzyl alcohol moiety improved
the potential of benzyl benzoate. In addition, electroneg-
ative substituents on ortho–portion of benzoate moiety
were not thought to disturb the biological activity of 19.
Then, we tried to analyze the biological activities in terms
of various parameters (electronegativity of the substitu-
ents, hydrophobicity and bulkiness) by numerical meth-
ods, but did not find any significant relationships.
Meanwhile, we also synthesized b-phenylpropiophenone,
which possesses alkyl-ketone connectivity between two
aromatic rings, as a derivative of benzyl benzoate. It also
inhibited Ang II-induced Ca2+ uptake in mAT1a(HA)/
293T cells (IC50 value: 38.0 lg/ml, supplementary data).
Thus, two aromatic rings in a molecule may be essential
for its biological activity.
pounds are sought to obtain a better understanding of
the binding between Ang II and its receptors and to
achieve better pharmacological profiles and lower cost.
Recently, attempts have been made to design, synthe-
size, and evaluate several kinds of optimized compounds
using computer modeling software. Among them, (5S)-
1-benzylo-5-(1H
imidazol-lylo-methylo-)-2-pyrrolidi-
none (MM1) and 2-metylsulfanyl-3-[20-(2H-tetrazol-5-
yl)biphenyl-4-ylmethyl]-3H-quinazolin-4-one were re-
ported to show good activities similar to losartan.20–22
Compared to these synthesized compounds, benzyl ben-
zoate and its derivatives have much simpler structures.
They also do not have as potent a biological activity
as losartan. However, the modification of substituents
in benzyl benzoate can enhance the activity to at least
10-fold stronger than that of the mother molecule. This
result suggests that it may be possible to synthesize
much stronger derivatives from benzyl benzoate. In
addition, benzyl benzoate is a natural product. Thus,
plant extracts which contain benzyl benzoate may them-
selves have an anti-hypertensive effect.
The inhibitory effects observed with regard to Ang II-in-
duced intracellular Ca2+ uptake do not necessarily mean
antagonistic activity toward Ang II receptors. Thus, a
receptor binding assay was carried out by competitive
displacement of the binding of specific ligands of AT1
or AT2 receptor to their receptors. This experiment
was performed for benzyl benzoate. The results con-
As mentioned above, many kinds of Ang II antagonists
have been developed and are used clinically. Some of
them show adequate potential, but improved com-

O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
7847
Table 1. Synthetic derivatives of benzyl benzoate and their IC50 values
against Ang II-stimulated Ca2+ uptake in mAT1a(HA)/293T cells
*
*
Compound
R1
R2
lg/ml
200
150
100
50
1
2
H
—
H
—
107.2
47.2
70.8
98.6
85.3
40.7
53.7
89.1
97.1
87.1
52.5
186.2
135.8
280.4
229.1
120.2
70.8
20.4
11.2
20.9
117.4
39.2
42.3
56.2
81.3
81.3
50.1
24.0
32.4
26.3
24.5
44.7
34.7
56.2
56.2
>100
11.2
91.2
93.3
42.7
24.5
20.9
26.3
2.4
##
3
2-F
3-F
H
4
H
5
4-F
2-Cl
H
6
H
7
3-Cl
4-Cl
H
8
H
9
3-Br
4-Br
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Losartan
H
3-I
H
2-NO2
3-NO2
4-NO2
2-NHAc
3-NHAc
4-NHAc
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-Et
H
H
H
H
H
H
H
0
H
0
2
10
H
Benzyl benzoate (mg/kg)
H
H
Figure 4. Anti-hypertensive effect of benzyl benzoate in Ang II-
induced hypertensive mice. Male Std:ddY mice (6 weeks old) were
injected with the indicated concentrations of benzyl benzoate by oral
gavage, and then, 30 min later, received an intraperitoneal injection of
100 lg/kg Ang II. Systolic arterial pressure was then measured before
(solid column) and after (open column) Ang II treatment as described
in Section 5. Values are means SD (n = 5 per group). *p < 0.05 versus
vehicle group without benzyl benzoate. ##p < 0.005 versus before Ang
II treatment.
H
H
4-Et
H
4-i-Pr
3,4-(OMe)2
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
3-Me
—
H
H
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-Cl
3-Cl
4-C1
2-NO2
3-NO2
4-NO2
4-SO2NH2
3-OH
4-OH
3,4-(OH)2
—
tion, benzyl benzoate reduced high blood pressure in
Ang II-induced hypertensive mice (Fig. 4). In conclu-
sion, benzyl benzoate, its derivatives, and benzyl cinna-
mate may suppress hypertension, and therefore they and
plant components containing benzyl benzoate or benzyl
cinnamate may have therapeutic potential for the treat-
ment of hypertension.
4. Conclusion
In the present study we constructed a new screening sys-
tem for compounds that inhibit the function of Ang II,
and isolated benzyl benzoate and benzyl cinnamate from
the resin of sweet gum L. styraciflua, as antagonist-like
compounds of Ang II receptors. Those compounds were
shown to inhibit the function of Ang II in a dose-depen-
dent manner without showing cytotoxicity. Next, we syn-
thesized derivatives of benzyl benzoate, and tested their
biological activities. Among them, the meta-methyl (19)
and 3-methylbenzyl 20-nitrobenzoate derivatives (37)
showed about 10-fold higher activity than benzyl benzo-
ate itself. We confirmed that benzyl benzoate and one of
its derivatives block the binding of Ang II to AT1 recep-
tors selectively. Benzyl benzoate showed an anti-hyper-
tensive effect in Ang II-induced hypertensive mice.
Thus, benzyl benzoate, its derivatives, and benzyl cinna-
mate were shown to suppress the function of Ang II,
and therefore they and plant components containing ben-
zyl benzoate or benzyl cinnamate may have therapeutic
potential for the treatment of hypertension.
Table 2. Antagonistic activity of benzyl benzoate and its derivative on
Ang II receptors
Benzyl benzoate
Derivative 9
100 lM 300 lM
100 lM
300 lM
AT1 receptor (%)
AT2 receptor (%)
21
0
62
9
50
10
72
23
firmed that benzyl benzoate blocked the binding of Ang
II to AT1 receptors selectively (Table 2). One of the
derivatives of benzyl benzoate (9) also inhibited the
binding of Ang II to AT1 receptors. It was determined
that a substituent on meta-portion of benzyl alcohol in
benzyl benzoate did not diminish the inhibitory activity
of benzyl benzoate against Ang II-AT1 receptors bind-
ing. Based on these results, benzyl benzoate and its
derivatives were expected to have an anti-hypertensive
effect in an animal model. Consistent with this expecta-

7848
O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
5. Experimental
2000 Ci/mmol, was purchased from Amersham Pharma-
cia Biotech (Uppsala, Sweden). The cells in 24-well
plates were washed with an incubation buffer (50 mM
Tris–HCl, pH 7.4, 5 mM MgCl2 0.1%BSA, 1 mM
EDTA). The cells were incubated in incubation buffer
with various concentrations of radio-labeled Ang II at
20 ꢁC for 45 min. After the cells were washed twice with
the incubation buffer, the cells were lysed in 1 N NaOH
and cell-bound radioactivity was counted with a c-ray
counter (Aloka). Non-specific binding was determined
by the addition of 100-fold unlabeled Ang II to the reac-
tions. Specific binding was calculated as total binding
minus non-specific binding. The same experiments were
performed using [125I] CGP-42112A, a specific radioli-
gand of AT2 receptors, and HeLa cells that stably ex-
pressed AT2 receptors.
5.1. Materials
Ang II and benzyl benzoate were purchased from Nac-
alai Tesque, Inc. (Kyoto, Japan). Fura 2-AM was pro-
cured from Wako (Tokyo, Japan). Losartan was
kindly provided by Dr. T. Fukami, Banyu Pharmaceuti-
cal Co., Ltd. Benzyl cinnamate was purchased from To-
kyo Chemical Industry Co. (Tokyo, Japan). All other
substrates (substituted benzyl alcohols and benzoic
acids) used to synthesize derivatives of benzyl benzoate
were obtained from Sigma–Aldrich Co. (St. Louis, MO).
5.2. Cell culture
mAT1a(HA)/293T cells were prepared as described pre-
viously.23 mAT1a(HA)/293T cells were cultured at 37 ꢁC
in a humidified atmosphere of CO2–95% air in DMEM
supplemented with 10% heat-inactivated fetal bovine
serum (FBS; JRH Biosciences, Lenexa, KS), 2.25 mg/
ml NaHCO3, and 0.3 lg/ml hygromycin (Sigma–Aldrich
Co.).
5.6. In vivo experiments
Male Std:ddY mice (6 weeks old, SLC, Tokyo, Japan)
were used to examine in vivo anti-Ang II effects. They
were housed in standard cages (21.5 · 32 · 14 cm, five
mice/cage) under controlled conditions of temperature
(24 1 ꢁC), humidity (50 2%), and lighting (lights on
from 08:00 to 20:00). Animal studies were performed
in accordance with the 1980 guidelines, Notification
No. 6 of the Prime Minister’s Office of Japan.
5.3. Measurement of intracellular Ca2+ concentration
mAT1a(HA)/293T cells were harvested and washed
twice with solution A [9.8 g/L Hanks’ solution (Nissui,
Tokyo, Japan) supplemented with 5 mM HEPES (pH
7.4)]. The cells were then incubated with solution A
containing 4 lg/ml fura 2-AM and 0.2% bovine serum
albumin (BSA) for 1 h at 37 ꢁC. After being washed
twice with solution A, fura 2-AM-loaded cells were
prepared at a concentration of 5 · 106 cells/ml and
each group was used for fluorometry. Fluorescence
was measured with a CAF-110 spectrofluorometer (Ja-
pan Spectroscopic Co., Tokyo, Japan) that detected
500 nm fluorescence emitted by excitation at 340 nm
(F340) and 380 nm (F380) alternately at a frequency
of 128 Hz, and the F340/F380 ratio from successive
excitation periods was calculated. The increase in the
F340/F380 ratio (% of control) was treated as the
intracellular Ca2+ uptake rate. This was also calcu-
lated and transformed to the corresponding levels of
Ca2+ concentration as described by Grynkiewicz and
coworkers.24 The control level in Figure 2A was calcu-
lated to be 306.8 53.6 nM.
After mice received a standard laboratory diet (Oriental
Yeast, Tokyo, Japan) and water ad libitum for 1 week,
they were divided into three groups. Mice were injected
with several concentrations of benzyl benzoate by oral
gavage, and then, 30 min later, received intraperitoneal
injection of 100 lg/kg Ang II in 1% aqueous ethanol.
After 4 min, systolic arterial pressure in trained con-
scious mice was recorded using an automated tail-cuff
device (Model MK-2000, Muromachi Kikai Co., Ltd,
Tokyo, Japan). The values from five consecutive read-
ings were averaged and compared with ordinary blood
pressure values obtained from each mouse before Ang
II treatment.
5.7. Synthesis of derivatives of benzyl benzoate
5.7.1. Synthesis of 2-chlorobenzyl benzoate (6) (typical
procedure with benzyl alcohol and benzoyl chloride;
method A). To a solution of 2-chlorobenzyl alcohol
(143 mg, 1.0 mmol) in pyridine (4.0 ml) was added
benzoyl chloride (127 ll, 1.1 mmol) at 0 ꢁC under a
nitrogen atmosphere. The reaction mixture was kept
at the same temperature for 1.5 h, and the excess re-
agent was destroyed by adding water (10 ml). The
reaction mixture was extracted with ethyl acetate,
and the organic layer was washed with saturated aq
ammonium chloride followed by brine, and then evap-
orated. Purification of the residue by column chroma-
tography (silicagel 60) with hexane/EtOAc (39:1) as an
5.4. Trypan blue dye exclusion
mAT1a(HA)/293 T cells were seeded at 1 · 105 cells/well
in 24-well plates (Iwaki, Tokyo, Japan) and cultured
overnight. The cells were treated with various concentra-
tions of benzyl benzoate and its derivatives for 24 h.
They were then stained with trypan blue, and the num-
ber of stained cells was counted.
5.5. Binding assay
eluent yielded the benzyl benzoate derivative
(155 mg) as a colorless oil.
6
Ang II receptor binding assays were performed by MDS
Pharma Services-Taiwan Ltd (Taipei, Taiwan) accord-
Yield 73%; Rf 0.83 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 7.4 Hz, 2H), 7.56 (t,
J = 7.4 Hz, 1H), 7.51–7.39 (m, 4H), 7.29–7.25 (m, 2H),
ing to the method reported previously.25,26 125I] (Sar1,
[
Ile8) Ang II, which had a specific radioactivity of

O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
7849
5.46 (s, 2H); HR-ESIMS m/z 269.0328 [M+Na]+, calcd
for C14H11ClO2Na, 269.0345.
5.7.10. 2-Nitrobenzyl benzoate (12) [method A]. Yield
75%; Rf 0.46 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 8.12 (d, J = 8.0 Hz, 1H), 8.08 (d,
J = 7.5 Hz, 2H), 7.67–7.63 (m, 2H), 7.58 (t, J = 7.5 Hz
1H), 7.50–7.45 (m, 3H), 5.77 (s, 2H); IR (CDCl3)
1724, 1529 cmꢁ1; HR-ESIMS m/z 280.0576 [M+Na]+,
calcd for C14H11NO4Na, 280.0586.
1
5.7.2. 2-Fluorobenzyl benzoate (3) [method A]. H NMR
(600 MHz, CDCl3) d 8.06 (d, J = 7.5 Hz, 2H), 7.54 (t,
J = 7.7 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.42 (t,
J = 7.5 Hz, 2H), 7.34–7.31 (m, 1H), 7.15 (dd, J = ꢀ6,
ꢀ7 Hz, 1H), 7.09 (dd, J = ꢀ7, ꢀ9 Hz, 1H), 5.43 (s,
2H); HR-ESIMS m/z 253.0651 [M+Na]+, calcd for
C14H11FO2Na, 253.0641.
5.7.11. 3-Nitrobenzyl benzoate (13) [method A]. Yield
100%; Rf 0.45 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 8.31 (s, 1H), 8.19 (d, J = 7.7 Hz,
1H), 8.07 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H),
7.59–7.54 (m, 2H), 7.45 (t, J = 7.9 Hz, 2H), 5.44 (s,
2H); IR (CDCl3) 1719, 1534 cmꢁ1; HR-ESIMS m/z
280.0576 [M+Na]+, calcd for C14H11NO4Na, 280.0586.
1
5.7.3. 3-Fluorobenzyl benzoate (4) [method A]. H NMR
(400 MHz, CDCl3) d 8.09 (d, J = 7.4 Hz, 2H), 7.58 (t,
J = 7.4 Hz, 1H), 7.46 (t, J = 7.4 Hz, 2H), 7.38–7.33 (m,
1H), 7.22 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 9.6 Hz, 1H),
7.03 (dd, J = ꢀ7, ꢀ9 Hz, 1H), 5.36 (s, 2H); HR-ESIMS
m/z 253.0644 [M+Na]+, calcd for C14H11FO2Na,
253.0641.
5.7.12. 4-Nitrobenzyl benzoate (14) [method A]. Yield
94%; Rf 0.40 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 8.24 (d, J = 8.3 Hz, 2H), 8.07 (d,
J = 8.2 Hz, 2H), 7.57–7.60 (m, 3H), 7.46 (t, J = 8.2 Hz,
1
5.7.4. 4-Fluorobenzyl benzoate (5) [method A]. H NMR
(400 MHz, CDCl3) d 8.07 (d, J = 7.4 Hz, 2H), 7.57 (t,
J = 7.4 Hz, 1H), 7.46–7.42 (m, 4H), 7.07 (dd, J = ꢀ7,
ꢀ9 Hz, 2H), 5.33 (s, 2H); HR-ESIMS m/z 253.0645
[M+Na]+, calcd for C14H11FO2Na, 253.0641.
2H), 5.45 (s, 2H); IR (CDCl3) 1721, 1605, 1525 cmꢁ1
;
HR-ESIMS m/z 280.0575 [M+Na]+, calcd for
C14H11NO4Na, 280.0586.
5.7.13. 2-Methylbenzyl benzoate (18) [method A]. Yield
85%; Rf 0.55 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.07 (d, J = 7.8 Hz, 2H), 7.56 (t,
J = 7.8 Hz, 1H), 7.46–7.42 (m, 3H), 7.27–7.23 (m, 3H),
5.38 (s, 2H), 2.42 (s, 3H); HR-ESIMS m/z 249.0899
[M+Na]+, calcd for C15H14O2Na, 249.0891.
5.7.5. 3-Chlorobenzyl benzoate (7) [method A]. Yield
78%; Rf 0.73 (hexane/EtOAc = 1:1); 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 8.0 Hz, 2H), 7.58 (t,
J = 8.0 Hz, 1H), 7.47–7.43 (m, 3H), 7.32 (m, 3H), 5.34
(s, 2H); HR-ESIMS m/z 269.0336 [M+Na]+, calcd for
C14H11ClO2Na, 269.0345.
5.7.14. 3-Methylbenzyl benzoate (19) [method A]. Yield
98%; Rf 0.55 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.07 (d, J = 7.8 Hz, 2H), 7.56 (t,
J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 2H), 7.27–7.23 (m,
3H), 7.14 (d, J = 6.8 Hz, 1H), 5.38 (s, 2H), 2.42 (s,
3H); HR-ESIMS m/z 249.0896 [M+Na]+, calcd for
C15H14O2Na, 249.0891.
5.7.6. 4-Chlorobenzyl benzoate (8) [method A]. Yield
87%; Rf 0.75 (hexane/EtOAc = 1:1); 1H NMR
(400 MHz, CDCl3) d 8.06 (d, J = 7.3 Hz, 2H), 7.57 (t,
J = 7.3 Hz, 1H), 7.46–7.34 (m, 6H), 5.33 (s, 2H); HR-
ESIMS m/z 269.0330 [M+Na]+, calcd for C14H11ClO2-
Na, 269.0345.
5.7.7. 3-Bromobenzyl benzoate (9) [method A]. Yield
94%; Rf 0.67 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 7.1 Hz, 2H), 7.60–
7.56 (m, 2H), 7.49–7.44 (m, 3H), 7.29 (d, J = 7.8 Hz,
1H), 7.26 (t, J = 7.8 Hz, 1H), 5.33 (s, 2H); IR (CDCl3)
1718, 1572, 1271 cmꢁ1; HR-ESIMS m/z 312.9826 [M +
Na]+, calcd for C14H11BrO2Na, 312.9840.
5.7.15. 4-Methylbenzyl benzoate (20) [method A]. Yield
97%; Rf 0.85 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 7.56 (d, J = 7.3 Hz, 2H), 7.54 (t,
J = 7.3 Hz, 1H), 7.42 (t, J = 7.3 Hz, 2H), 7.34 (d,
J = 8.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 5.32 (s, 2H),
2.36 (s, 3H); HR-ESIMS m/z 249.0903 [M+Na]+, calcd
for C15H14O2Na, 249.0891.
5.7.8. 4-Bromobenzyl benzoate (10) [method A]. Yield
93%; Rf 0.66 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 7.92 (d, J = 7.5 Hz, 2H), 7.56
(d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.38
(t, J = 7.5 Hz, 2H), 7.35–7.33 (m, 1H), 5.34 (s,
2H); IR (CDCl3) 1718, 1590, 1269 cmꢁ1; HR-ESIMS
m/z 312.9852 [M+Na]+, calcd for C14H11BrO2Na,
312.9840.
5.7.16. 2-Methoxybenzyl benzoate (21) [method A]. Yield
92%; Rf 0.59 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 8.08 (d, J = 7.3 Hz, 2H), 7.54 (t,
J = 7.3 Hz, 1H), 7.43–7.41 (m, 3H), 7.31 (t, J = 8.0 Hz,
1H), 6.97 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H),
5.42 (s, 2H), 3.85 (s, 3H); IR (CDCl3) 1714, 1613,
1515 cmꢁ1; HR-ESIMS m/z 265.0835 [M+Na]+, calcd
for C15H14O3Na, 265.0841.
5.7.9. 3-Iodobenzyl benzoate (11) [method A]. Yield
5.7.17. 3-Methoxybenzyl benzoate (22) [method A]. Yield
100%; Rf 0.73 (hexane/EtOAc = 5:1); 1H NMR
(600 MHz, CDCl3) d 8.09 (d, J = 7.3 Hz, 2H), 7.55
(t, J = 7.3 Hz, 1H), 7.43 (t, J = 7.3 Hz, 2H), 7.30
(t, J = 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 7.00
(s, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.35 (s, 2H), 3.81
(s, 3H); IR (CDCl3) 1715, 1602, 1490 cmꢁ1; HR-
100%; 1H NMR (600 MHz, CDCl3)
d 8.07 (d,
J = 7.3 Hz, 2H), 7.79 (s, 1H), 7.66 (d, J = 7.7 Hz,
1H), 7.55 (t, J = 7.3 Hz, 1H), 7.44–7.39 (m, 3H),
7.10 (t, J = 7.7 Hz, 1H), 5.28 (s, 2H); HR-ESIMS
m/z 360.9705 [M+Na]+, calcd for C14H11IO2Na,
360.9701.

7850
O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
ESIMS
C15H14O3Na, 265.0841.
m/z
265.0840
[M+Na]+,
calcd
for
5.7.26. 3-Methylbenzyl 20-methoxybenzoate (31) [method
A]. Yield 82%; Rf 0.40 (hexane/EtOAc = 5:1); H NMR
1
(400 MHz, CDCl3) d 7.75 (d, J = 7.6 Hz, 1H), 7.38 (t,
J = 7.6 Hz, 1H), 7.19–7.17 (m, 3H), 7.05 (br s, 1H),
6.88 (t, J = 7.8 Hz, 2H), 5.23 (s, 2H), 3.83 (s, 3H), 2.29
(s, 3H); HR-ESIMS m/z 279.0992 [M+Na]+, calcd for
C16H16O3Na, 279.0997.
5.7.18. 4-Methoxybenzyl benzoate (23) [method A]. Yield
84%; 1H NMR (400 MHz, CDCl3) d 8.09 (d, J = 7.3 Hz,
2H), 7.55 (t, J = 7.3 Hz, 1H), 7.45–7.40 (m, 4H), 6.94 (t,
J = 8.5 Hz, 2H), 5.33 (s, 2H), 3.81 (s, 3H); IR (CDCl3)
1714, 1603, 1495 cmꢁ1
; HR-ESIMS m/z 265.0834
[M+Na]+, calcd for C15H14O3Na, 265.0841.
5.7.27. 3-Methylbenzyl 30-methoxybenzoate (32) [method
A]. Yield 74%; Rf 0.53 (hexane/EtOAc = 5:1); 1H
5.7.19. 2-Ethylbenzyl benzoate (24) [method A]. Yield
89%; Rf 0.75 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.05 (d, J = 7.5 Hz, 2H), 7.54 (t,
J = 7.5 Hz, 1H), 7.44–7.40 (m, 3H), 7.33–7.20 (m, 3H),
5.40 (s, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.26 (t,
J = 7.6 Hz, 3H); HR-ESIMS m/z 263.1074 [M+Na]+,
calcd for C16H16O2Na, 263.1048.
NMR (400 MHz, CDCl3) d 7.69 (d, J = 7.6 Hz,
1H), 7.61 (s, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.31–
7.26 (m, 3H), 7.16 (d, J = 6.8 Hz, 1H), 7.11 (dd,
J = 8.0, 2.7 Hz, 1H), 5.34 (s, 2H), 3.85 (s, 3H),
2.38 (s, 3H); HR-ESIMS m/z 279.0998 [M+Na]+,
calcd for C16H16O3Na, 279.0997.
5.7.28. 3-Methylbenzyl 40-methoxybenzoate (33) [method
A]. Yield 86%; Rf 0.55 (hexane/EtOAc = 5:1); H NMR
1
5.7.20. 4-Ethylbenzyl benzoate (25) [method A]. Yield
100%; Rf 0.73 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 8.4 Hz, 2H), 7.55 (t,
J = 7.4 Hz, 1H), 7.45–7.37 (m, 4H), 7.23 (d,
J = 8.0 Hz, 2H), 5.34 (s, 2H), 2.67 (q, J = 7.6 Hz, 2H),
1.25 (t, J = 7.6 Hz, 3H); HR-ESIMS m/z 263.1068
[M+Na]+, calcd for C16H16O2Na, 263.1048.
(400 MHz, CDCl3) d 7.95 (d, J = 8.8 Hz, 2H), 7.19–7.15
(m, 3H), 7.06 (d, J = 6.8 Hz, 1H), 6.83 (d, J = 8.8 Hz,
2H), 5.22 (s, 2H), 3.77 (s, 3H), 2.93 (s, 3H); HR-ESIMS
m/z 279.1012 [M+Na]+, calcd for C16H16O3Na,
279.0997.
5.7.29. 3-Methylbenzyl 20-chlorobenzoate (34) [method
5.7.21. 4-Isopropylbenzyl benzoate (26) [method A]. Yield
79%; Rf 0.70 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.08 (d, J = 8.1 Hz, 2H), 7.54 (t,
J = 8.1 Hz, 1H), 7.44–7.37 (m, 4H), 7.24 (d,
J = 8.3 Hz, 2H), 5.34 (s, 2H), 2.92 (sept, J = 6.9 Hz,
1H), 1.26 (d, J = 6.9 Hz, 6H); HR-ESIMS m/z
277.1204 [M+Na]+, calcd for C17H18O2Na, 277.1204.
A]. Yield 100%; Rf 0.53 (hexane/EtOAc = 5:1); 1H
NMR (400 MHz, CDCl3)
d 7.77 (d, J = 7.8 Hz,
1H), 7.38–7.30 (m, 2H), 7.24–7.17 (m, 4H), 7.07
(d, J = 6.4 Hz, 1H), 5.26 (s, 2H), 2.29 (s, 3H);
HR-ESIMS m/z 283.0494 [M+Na]+, calcd for
C15H13ClO2Na, 283.0502.
5.7.30. 3-Methylbenzyl 30-chlorobenzoate (35) [method
A]. Yield 88%; Rf 0.78 (hexane/EtOAc = 5:1); H NMR
1
5.7.22. 3,4-Dimethoxybenzyl benzoate (27) [method A].
Yield 81%; Rf 0.58 (hexane/EtOAc = 2:1); 1H NMR
(400 MHz, CDCl3) d 8.06 (d, J = 7.1 Hz, 2H), 7.57–
7.53 (m, 1H), 7.43 (t, J = 7.1 Hz, 2H), 7.04–6.98 (m,
2H), 6.88 (d, J = 8.1 Hz, 1H), 5.30 (s, 2H), 3.90 (s,
3H), 3.89 (s, 3H); HR-ESIMS m/z 295.0946 [M+Na]+,
calcd for C16H16O4Na, 295.0946.
(400 MHz, CDCl3) d 8.05 (s, 1H), 7.97 (d, J = 7.7 Hz,
1H), 7.53 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H),
7.32–7.24 (m, 3H), 7.19 (d, J = 7.1 Hz, 1H), 5.34 (s,
2H), 2.39 (s, 3H); HR-ESIMS m/z 283.0497 [M+Na]+,
calcd for C15H13ClO2Na, 283.0502.
5.7.31. 3-Methylbenzyl 40-chlorobenzoate (36) [method
A]. Yield 96%; Rf 0.65 (hexane/EtOAc = 5:1); H NMR
1
5.7.23. 3-Methylbenzyl 20-methylbenzoate (28) [method
A]. Yield 100%; Rf 0.78 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 7.87 (d, J = 7.3 Hz, 1H), 7.30 (t,
J = 7.3 Hz, 1H), 7.21–7.12 (m, 5H), 7.06 (d,
J = 6.6 Hz, 1H), 5.22 (s, 2H), 2.52 (s, 3H), 2.28 (s,
3H); HR-ESIMS m/z 263.1029 [M+Na]+, calcd for
C16H16O2Na, 263.1048.
(400 MHz, CDCl3) d 8.02 (d, J = 8.5 Hz, 2H), d 7.41 (d,
J = 8.5 Hz, 2H), 7.31–7.28 (m, 3H),
d 7.17 (d,
J = 7.1 Hz, 1H), 5.33 (s, 2H), 2.39 (s, 3H); HR-ESIMS
m/z 283.0521 [M+Na]+, calcd for C15H13ClO2Na,
283.0502.
5.7.32. 3-Methylbenzyl 20-nitrobenzoate (37) [method A].
Yield 92%; Rf 0.60 (hexane/EtOAc = 2:1); 1H NMR
(400 MHz, CDCl3) d 7.83 (d, J = 8.0 Hz, 1H), 7.67 (d,
J = 7.3 Hz, 1H), 7.60–7.51 (m, 2H), 7.21–7.07 (m, 4H),
5.24 (s, 2H), 2.29 (s, 3H); HR-ESIMS m/z 294.0743
[M+Na]+, calcd for C15H13NO4Na, 294.0742.
5.7.24. 3-Methylbenzyl 30-methylbenzoate (29) [method
A]. Yield 100%; Rf 0.75 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 7.81 (m, 2H), 7.29–7.16 (m, 5H),
7.07 (d, J = 6.8 Hz, 1H), 5.24 (s, 2H), 2.31 (s, 3H),
2.29 (s, 3H); HR-ESIMS m/z 263.1036 [M+Na]+, calcd
for C16H16O2Na, 263.1048.
5.7.33. 3-Methylbenzyl 30-nitrobenzoate (38) [method A].
Yield 93%; Rf 0.58 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.81 (s, 1H), 8.35–8.30 (m, 2H),
7.57 (t, J = 8.1 Hz, 1H), 7.24–7.17 (m, 3H), 7.11 (d,
J = 7.1 Hz, 1H), 5.30 (s, 2H), 2.31 (s, 3H); HR-ESIMS
m/z 294.0767 [M+Na]+, calcd for C15H13NO4Na,
294.0742.
5.7.25. 3-Methylbenzyl 40-methylbenzoate (30) [method
A]. Yield 83%; Rf 0.70 (hexane/EtOAc = 5:1); H NMR
(400 MHz, CDCl3) d 7.89 (d, J = 8.3 Hz, 2H), 7.22–7.14
(m, 5H), 7.07 (d, J = 6.8 Hz, 1H), 5.24 (s, 2H), 2.32 (s,
3H), 2.29 (s,3H); HR-ESIMS m/z 263.1065 [M+Na]+,
calcd for C16H16O2Na, 263.1048.
1

O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
7851
5.7.34. 3-Methylbenzyl 40-nitrobenzoate (39) [method A].
Yield 87%; Rf 0.75 (hexane/EtOAc = 5:1); 1H NMR
(400 MHz, CDCl3) d 8.22–8.15 (m, 4H), 7.24–7.17 (m,
3H), 7.11 (d, J = 7.1 Hz, 1H), 5.29 (s, 2H), 2.31 (s,
3H); HR-ESIMS m/z 294.0746 [M+Na]+, calcd for
C15H13NO4Na, 294.0742.
3H); HR-ESIMS m/z 328.0640 [M+Na]+, calcd for
C15H15NO4SNa, 328.0619.
5.7.39. Synthesis of 3-methylbenzyl 30-hydroxybenzoate
(41) (typical procedure with benzyl chloride and hydroxy-
benzoic acid; method C). To a solution of 3-hydroxyben-
zoic acid (27.6 mg, 0.2 mmol) in DMF (0.5 ml) were
added 3-methylbenzyl chloride (15.8 ll, 0.24 mmol)
and potassium fluoride 11.2 mg (0.2 mmol) under a
nitrogen atmosphere. The reaction mixture was re-
fluxed for 4 h. The reaction mixture was filtered
through a cotton pad after being diluted with ethyl ace-
tate, and washed with saturated aq ammonium chlo-
ride followed by brine, and then evaporated.
Purification of the residue by column chromatography
(silicagel 60) with hexane/EtOAc (39:1) as an eluent
yielded the benzyl benzoate derivative 41 (155 mg) as
a colorless oil.
5.7.35. Synthesis of 2-acetamidobenzyl benzoate (15)
(typical procedure by reductive acetylation of the corre-
sponding nitro derivative; method B). To a solution of 2-
nitrobenzyl benzoate (12, 47.5 mg, 0.2 mmol) in ethyl
acetate (4.0 ml) was added 10% palladium on carbon
(10 mg). The reaction mixture was stirred under a
hydrogen atmosphere for 3 h after the addition of acetic
anhydride 0.038 ml (0.4 mmol). The reaction mixture
was filtered through a Celite (hi-flo super cel) pad, and
concentrated. Purification of the residue by column
chromatography (silicagel 60) with hexane/EtOAc (1:1)
as an eluent yielded the benzyl benzoate derivative 15
(10.4 mg) as a crystalline solid.
Yield 73%; Rf 0.48 (hexane/EtOAc = 3:1); 1H NMR
(400 MHz, CDCl3) d 7.65–7.62 (m, 2H), 7.32–7.06 (m,
6H), 6.26 (br s, 1H), 5.32 (s, 2H), 2.37 (s, 3H); HR-
ESIMS m/z 265.0864 [M+Na]+, calcd for C15H14O3Na,
265.0841.
Yield 20%; Rf 0.43 (hexane/EtOAc = 1:1); 1H NMR
(400 MHz, CDCl3) d 8.96 (br s, 1H), 8.11–7.98 (m,
3H), 7.58 (t, J = 7.6 Hz, 1H), 7.49–7.43 (m, 3H),
7.38 (t, J = 7.3 Hz, 1H), 7.16 (t, J = 7.3 Hz, 1H),
5.37 (s, 2H), 2.27 (s, 3H); HR-ESIMS m/z
292.0967 [M+Na]+, calcd for C16H15NO3Na,
292.0950.
5.7.40. 3-Methylbenzyl 40-hydroxybenzoate (42) [method
C]. Yield 58%; Rf 0.45 (hexane/EtOAc = 3:1); H NMR
1
(400 MHz, CDCl3) d 7.90 (d, J = 8.8 Hz, 2H), 7.21–7.14
(m, 3H), 7.07 (d, J = 7.1 Hz, 1H), 6.78 (d, J = 8.8 Hz,
2H), 6.22 (br s, 1H), 5.23 (s, 2H), 2.28 (s, 3H); HR-
ESIMS m/z 265.0841 [M+Na]+, calcd for C15H14O3Na,
265.0841.
5.7.36. 3-Acetamidobenzyl benzoate (16) [method B].
Yield 50%; Rf 0.33 (hexane/EtOAc = 1:1); 1H NMR
(400 MHz, CDCl3) d 8.07 (d, J = 7.3 Hz, 2H), 7.58–
7.41 (m, 6H), 7.32 (t, J = 7.8 Hz, 1H), 7.18 (d,
J = 7.3 Hz, 1H), 5.33 (s, 2H), 2.16 (s, 3H); HR-ESIMS
m/z 292.0953 [M+Na]+, calcd for C16H15NO3Na,
292.0950.
5.7.41. 3-Methylbenzyl 30,40-dihydroxybenzoate (43)
[method C]. Yield 53%; Rf 0.25 (hexane/EtOAc = 3:1);
1H NMR (400 MHz, CDCl3) d 7.59–7.57 (m, 2H),
7.28–7.15 (m, 6H), 6.88 (d, J = 8.5 Hz, 1H), 5.29 (s,
2H), 2.38 (s, 3H); HR-ESIMS m/z 281.0818 [M+Na]+,
calcd for C15H14O4Na, 281.0790.
5.7.37. 4-Acetamidobenzyl benzoate (17) [method B].
Yield 74%; Rf 0.50 (hexane/EtOAc = 1:1); 1H NMR
(400 MHz, CDCl3) d 8.03 (d, J = 7.3 Hz, 2H), 7.77 (br
s, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.42–7.35 (m, 5H),
5.28 (s, 2H), 2.14 (s, 3H); HR-ESIMS m/z 292.0961
[M+Na]+, calcd for C16H15NO3Na, 292.0950.
Acknowledgments
The authors thank Dr. T. Fukami, Banyu Pharmaceuti-
cal Co., Ltd, for providing losartan. This work was sup-
ported in part by a Grant-in-Aid for Creative Scientific
Research from the Ministry of Education, Science, Cul-
ture, and Sports of Japan, the 21st Century Center of
Excellence (COE) program, and the Global-COE pro-
gram in Chemistry, Nagoya University.
5.7.38. Synthesis of 3-methylbenzyl 40-sulfonamoylbenzo-
ate (40). To a solution of 4-carboxybenzene sulfonamide
(20 mg, 0.1 mmol) in DMF (0.25 ml) were added, sequen-
tially, 3-methylbenzyl alcohol (28 mg, 0.23 mmol), 1-
hydroxybenzotriazole (16.8 mg, 0.11 mmol), and N,N0-
diisopropylcarbodiimide (16.6 ll, 0.11 mmol) at room
temperature under a nitrogen atmosphere, and the mix-
ture was continuously stirred for 12 h. The reaction mix-
ture was diluted with saturated aq NH4Cl and extracted
with ethyl acetate, and the organic layer was washed with
brine and evaporated. Purification of the residue by col-
umn chromatography (silicagel 60) with toluene/acetone
(4:1) as an eluent yielded the benzyl benzoate derivative
40 (15.7 mg) as a colorless oil.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.03.056.
Yield 51%; Rf 0.45 (toluene/acetone = 5:1); 1H NMR
(400 MHz, CD3OD) d 8.08 (d, J = 10.0 Hz, 2H), 7.90
(d, J = 10.0 Hz, 2H), 7.18–7.17 (m, 3H), 7.07 (d,
J = 4.0 Hz, 1H), 5.38 (s, 2H), 5.25 (s, 2H), 2.26 (s,
References and notes
1. Burnier, M.; Brunner, H. R. J. Am. Soc. Nephrol. 1999, 10,
S278.

7852
O. Ohno et al. / Bioorg. Med. Chem. 16 (2008) 7843–7852
2. Gavras, H.; Brunner, H. R.; Turini, G. A.; Kershaw, G.
15. Matsoukas, J. M.; Polevaya, L.; Ancans, J.; Mavromou-
stakos, T.; Kolocouris, A.; Roumelioti, P.; Vlahakos, D.
V.; Yamdagni, R.; Wu, Q.; Moore, G. J. Bioorg. Med.
Chem. 2000, 8, 1.
16. McClellan, K. J.; Goa, K. L. Drugs 1998, 56, 847.
17. McClellan, K. J.; Balfour, J. A. Drugs 1998, 55, 713.
18. Markham, A.; Spencer, C. M.; Jarvis, B. Drugs 2000, 59,
1187.
R.; Tifft, C. P.; Cuttelod, S.; Gavras, I.; Vukovich, R. A.;
McKinstry, D. N. N. Engl. J. Med. 1978, 298, 991.
3. Corvol, P. Clin. Exp. Hypertens. A 1989, 11, 463.
4. Brunner, H. R.; Nussberger, J.; Waeber, B. Adv. Nephrol.
Necker. Hosp. 1993, 22, 305.
5. Goodfriend, T. L.; Elliott, M. E.; Catt, K. J. N. Engl. J.
Med. 1996, 334, 1649.
6. Whitebread, S.; Mele, M.; Kamber, B.; de Gasparo, M.
Biochem. Biophys. Res. Commun. 1989, 163, 284.
7. Wu, X.; Wan, Y.; Mahalingam, A. K.; Murugaiah, A.
19. Goa, K. L.; Wagstaff, A. J. Drugs 1996, 51, 820.
20. Moutevelis-Minakakis, P.; Gianni, M.; Stougiannou, H.;
Zoumpoulakis, P.; Zoga, A.; Vlahakos, A. D.; Iliodrom-
itis, E.; Mavromoustakos, T. Bioorg. Med. Chem. Lett.
2003, 13, 1737.
21. Mavromoustakos, T.; Moutevelis-Minakakis, P.; Koko-
tos, C. G.; Kontogianni, P.; Politi, A.; Zoumpoulakis, P.;
Findlay, J.; Cox, A.; Balmforth, A.; Zoga, A.; Iliodrom-
itis, E. Bioorg. Med. Chem. 2006, 14, 4353.
´
M.; Plouffe, B.; Botros, M.; Karlen, A.; Hallberg, M.;
Gallo-Payet, N.; Alterman, M. J. Med. Chem. 2006,
49, 7160.
8. Basso, N.; Terragno, N. A. Hypertension 2001, 38, 1246.
9. Singh, R. K.; Barker, S. Curr. Opin. Invest. Drugs 2005, 6,
269.
10. Mancia, G.; Seravalle, G.; Grassi, G. Am. J. Hypertens.
2003, 16, 1066.
22. Ismail, M. A.; Barker, S.; Abou el-Ella, D. A.; Abouzid,
K. A.; Toubar, R. A.; Todd, M. H. J. Med. Chem. 2006,
49, 1526.
23. Ishida, J.; Asada, S.; Daitoku, H.; Fujiwara, K.; Kon, Y.;
Sugaya, T.; Murakami, K.; Nakajima, T.; Kasuya, Y.;
Fukamizu, A. Int. J. Mol. Med. 1999, 3, 263.
24. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem.
1985, 260, 3440.
25. Webb, M. L.; Liu, E. C.; Monshizadegan, H.; Hedberg,
A.; Misra, R. N.; Goldenberg, H.; Harris, D. N.
J. Pharmacol. Exp. Ther. 1993, 264, 1387.
26. Whitebread, S. E.; Taylor, V.; Bottari, S. P.; Kamber, B.;
de Gasparo, M. Biochem. Biophys. Res. Commun. 1991,
181, 1365.
11. Adang, A.; Hermkens, P.; Linders, J.; Ottenheijm, H.;
Staveren, C. J. R. Neth. Chem. Soc. 1994, 113, 63.
12. Carini, D. J.; Duncia, J. V.; Johnson, A. L.; Chiu, A. T.;
Price, W. A.; Wong, P. C.; Timmermans, P. B. J. Med.
Chem. 1990, 33, 1330.
13. Duncia, J. V.; Carini, D. J.; Chiu, A. T.; Johnson, A. L.;
Price, W. A.; Wong, P. C.; Wexler, R. R.; Timmermans, P.
B. Med. Res. Rev. 1992, 12, 149.
14. Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.;
Panagiotopoulos, D.; Yamdagni, R.; Wu, Q.; Mavromou-
stakos, T.; Maia, H. L.; Ganter, R.; Moore, G. J. J. Med.
Chem. 1995, 38, 4660.