Design, synthesis and biological evaluation of 6-substituted aminocarbonyl benzimidazole derivatives as nonpeptidic angiotensin II AT1 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2012.01.009

A series of 6-substituted aminocarbonyl benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. The preliminary pharmacological evaluation revealed nanomolar AT₁ receptor binding affinity and good AT₁ receptor selectivity over AT₂ receptor for all compounds of the series, a potent antagonistic activity in isolated rabbit aortic strip functional assay for compounds 6b, 6d and 6i was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6i is an orally active AT₁ receptor antagonist with low toxicity.

Full text of DOI:10.1016/j.ejmech.2012.01.009

European Journal of Medicinal Chemistry 49 (2012) 183e190
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of 6-substituted aminocarbonyl
benzimidazole derivatives as nonpeptidic angiotensin II AT1 receptor antagonists
,
a
,
a
a
a
a
Jin-Liang Wang a, Jun Zhang a , Zhi-Ming Zhou , Zhi-Huai Li , Wei-Zhe Xue , Di Xu , Li-Ping Hao ,
*
*
Xiao-Feng Han a, Fan Fei a, Ting Liu b, Ai-Hua Liang b
a R&D Center for Pharmaceuticals, School of Chemical Engineering & the Environment, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing 100081, China
b Institute of Chinese Materia Medica, China Academy of Chinese Medical Science, Beijing 100029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-substituted aminocarbonyl benzimidazole derivatives were designed and synthesized as
nonpeptidic angiotensin II AT1 receptor antagonists. The preliminary pharmacological evaluation
revealed nanomolar AT1 receptor binding affinity and good AT1 receptor selectivity over AT2 receptor for
all compounds of the series, a potent antagonistic activity in isolated rabbit aortic strip functional assay
for compounds 6b, 6d and 6i was also demonstrated. Furthermore, evaluation in spontaneous hyper-
tensive rats and a preliminary toxicity evaluation showed that compound 6i is an orally active AT1
receptor antagonist with low toxicity.
Received 15 September 2011
Received in revised form
29 December 2011
Accepted 5 January 2012
Available online 17 January 2012
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Angiotensin II AT1 receptor antagonists
Aminocarbonyl benzimidazole
Hypertension
1. Introduction
these drugs is not feasible because of their low oral bioavailability
and short half-life [6]. The first nonpeptidic Ang II receptor antag-
It is well known that the renin-angiotensin aldostenone system
(RAAS) plays a key role in fluid/electrolyte homeostasis and blood
pressure regulation [1,2]. Angiotensin II (Ang II) is an active octa-
peptide and a potent vasoconstrictor in the RAAS, which is
produced in vivo from angiotensin I by the angiotensin-converting
enzyme (ACE). Regulators of the RAAS have been found to be
effective for the treatment of hypertension and congestive heart
failure, and they continue to be one of the most active areas of drug
discovery [3]. Among them, ACE inhibitors, such as captopril and
enalapril, are useful in the treatment of hypertension, but may
cause side effects, such as hypotension, angioedema and dry cough
[4]. On the other hand, Ang II receptor antagonists directly block the
effect of Ang II at the receptor level, without affecting kininase II-
related factors, such as the bradykinin system. This provides
a more specific way to the inhibition of the activity of the RAAS and
has been considered a reliable alternative to inhibitors of ACE and
renin [5].
onists, N-benzylimidazole-5-acetic acid derivatives, were firstly
reported by the Takeda laboratories [7]. Since then, this imidazole
lead has been developed into a series of potent, selective, and orally
active AT1 receptor antagonists. Losartan is the most widely used
drug of this series, and numerous modifications to its chemical
structure have generated a large number of Ang II antagonists
[8e12], including candesartan, tasosartan, telmisartan, irbesartan
and olmesartan (Fig. 1), all of which have been used clinically.
Data available from the literature [10] indicated that a benz-
imidazole scaffold was the most using core for good binding affinity
with AT1 receptor. Various substitutions in the benzimidazole
nucleus at positions 5e7 havebeen studied for further modifications
[9,10,13].
40-[[6-Carboxyl-4-methyl-2-propropyl-1H-benzimida-
zolyl]methyl]-2-biphenyl carboxylicacid (1, Fig. 2) was previously
synthesized in our laboratory and exhibited moderate activity [14].
Thus we envisaged that the insertion of an aminocarbonyl moiety
with lipophilic substituents at the 6-position of the benzimidazole
nucleus as hydrogen-bond acceptor would produce significant
effects on the binding affinities to the AT1 receptor. In this paper,
We describe the design, synthesis and biological evaluation of
6-substituted aminocarbonyl benzimidazole derivatives. A molec-
ular modelling simulation was also performed to support our efforts
in optimising the biological activity of the prepared compounds and
obtaining a better understanding of the experimental results.
Peptidic Ang II antagonists, such as saralasin, were found to
reduce blood pressure in hypertensive patients with high renin
levels. Unfortunately, long-term antihypertensive treatment with
* Corresponding authors. Tel./fax: þ86 10 68918982.
E-mail addresses: zhangjunbit@sina.com (J. Zhang), zzm@bit.edu.cn
(Z.-M. Zhou).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.009

184
J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
Scheme 1. Reagents and conditions: (a) thionyl dichloride, reflux, 3 h; (b) various
substituted amines, Et3N, DCM, rt,
h; (c) methyl-40-bromomethylbiphenyl-2-
6
carboxylic acid, t-BuOK, DMF, rt, 12 h; (d) NaOH, MeOH, H2O, reflux, 3 h.
Fig. 1. Structures of several AT1 receptor antagonists.
expressed as IC50 values, which is the concentration of a compound
that inhibits [125I] Ang II binding to the receptor by 50%. The
antagonistic activity of the more potent compounds (6b, 6d and 6i)
were also investigated in vitro using the contractile response of
isolated rabbit aortic strips in a functional assay [17]. Losartan was
taken as a positive control drug in the assays. At last, the most
potent compound 6i was evaluated in an in vivo model.
Furthermore, a preliminary evaluation of toxicity to luminescent
bacteria was performed on the prepared compounds according to
the standard microtox test using luminescent bacteria, which is
considered to be a useful tool, providing quick and reliable data
[18].
2. Chemistry
The target compounds 6ae6k were synthesized according to
the route described in Scheme 1, and the starting material
4-methyl-2-propyl-1H-benzimidazole6-carboxylic acid (2) used in
this scheme was prepared from 3-methyl-4-nitrobenzoic acid
according to the reported methods [14]. This compound was con-
verted to the acyl chloride with thionyl dichloride under reflux
conditions; the acyl chloride was then coupled with different
amines to give the acylamide compounds 4ae4k in 70e80% yield.
The acylamides were then alkylated with methyl 40-(bromomethyl)
biphenyl-2-carboxylate using potassium tert-butoxide in DMF to
give the corresponding products in yields of 70e90% yield. The last
step, hydrolysis of 5ae5k using aqueous sodium hydroxide in
boiling MeOH, was accomplished with a greater than 70% yield.
All target compounds were identified by IR, 1H NMR, 13C NMR
and HRMS. To provide confirmatory evidence on the regiochemis-
try of our target molecules, a Nuclear Overhauser Effect (NOE)
experiment was conducted on one of the alkylation products,
compound 5i (Fig. 3). In DMSO-d6, 5i showed an NOE between the
4. Molecular modelling
In the molecular modelling simulation study, six clinically used
sartans (Fig. 1) were selected as the training set, and the pharma-
cophore hypothesis generation was performed using the HipHop
module of the Discovery Studio software [19]. Though the 3D
structure of AT1 receptor is unknown until now, an ideal pharma-
cophore hypothesis can be identified [20]. Accordingly, we used the
method as a template, which represents the geometry of the
receptor sites as a collection of functional groups in space. The fit
values of the synthesised compounds were determined on the
selected hypothesis of the AT1 receptor antagonist using the best fit
algorithm as described in the literature [21].
hydrogen of the methyl at C-4 (
an NOE between the protons on the methylene adjacent to N-1
5.60) and the proton at C-7 ( 7.95), and an NOE between the
protons on the methylene adjacent to N-1 ( 5.60) and the proton at
d 2.59) and the proton at C-5 (d 7.60),
(d
d
d
the methylene adjacent to C-2. Furthermore, the structure of
compound 5e was confirmed by X-ray crystallography [15] (Fig. 4).
Taken together, these indicate that the N1 alkylation occurred
rather than the N3 alkylation.
5. Results and discussion
As summarised in Table 1, the binding assay and the antagonistic
activity results showed that all compounds displayed a certain
extent of inhibition activity as expected. Most of compounds
showed high selectivity for the AT1 receptor over AT2 receptor,
given that the binding affinities were in the submicromolar range
for the AT1 receptor and in the micromolar range for the AT2
receptor. Of all the synthesised compounds, compound 6i was the
most potent compound (AT1 IC50 ¼ 1.3 nM, AT2 IC50 ¼ 1400 nM),
which exhibited almost the same binding affinity as the marketed
AT1 receptor antagonist telmisartan (IC50 ¼ 1.0 nM, 0.33 nM [23]),
3. Biological evaluation
The prepared compounds were evaluated for their in vitro Ang II
receptor binding affinity in the competitive inhibition of [125I] Ang
II binding to the AT1 and AT2 receptors by a conventional ligand-
binding assay as described previously [16]. The results are
Fig. 2. Structure of compound 1.
Fig. 3. NOESY of compound 5i.

J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
185
connect the acylamino moiety and the phenyl ring) and the
substitution pattern on the aromatics played key roles in binding
affinity.
Based on the results of in vitro AngII-binding assay and func-
tional antagonism, 6i were selected for further evaluation of in vivo
models. When evaluated orally in conscious spontaneously
hypertensive rats (SHR), compound 6i at doses of 10 mg/kg
significantly decreased blood pressure by more than 30 mmHg
(Fig. 5), which is more efficacious than losartan.
In the molecular modelling simulation section, fit value indi-
cates how well the chemical features in the molecule map the
features in the pharmacophore [22]. The higher the fit value is, the
more active a compound is. In this study, there are five features in
the selected pharmacophore hypothesis, so the highest fit value is
5. Accordingly, the estimated activity could be classified according
to the fit value as highly active (fit value > 4, þþþ), moderately
active (2 < fit value<4, þþ) and low active (fit value < 4, þ). All
synthesized compounds of this study were also classified according
to the literature [21] by their activity as highly active
(<100 nM, þþþ), moderately active (100e10000 nM, þþ) and low
active (>10000 nM, þ). All highly active compounds were pre-
dicted correctly, only four moderately active compounds were
predicted to be highly active. The results showed that despite these
disparities, it could still to conclude that the pharmacophore
hypothesis was reasonable. Furthermore, the molecular modelling
simulation performed above reflected the interaction of ligands
with the AT1 receptor. Unexpectedly, it was the benzene ring in the
phenylethyl, rather than the propyl at the 2-position of the benz-
imidazole ring that mapped onto the hydrophobic aliphatic feature.
The same binding modality was also observed in the mapping of
telmisartan with the pharmacophore (Fig. 6), in which it was the
methyl in the second benzimidazole ring and not the propyl that
mapped onto the hydrophobic aliphatic feature. However, in the
case of losartan, it was the alkyl substituent that mapped onto the
Fig. 4. Crystal structure of compound 5e.
and about 12-fold the affinity of losartan (IC50 ¼ 16.2 nM, 150 nM
[24], 6.7 nM [17,25]). The pA2 8.50 also shows that this compound
contains more potent antagonist activity than the positive drug
losartan (pA2 ¼ 7.9). Compounds 6b (AT1 IC50 ¼ 2.4 nM, AT2
IC50 ¼ 1100 nM) and 6d (AT1 IC50 ¼ 4.3 nM, AT2 IC50 ¼ 3600 nM)
were also much more active than losartan. The pA2 of compound 6b
and 6d are 8.16 and 8.32 respectively, which are higher than that of
losartan. In addition, compounds 6a, 6e, 6f and 6h showed the
same activity level as losartan (10 nM < AT1 IC50 < 100 nM). These
results suggested that both the length of aliphatic chains (which
Table 1
Fit values on the selected pharmacophore and experimental data of the target compounds.
N
N
H
N
R
n
O
H
COO
a-
6 6k
No.
n
R
Fit value
IC50(nM)
AT1
est. act. scalea
act.(AT1R) scaleb
pA2
LC50 (mg Lꢀ1
)
AT2
6a
6b
6c
6d
6e
6f
6g
6h
6i
1
1
1
1
1
2
2
2
2
2
2
H
4.78
4.70
4.77
4.87
4.90
4.90
4.84
4.81
4.85
4.71
4.91
4.76
4.91
52.5
2.4
326
4.3
30.6
30.8
214
57.6
1.3
106
198
16.2
1.0
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþ
78
>100c
>100
>100
>100
72
>100
>100
>100
65
2-OMe
3-OMe
4-OMe
3,4-di-OMe
3-OMe
4-OMe
3,4-di-OMe
2,5-di-OMe
2-F
1100
>10,000
3600
>10,000
>10,000
1500
1200
1400
NT
1300
8.16
8.32
þþþ
þþþ
þþþ
þþ
þþþ
þþþ
þþ
8.50
6j
6k
4-F
Losartan
Telmisartan
þþ
89
210
>100
>10,000
þþþ
þþþ
a
Estimated activity scale: highly active (fit value > 4, þþþ), moderately active (2 < fit value < 4, þþ) and low active (fit value < 4, þ).
Activity scale: highly active (<100 nM, þþþ), moderately active (100e10000 nM, þþ) and low active (>10000 nM, þ) [21].
b
c
No LC50 value could be determined because it did not produce more than 50% inhibition of luminescence of photobacterium phosphoreum at the saturated concentration
level.

186
J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
spectra were recorded on a PerkinElmer Spectrometer with KBr
pellets, Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were recorded on a Bruker NMR spectrometer (400 MHz) using
TMS as internal standard (chemical shift in ppm). High-resolution
mass spectrums were recorded on a Fourier Transform Ion Cyclo-
tron Resonance Mass Spectrometer. Silica gel column chromatog-
raphy was performed with silica gel (200e300 mesh) and F254 pre-
coated silica gel TLC plate.
7.1.1. 4-Methyl-2-propyl-1H-benzimidazole-6-carboxylic
chloride (3)
A
mixture
of
4-methyl-2-propyl-1H-benzimidazole-6-
carboxylic acid (2) (5.2 g, 23.8 mmol) and thionyl dichloride
(30 mL) was refluxed for 4 h, then thionyl dichloride was evapo-
rated under reduced pressure to obtain off-white solid (5.64 g). It
was used for the next step directly.
Fig. 5. Effects of 6i and losartan (10 mg/kg po) on mean arterial pressure in conscious
SHR after oral administration.
7.1.2. General procedure for the preparation of acylamides
(compound 4ae4k)
hydrophobic aliphatic feature (Fig. 7). This difference implied that
the interaction modality of 6i, like that of telmisartan, was not
exactly the same as that of losartan. It is worth noting that the
different interaction modalities of telmisartan and losartan have
been reported in the literature [26] and were obtained through the
docking of ligands with the homology modelled AT1 receptor.
In the toxicity assay, the results revealed that the LC50 value of
most our synthetic compounds and telmisartan are higher than
100 mg Lꢀ1, for they did not produce more than 50% inhibition of
bacterial luminescence at the saturated concentration level. It is
A mixture of amines (1.1 mmol), dichloromethane (30 mL) and
triethylamine (0.3 g, 3 mmol) was placed in a three-necked bottle
and cooled with ice. Then 4-methyl-2-propyl-1H-benzimidazole-6-
carboxylic chloride (3) (0.24 g, 1 mmol) was added slowly. After the
addition, the mixture was stirred at room temperature for 6 h. The
white precipitate formed in the mixture was filtered, the filtrate
was evaporated and the solid obtained was washed with saturated
sodium bicarbonate and brine. The obtained acylamides was
recrystallized from ethanol.
worth mentioning that the LC50 value of losartan is 331 mg Lꢀ1
,
which is almost the same as that (210 mg Lꢀ1) obtained in another
paper when the algae was used [27]. This experiment provide
a choice to evaluate the preliminary toxicity of these compounds
and the results showed that the toxicity of most compounds is as
low as that of telmisartan.
7.1.2.1. N-Benzyl-[(4-methyl-2-propyl-1H-benzimidazole)-6-yl]
amide (4a). White solid (0.22 g, 72.3%), mp: 134e136 ꢁC. 1H NMR
(400 MHz, DMSO)
d
: 0.96 (t, J ¼ 7.2 Hz, 3H), 1.76 (m, 2H), 2.57 (s,
3H), 2.84 (t, J ¼ 7.1 Hz, 2H), 4.32 (s, 2H), 7.06e7.21 (m, 5H), 7.61
(s, 1H), 7.82 (s, 1H), 8.45 (s, 1H).
7.1.2.2. N-(2-Methoxybenzyl)-[4-methyl-2-propyl-1H-benzimid-
6. Conclusions
azole)-6-yl]amide (4b). White solid (0.26 g, 77.9%),mp: 129e131 ꢁC.
1H NMR (400 MHz, DMSO)
d
: 0.94 (t, J ¼ 7.3 Hz, 3H), 1.76 (m, 2H),
In summary, a series of 6-substituted aminocarbonyl benz-
imidazole derivatives were designed and synthesized as non-
peptidic AT1 receptor antagonists. Compounds 6i was found to be
the most potent AT1-selective AII receptor antagonists with low
toxicity, it could be used as lead compounds for the further design
and synthesis of more potent nonpeptidic angiotensin II AT1
receptor antagonists.
2.56 (s, 3H), 2.83 (t, J ¼ 7.1 Hz, 2H), 3.47 (s, 3H), 4.45 (s, 2H),
7.09e7.31 (m, 4H), 7.57 (s, 1H), 7.88 (s, 1H), 8.48 (s, 1H).
7.1.2.3. N-(3-Methoxybenzyl)-[4-methyl-2-propyl-1H-benzimid-
azole)-6-yl]amide (4c). White solid (0.25 g, 74.8%), mp: 132e134 ꢁC.
1H NMR (400 MHz, DMSO)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.77 (m, 2H),
2.55 (s, 3H), 2.86 (t, J ¼ 7.1 Hz, 2H), 3.72 (s, 3H), 4.42 (s, 2H),
6.78e7.24 (m, 4H), 7.58 (s, 1H), 7.87 (s, 1H), 8.47 (s, 1H).
7. Experimental section
7.1.2.4. N-(4-Methoxy
azole)-6-yl]amide (4d). White solid (0.24 g, 71.4%), mp: 133e135 ꢁC.
1H NMR (400 MHz, DMSO)
benzyl)-[4-methyl-2-propyl-1H-benzimid-
7.1. Chemistry
d: 0.94 (t, J ¼ 7.2 Hz, 3H), 1.79 (m, 2H),
The melting points were determined on an XT-4A melting point
apparatus and are uncorrected. All the target compounds were
characterised by IR, 1H NMR, 13C NMR and HRMS. Infrared (IR)
2.56 (s, 3H), 2.87 (t, J ¼ 7.2 Hz, 2H), 3.78 (s, 3H), 4.47 (s, 2H),
6.82e7.29 (m, 4H), 7.55 (s, 1H), 7.82 (s, 1H), 8.46 (s, 1H).
Fig. 6. (a) The selected pharmacophore hypothesis; (b) Mapping of 6i with the selected hypothesis; (c) Mapping of telmisartan with the selected hypothesis.

J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
187
into 30 mL water then extracted with ethyl ester. The extract was
washed with water and dried (MgSO4). After the evaporation of
the solvent in vacuo, the residue was purified by flash column
chromatography.
7.1.3.1. Methyl 40-[(6-benzylaminocarboxyl-4-methyl-2-propyl-1H-
benzimidazolyl)methyl]biphenyl-2- carboxylate (5a). White solid
(801 mg, 75.3%), mp: 122e124 ꢁC. 1H NMR (400 MHz, CDCl3)
d: 0.98
(t, J ¼ 7.4 Hz, 3H), 1.80 (m, 2H), 2.65 (s, 3H), 2.84 (t, J ¼ 7.6 Hz, 2H),
3.58 (s, 3H), 4.59 (t, J ¼ 5.6 Hz, 2H), 5.33 (s, 2H), 6.99e7.80 (m, 15H),
8.44 (s, 1H); ESIMS(m/z): 532.3(M þ H)þ.
Fig. 7. Mapping of losartan with the selected hypothesis.
7.1.2.5. N-(3,4-Dimethoxy benzyl) -[4-methyl-2-propyl-1H-benz-
7.1.3.2. Methyl 40-[[6-(20-methoxy)benzylaminocarboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]biphenyl-2- carboxylate (5b). White
solid (940 mg, 83.6%), mp: 163e164 ꢁC. 1H NMR (400 MHz, CDCl3)
imidazole)-6-yl]amide (4e). White solid (0.28 g, 75.0%), mp:
128e130 ꢁC. 1H NMR (400 MHz, DMSO)
d
: 0.81 (t, J ¼ 7.3 Hz, 3H),
1.72 (m, 2H), 2.50 (s, 3H), 2.81 (t, J ¼ 7.2 Hz, 2H), 3.71 (s, 3H),
d
: 0.97 (t, J ¼ 7.2 Hz, 3H), 1.74 (m, 2H), 2.55 (s, 3H), 2.84 (t, J ¼ 7.6 Hz,
3.79 (s, 3H), 4.43 (s, 2H), 6.99e7.01 (m, 3H), 7.68 (s, 1H), 7.81 (s, 1H).
2H), 3.47 (s, 3H), 3.69 (s, 3H), 4.45 (s, 2H), 5.55 (s, 2H), 7.12e7.86 (m,
14H), 8.45 (s, 1H); ESIMS(m/z):562.3(M þ H)þ.
7.1.2.6. N-(3-Methoxyphenethyl)-[(4-methyl-2-propyl-1H-benzimid-
azole)-6-yl]amide (4f). White solid (0.27 g, 75.7%), mp: 148e150 ꢁC.
7.1.3.3. Methyl 40-[[6-(30-methoxy)benzylaminocarboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]biphenyl-2- carboxylate (5c). White
solid (1007 mg, 89.7%), mp: 124e125 ꢁC. 1H NMR (400 MHz, CDCl3)
1H NMR (400 MHz, DMSO)
d
: 0.81 (t, J ¼ 7.2 Hz, 3H), 1.71 (m, 2H),
2.49 (s, 3H), 2.79 (t, J ¼ 7.2 Hz, 2H), 2.89 (t, J ¼ 7.2 Hz, 2H), 3.65
(m,2H), 3.77 (s, 3H), 6.77e7.20 (m, 4H), 7.67 (s, 1H), 7.78 (s, 1H).
d
: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.76 (m, 2H), 2.56 (s, 3H), 2.79 (t, J ¼ 7.2 Hz,
2H), 3.68 (s, 3H), 3.73 (s, 3H), 4.41 (s, 2H), 5.56 (s, 2H), 7.08e7.92 (m,
14H), 8.43 (s, 1H); ESIMS(m/z): 562.3(M þ H)þ.
7.1.2.7. N-Benzyl-[(4-methyl-2-propyl-1H-benzimidazole)-6-yl]
amide (4g). White solid (0.27 g, 76.6%), mp: 149e151 ꢁC. 1H NMR
(400 MHz, DMSO)
3H), 2.77 (t, J ¼ 7.2 Hz, 2H), 2.88 (t, J ¼ 7.1 Hz, 2H), 3.40 (m, 2H),
3.67 (s, 3H), 6.84e7.32 (m, 4H), 7.45 (s, 1H), 7.84 (s, 1H).
d
: 0.98 (t, J ¼ 7.3 Hz, 3H), 1.81 (m, 2H), 2.57 (s,
7.1.3.4. Methyl 40-[[6-(40-methoxy)benzylaminocarboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]biphenyl-2- carboxylate (5d). White
solid (1028 mg, 91.5%), mp: 147e148 ꢁC. 1H NMR (400 MHz, CDCl3)
d
: 0.96 (t, J ¼ 7.2 Hz, 3H), 1.75 (m, 2H), 2.55 (s, 3H), 2.79 (t, J ¼ 7.2 Hz,
7.1.2.8. N-(3,4-Dimethoxyphenethyl)-[(4-methyl-2-propyl-1H-benz-
imidazole)-6-yl]amide (4h). White solid (0.30 g, 78.7%), mp:
2H), 3.69 (s, 3H), 3.77 (s, 3H), 4.46 (s, 2H), 5.56 (s, 2H), 7.09e7.90 (m,
14H), 8.44 (s, 1H); ESIMS(m/z): 562.3(M þ H)þ.
147e149 ꢁC. 1H NMR (400 MHz, DMSO)
1.78 (m, 2H), 2.56 (s, 3H), 2.77 (t, J ¼ 7.2 Hz, 2H), 2.87 (t,
J ¼ 7.2 Hz, 2H), 3.47 (m, 2H), 3.72 (s, 3H), 3.78 (s, 3H), 6.98e7.15
(m, 3H), 7.72 (s, 1H), 7.89 (s, 1H).
d
: 0.96 (t, J ¼ 7.2 Hz, 3H),
7.1.3.5. Methyl 40-[[6-(30,40-dimethoxy)benzylaminocarboxyl-4-methyl-
2-propyl-1H-benzimidazolyl]methyl]biphenyl-2- carboxylate (5e). White
solid (927 mg, 78.3%), mp: 126e127 ꢁC. 1H NMR (400 MHz, CDCl3)
d:
0.96 (t, J ¼ 7.2 Hz, 3H), 1.75 (m, 2H), 2.55 (s, 3H), 2.79 (t, J ¼ 7.2 Hz, 2H),
3.67 (s, 3H), 3.72 (s, 3H), 3.80 (s, 3H), 4.42 (s, 2H), 5.55 (s, 2H), 7.07e7.93
(m, 13H), 8.43 (s, 1H)；ESIMS(m/z): 592.3(M þ H)þ.
7.1.2.9. N-(2,5-Dimethoxyphenethyl)-[(4-methyl-2-propyl-1H-benz-
imidazole)-6-yl]amide (4i). White solid (0.28 g, 72.4%), mp:
145e147 ꢁC. 1H NMR (400 MHz, DMSO)
1.76 (m, 2H), 2.55 (s, 3H), 2.78 (t, J ¼ 7.2 Hz, 2H), 2.86 (t,
J ¼ 7.2 Hz, 2H), 3.48 (m, 2H), 3.70 (s, 3H), 3.76 (s, 3H), 6.94e7.12
(m, 3H), 7.70 (s, 1H), 7.86 (s, 1H).
d
: 0.95 (t, J ¼ 7.2 Hz, 3H),
7.1.3.6. Methyl 40-[[6-(30-methoxy)phenethylaminocarboxyl-4-methyl-
2-propyl-1H-benzimidazolyl]methyl]biphenyl-2- carboxylate (5f). White
solid (832 mg, 72.3%), mp: 110e112 ꢁC. 1H NMR (400 MHz, CDCl3)
d:
1.02 (t, J ¼ 7.4 Hz, 3H), 1.80e1.86 (m, 2H), 2.68 (s, 3H), 2.87e2.92 (m,
4H), 3.61 (s, 3H), 3.67e3.72 (m, 2H), 3.77 (s, 3H), 5.40 (s, 2H), 6.21（s,
1H）, 6.77e7.62 (m, 13H), 7.81 (d, J ¼ 0.8 Hz, 1H); ESIMS (m/z):
576.3(M þ H)þ.
7.1.2.10. N-(2-Fluorophenethyl-[(4-methyl-2-propyl-1H-benzimid-
azole)-6-yl]amide (4j). White solid (0.26 g, 76.3%), mp: 133e135 ꢁC.
1H NMR (400 MHz, DMSO)
d: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.67 (m, 2H),
2.55 (s, 3H), 2.75 (t, J ¼ 7.2 Hz, 2H), 2.86 (t, J ¼ 7.2 Hz, 2H), 3.53
(m, 2H), 6.93e7.25 (m, 4H), 7.47 (s, 1H), 7.82 (s, 1H).
7.1.3.7. Methyl 40-[[6-(40-methoxy)phenethylaminocarboxyl-4-methyl-
2-propyl-1H-benzimidazolyl]methyl]biphenyl-2-carboxylate (5g). White
7.1.2.11. N-(4-Fluorophenethyl-[(4-methyl-2-propyl-1H-benzimid-
solid (931 mg, 81.0%), mp: 113e114 ꢁC. 1H NMR (400 MHz, CDCl3)
d:
azole)-6-yl]amide (4k). White solid (0.25 g, 74.4%), mp: 137e139 ꢁC.
0.97 (t, J ¼ 7.2 Hz, 3H), 1.73 (m, 2H), 2.56 (s, 3H), 2.78 (t, J ¼ 7.2 Hz, 2H),
2.87 (t, J ¼ 7.1 Hz, 2H), 3.47 (m, 2H), 3.58 (s, 3H), 3.69 (s, 3H), 5.56 (s, 2H),
7.10e7.90 (m, 14H), 8.45 (s, 1H); ESIMS (m/z): 576.3(M þ H)þ.
1H NMR (400 MHz, DMSO)
2.56 (s, 3H), 2.78 (t, J ¼ 7.3 Hz, 2H), 2.87 (t, J ¼ 7.2 Hz, 2H), 3.56
(m, 2H), 6.97e7.23 (m, 4H), 7.46 (s, 1H), 7.84 (s, 1H).
d
: 0.96 (t, J ¼ 7.2 Hz, 3H), 1.69 (m, 2H),
7.1.3.8. Methyl 40-[[6-(30,40-dimethoxy)phenethylaminocar-boxyl-4-
methyl-2-propyl-1H-benzimidazolyl]methyl] biphenyl-2-carboxylate
(5h). White solid (1016 mg, 84.0%), mp: 145e146 ꢁC. 1H NMR
7.1.3. General procedure for the preparation of alkylated products
(compound 5ae5k)
A mixture of amide compound (one of compound in 4ae4k)
(2 mmol) and 3 mL DMF was placed in a three-necked bottle and-
cooled with ice, then potassium tert-butoxide 354 mg (3 mmol) was
added. After stirring at the same temperature for 30 min, methyl-40-
bromomethyl-2-biphenylcarboxylic acid 610 mg (2.1 mmol) was
added. After addition of all reagents, the ice bath was removed, and
the reaction mixture was stirred overnight. The mixture was poured
(400 MHz, CDCl3)
d
: 0.94 (t, J ¼ 7.2 Hz, 3H),1.77 (m, 2H), 2.56 (s, 3H),
2.77 (t, J ¼ 7.2 Hz, 2H), 2.87 (t, J ¼ 7.2 Hz, 2H), 3.49 (m, 2H), 3.61 (s,
3H), 3.73 (s, 3H), 3.79 (s, 3H), 5.56 (s, 2H), 7.10e7.91 (m, 13H), 8.44
(s, 1H); ESIMS (m/z): 606.3(M þ H)þ.
7.1.3.9. Methyl 40-[[6-(20,50-dimethoxy)phenethylaminocarboxyl-4-
methyl-2-propyl-1H-benzimidazolyl]methyl] biphenyl-2- carboxylate

188
J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
(5i). White solid (1098 mg, 90.7%), mp: 130e132 ꢁC. 1H NMR
(400 MHz, CDCl3)
2.80 (t, J ¼ 7.2 Hz, 2H), 2.85 (t, J ¼ 7.2 Hz, 2H), 3.48 (m, 2H), 3.60 (s,
3H), 3.71 (s, 3H), 3.77 (s, 3H), 5.55 (s, 2H), 7.07e7.87 (m, 13H), 8.43
(s, 1H); ESIMS (m/z): 606.3(M þ H)þ.
7.1.4.4. 40-[(4-Methyl-6-40-methoxybenzylaminocarboxyl-2-propyl-
1H-benzimidazolyl)methyl]-2-biphenylcarboxylic acid (6d). White
solid (174 mg, 79.3%), mp >250 ꢁC, IR (KBr): 3357, 1652, 1593, 1533,
1511, 1454, 1293, 1245, 1041, 880, 818, 756. 1H NMR (400 MHz,
d
: 0.96 (t, J ¼ 7.3 Hz, 3H),1.75 (m, 2H), 2.55 (s, 3H),
DMSO-d6)
J ¼ 7.2 Hz, 2H), 3.77 (s, 3H), 4.46 (s, 2H), 5.56 (s, 2H), 7.09e7.90 (m,
14H), 8.44 (s, 1H), 12.72 (br, 1H). 13C NMR (100 MHz, DMSO-d6)
d:
d
: 0.96 (t, J ¼ 7.2 Hz, 3H),1.75 (m, 2H), 2.55 (s, 3H), 2.79 (t,
7.1.3.10. Methyl 40-[[6-(20-fluoro)phenethylaminocarboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]biphenyl-2-carboxylate (5j). White
solid (784 mg, 69.6%), mp: 140e142 ꢁC. 1H NMR (400 MHz, CDCl3)
13.98, 16.63, 20.67, 28.89, 42.30, 46.00, 55.20, 56.18, 107.51, 113.81,
121.25, 126.17, 127.47, 127.58, 128.14, 128.79, 128.89, 129.24, 130.69,
131.00, 132.03, 132.36, 134.85, 136.07, 140.21, 140.57, 143.95, 156.76,
158.31, 166.64, 169.63. HRMS: calcd for C34H34N3O4 (MHþ)
548.2544, found 548.2533.
d
: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.74 (m, 2H), 2.55 (s, 3H), 2.79 (t, J ¼ 7.2 Hz,
2H), 2.85 (t, J ¼ 7.1 Hz, 2H), 3.53 (m, 2H), 3.66 (s, 3H), 5.55 (s, 2H),
7.13e7.85 (m, 14H), 8.44 (s, 1H); ESIMS(m/z): 564.3(M þ H)þ.
7.1.3.11. Methyl 40-[[6-(40-fluoro)phenethylaminocarboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]biphenyl-2-carboxylate (5k). White
7.1.4.5. 40-[(6-30,40-Dimethoxybenzylaminocarboxyl-4-methyl-2-propyl-
1H-benzimidazolyl)methyl]-2-biphenyl carboxylic acid (6e). White solid
(166 mg, 71.7%), mp >250 ꢁC, IR (KBr): 3346, 1653, 1593, 1513, 1462,
1418, 1257, 1215, 1149, 1034, 1012, 814, 754. 1H NMR (400 MHz, DMSO-
solid (953 mg, 84.7%), mp: 157e158 ꢁC. 1H NMR (400 MHz, CDCl3)
d:
0.96 (t, J ¼ 7.2 Hz, 3H), 1.73 (m, 2H), 2.56 (s, 3H), 2.78 (t, J ¼ 7.2 Hz,
2H), 2.86 (t, J ¼ 7.2 Hz, 2H), 3.56 (m, 2H), 3.69 (s, 3H), 5.55 (s, 2H),
7.08e7.96 (m, 14H), 8.45 (s, 1H)；ESIMS(m/z): 564.3(M þ H)þ.
d6)
d
: 0.96 (t, J ¼ 7.2 Hz, 3H),1.75 (m, 2H), 2.55 (s, 3H), 2.79 (t, J ¼ 7.2 Hz,
2H), 3.72 (s, 3H), 3.80 (s, 3H), 4.42 (s, 2H), 5.55 (s, 2H), 7.07e7.93 (m,
13H), 8.43 (s, 1H), 12.72 (br, 1H). HRMS: calcd for C35H36N3O5 (MHþ)
578.2650, found 578.2638.
7.1.4. General procedure for the preparation of target compounds
(compound 6ae6k)
Alkylated products (0.4 mmol) were added into a bottle, and
then 5 mL methanol and 2 mL of 2 N sodium hydroxide solution
were added. The mixture was heated and refluxed for 3 h. After the
completion of the hydrolysis, the mixture was evaporated to
remove methanol. Then the mixture system was acidified with 2N
aqueous hydrochloride to pH 6, and the white precipitate was
filtered and dried.
7.1.4.6. 40-[[4-Methyl-6-(2-(30-methoxyphenethylamino)carboxyl-2-
propyl-1H-benzimidazolyl]methyl]-2-biphenyl carboxylic acid (6f).
White solid (197 mg, 87.8%), mp 239e240 ꢁC, IR (KBr): 3368, 1654,
1593,1544,1451,1347,1279,1155,1042, 875, 763. 1H NMR (400 MHz,
DMSO-d6)
d
: 0.96(t, J ¼ 7.4 Hz, 3H), 1.72e1.82 (m, 2H), 2.56 (s, 3H),
2.80e2.86(m, 4H), 3.45e3.50 (m, 2H), 3.70 (s, 3H), 5.56 (s, 2H),
6.75e6.81 (m, 3H), 7.09e7.87(m,11H), 8.42 (t, J ¼ 5.4 Hz,1H),12.72 (s,
1H).13C NMR (100 MHz, DMSO-d6)
d: 13.98,16.64, 20.69, 28.88, 35.41,
7.1.4.1. 40-[(6-benzylaminocarboxyl-4-methyl-2-propyl-1H-benzimi-
dazolyl)methyl]-2-biphenyl carboxylic acid (6a). White solid
(147 mg, 71.2%), mp 266e268 ꢁC, IR (KBr): 3025, 2951, 1698, 1520,
45.99, 55.00, 56.18,107.39,111.73,114.39,121.06,121.16,126.17,127.48,
127.53, 128.39, 128.90, 129.25, 129.47, 130.60, 131.01, 132.35, 136.06,
140.23, 140.58, 141.37, 143.87, 156.70, 159.43, 166.75, 169.63.HRMS:
calcd for C35H36N3O4 (MHþ)562.2700, found 562.2690.
1271, 776. 1H NMR (400 MHz,DMSO-d6)
1.78 (m, 2H), 2.51 (s, 3H), 2.84 (t, J ¼ 7.3 Hz, 2H), 4.47 (s, 2H), 5.54 (s,
2H), 7.05e7.95 (m, 15H), 8.97 (s, 1H). HRMS: calcd for C33H31N3O3
(MHþ)518.2365, found 518.2359.
d
: 0.96 (t, J ¼ 7.2 Hz, 3H),
7.1.4.7. 40-[[4-Methyl-6-(2-(40-methoxyphenethylamino)carboxyl-
2-propyl-1H-benzimidazolyl]methyl]-2-biphenyl carboxylic acid
(6g). White solid (186 mg, 82.7%), mp 243e244 ꢁC, IR (KBr): 3377,
1673, 1644, 1599, 1533, 1511, 1483, 1455, 1349, 1269, 1243, 1029,
7.1.4.2. 40-[(4-Methyl-6-20-methoxy-benzylaminocarboxyl-2-propyl-
1H-benzimidazolyl)methyl]-2-biphenyl carboxylic acid (6b). White
solid (162 mg, 73.9%), mp 243e245 ꢁC, IR (KBr): 3395, 1655, 1598,
1540, 1490, 1459, 1358, 1243, 1122, 1026, 870, 818, 764. 1H NMR
820, 761. 1H NMR (400 MHz, DMSO-d6)
d
: 0.97 (t, J ¼ 7.2 Hz, 3H),
1.73 (m, 2H), 2.56 (s, 3H), 2.78 (t, J ¼ 7.2 Hz, 2H) 2.87 (t, J ¼ 7.1 Hz,
2H), 3.47 (m, 2H), 3.69 (s, 3H), 5.55 (s, 2H), 7.10e7.90(m, 14H),
(400 MHz, DMSO-d6)
d
: 0.97 (t, J ¼ 7.2 Hz, 3H), 1.74 (m, 2H), 2.55
8.45 (s, 1H), 12.72 (br, 1H). 13C NMR (100 MHz, DMSO-d6)
d:；
(s, 3H), 2.78 (t, J ¼ 7.2 Hz, 2H), 3.47 (s, 3H), 4.45 (s, 2H), 5.56
13.98, 16.65, 18.72, 20.69, 28.88, 34.54, 41.40, 46.00, 55.11, 107.39,
113.92, 121.16，126.17, 127.47, 127.52, 128.40, 128.90, 129.25,
129.73, 130.60, 131.00, 131.65, 132.36, 134.80, 136.06, 140.23,
140.57, 143.87, 156.69, 157.81, 166.70, 169.63. HRMS: calcd for
C35H36N3O4 (MHþ)562.2700, found 562.2691.
(s, 2H), 7.12e7.86 (m, 14H), 8.45 (s, 1H), 12.72 (br, 1H). 13C NMR
(100 MHz, DMSO-d6) d: 13.98, 16.64, 18.71, 20.69, 28.90, 46.01,
55.48, 56.18, 107.56, 110.58, 110.25, 121.30, 126.19, 127.27, 127.50,
127.61, 127.97, 128.12, 128.89, 129.24, 130.60, 131.00, 132.36,
134.85, 136.08, 140.22, 140.57, 143.98, 156.71, 156.77, 166.92,
169.62. HRMS: calcd for C34H34N3O4(MHþ) 548.2544, found
548.2533.
7.1.4.8. 40-[[4-Methyl-6-(2-(30,40-dimethoxy)phenethylamino)
carboxyl-2-propyl-1H-benzimidazolyl]methyl]-2-biphenyl carboxylic
acid (6h). White solid (178 mg, 75.3%), mp 240e241 ꢁC, IR (KBr):
3388, 1679, 1643, 1597, 1544, 1513, 1461, 1350, 1272, 1153, 1035, 889,
7.1.4.3. 40-[(4-Methyl-6-30-methoxybenzylaminocarboxyl-2-propyl-
1H-benzimidazolyl)methyl]-2-biphenylcarboxylic acid (6c). White
solid (181 mg, 82.8%), mp 228e229 ꢁC, IR (KBr): 3375, 1648, 1598,
1534, 1487, 1456, 1349, 1270, 1047, 853, 765. 1H NMR (400 MHz,
819, 760. 1H NMR (400 MHz, DMSO-d6)
1.77 (m, 2H), 2.56 (s, 3H), 2.77 (t, J ¼ 7.2 Hz, 2H), 2.87 (t, J ¼ 7.2 Hz,
2H), 3.49 (m, 2H), 3.73 (s, 3H), 3.79 (s, 3H), 5.55 (s, 2H), 7.10e7.91
(m, 13H), 8.44 (s, 1H), 12.72 (br, 1H). 13C NMR (100 MHz, DMSO-
d
: 0.94 (t, J ¼ 7.2 Hz, 3H),
DMSO-d6)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.76 (m, 2H), 2.56 (s, 3H),
2.79 (t, J ¼ 7.2 Hz, 2H), 3.73 (s, 3H), 4.41 (s, 2H), 5.55 (s, 2H),
d6) d: 13.98, 16.64, 18.72, 20.69, 28.89, 34.93, 41.32, 46.00, 55.47,
7.08e7.92 (m, 14H), 8.43 (s, 1H), 12.71 (br, 1H). 13C NMR
55.66, 56.18, 107.38, 112.09, 112.74, 120.62, 121.16, 126.16, 127.48,
127.52, 128.43, 128.90, 129.26, 130.61, 131.01, 132.29, 132.35, 134.41,
136.06, 140.23, 140.58, 143.86, 147.37, 148.76, 156.70, 166.72, 169.63.
HRMS: calcd for C36H38N3O5(MHþ) 592.2806, found 592.2793.
(100 MHz, DMSO-d6) d: 13.98, 16.64, 18.71, 20.68, 28.89, 42.80,
46.01, 55.11, 56.18, 107.55, 112.13, 113.18, 119.59, 121.26, 126.18,
127.47, 127.63, 128.06, 128.89, 129.24, 129.47, 130.60, 131.00,
132.35, 134.85, 136.06, 140.22, 140.58, 141.67, 144.00, 156.80,
159.44, 166.78, 169.63. HRMS: calcd for C34H34N3O4 (MHþ)
548.2544, found 548.2531.
7.1.4.9. 40-[[4-Methyl-6-(2-(20,50-dimethoxy)phenethylamino)
carboxyl-2-propyl-1H-benzimidazolyl]methyl]-2-biphenyl carboxylic

J.-L. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 183e190
189
acid (6i). White solid (194 mg, 82%), mp 231e233 ꢁC, IR (KBr):
O2 and 5% CO2. Each strip was connected to a force transducer, the
changes in isometric tension were recorded by a four-channel
recorder (Medlab-U/4c501, China). The tissues were allowed to
equilibrate for 1 h and were washed every 15 min. At the
beginning of the experiment, a 67 mM KCl solution was admin-
istered to check the sensitivity of the preparations as well as to
determine their maximal contractile response. After 30 min
washout, test substances or their respective vehicles were added.
Sixty minutes later, cumulative concentrationeresponse curves of
angiotensin II were obtained. Only one curve was obtained from
each strip, and the contractile response was expressed as
percentage (%) of the maximal contraction achieved with KCl. The
pA2 values were calculated using Schild’s plot [29]. Antagonist
potency was evaluated by the estimation of pA2 values.
3361, 1654, 1597, 1536, 1501, 1465, 1355, 1291, 1244, 1039,809, 764.
1H NMR (400 MHz, DMSO-d6)
d
: 0.96 (t, J ¼ 7.3 Hz, 3H),1.75 (m, 2H),
2.55(s, 3H), 2.80 (t, J ¼ 7.2 Hz, 2H), 2.85 (t, J ¼ 7.2 Hz, 2H), 3.48 (m,
2H), 3.71(s, 3H), 3.77 (s, 3H), 5.56 (s, 2H), 7.07e7.87 (m, 13H), 8.43
(s, 1H), 12.73(br, 1H). 13C NMR (100 MHz, DMSO-d6)
d:13.98, 16.64,
18.71, 20.68, 28.89, 30.09, 45.98, 55.38, 55.95, 56.18, 107.35, 111.73,
111.80, 116.38, 121.19, 126.16, 127.48, 128.44, 128.73, 128.90, 129.25,
130.60, 131.00, 132.36, 134.80, 136.07, 140.22, 140.58, 143.84, 151.55,
153.14, 156.67, 166.70, 169.63. HRMS: calcd for C36H38N3O5 (MHþ)
592.2806, found 592.2795.
7.1.4.10. 40-[[6-(2-(20-Fluoro)phenethylamino)carboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]-2-biphenyl carboxylic acid (6j).
White solid (169 mg, 76.9%), mp 233e234 ꢁC, IR (KBr): 3404, 1652,
1600, 1545, 1489, 1457, 1352, 1266, 1218, 1137, 822, 757. 1H NMR
7.4. Procedure for oral activity in the spontaneously hypertensive
rats (SHR)
(400 MHz, DMSO-d6)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H), 1.74 (m, 2H), 2.55 (s,
3H), 2.79 (t, J ¼ 7.2 Hz, 2H), 2.85 (t, J ¼ 7.1 Hz, 2H), 3.53 (m, 2H), 5.55
(s, 2H), 7.13e7.85 (m, 14H), 8.44 (s, 1H), 12.72 (br, 1H). 13C NMR
Male SHR,19e21 weeks old. Six animals served as controls. They
received vehicle (10 mL/kg). Hypertensive animals were divided
into two groups (n ¼ 6). Group 1 received losartan (standard drug:
10 mg/kg), group 2 was given the same doses of the tested
compound 6i. Both the vehicle and test compound were given by
oral administration [30]. Blood pressure and heart rate were
measured by tail plethysmography (BP-98A, Softron, Japan) after
a warming period in unanesthetized rats. The BP measurements
required only a few minutes per individual rat. All data were
expressed as means ꢂ SEM [13c].
(100 MHz, DMSO-d6) d: 13.98, 16.65, 18.72, 20.70, 28.88, 46.00,
56.18, 107.40, 115.15, 115.37, 121.16, 124.48, 124.52, 126.17, 126.23,
126.39, 127.48, 127.53, 128.31, 128.37, 128.45, 128.90, 129.25, 130.60,
131.00, 131.35, 131,40, 132.38, 134.79, 136.05, 140.23, 140.57, 143.90,
156.71, 159.69, 162.11, 166.78, 169.64. HRMS: calcd for
C34H33FN3O3(MHþ) 550.2501, found 550.2489.
7.1.4.11. 40-[[6-(2-(40-Fluoro)phenethylamino)carboxyl-4-methyl-2-
propyl-1H-benzimidazolyl]methyl]-2-biphenyl
carboxylic
acid
(6k). White solid (174 mg, 79.4%), mp >250 ꢁC, IR (KBr): 3386,
1645, 1598, 1573, 1508, 1455, 1351, 1272, 1217, 1157, 1083, 875, 820,
716. 1H NMR (400 MHz, DMSO-d6)
d
: 0.96 (t, J ¼ 7.2 Hz, 3H), 1.73 (m,
7.5. Procedure for preliminary toxicity evaluation
2H), 2.56 (s, 3H), 2.78 (t, J ¼ 7.2 Hz, 2H), 2.86 (t, J ¼ 7.2 Hz, 2H), 3.56
(m, 2H), 5.56 (s, 2H), 7.08e7.96 (m, 14H), 8.45 (s, 1H), 12.71 (br, 1H).
The preliminary toxicity evaluation was performed on a water
toxicity analyser (BHP 9511, Beijing Hamamatsu Inc.) by the inhi-
bition of Photobacterium phosphoreum (T3 mutation), which was
supplied by the Institute of Soil Science, Academia Sinica, Nanjing P.
R. China in the form of a freeze dried powder. For each test, a serial
dilution of the compounds was prepared in 2% saline water
according to the pre-test. In addition, a series of saline solutions
containing 106 colony units of Photobacterium phosphoreum was
prepared in glass cuvettes by pipetting 10 mL of the reconstituted
bacterial suspension into 500 mL of a 2% saline solution. These
solutions were incubated for 15 min and measured. The toxicity of
the treated compounds was recorded after 15 min of exposure to
the tested sample. The results of the Microtox test are expressed in
terms of LC50 values. LC50 is the concentration in water which
inhibits 50% of a test batch of Photobacterium phosphoreum as
exemplified by compound 6i and 6j in Fig. 8. The lower the LC50
value is, the greater the toxicity of the sample is [31].
13C NMR (100 MHz, DMSO-d6)
d: 13.98, 16.65, 18.72, 20.70, 28.88,
34.50, 46.00, 56.18, 107.40, 115.02, 115.23, 121.15, 126.16, 127.48,
127.55, 128.34, 128.90, 129.26, 130.52, 130.60, 131.00, 132.37, 134.80,
135.91, 135.94, 136.05, 140.24, 140.57, 143.89, 156.72, 159.76, 162.17,
166.77, 169.63. HRMS: calcd for C34H33FN3O3 (MHþ)550.2501,
found 550.2490.
7.2. Procedure for receptor binding assay
The AT1 receptor binding assay was carried out by competitive
displacement of the binding of [125I] Sar1 Ile8-Ang II with angio-
tensin AT1 receptor as described previously [16]. Each 180
incubate contained the following: [125I] Sar1 Ile8-Ang II (25pM),
AT1(AT2) receptor (25 g) and standard or test compounds. The
mL
m
binding was performed at 37 ꢁC for 180 min in 96-well filtration
plates (Costar, USA) and was terminated by rapid vacuum filtration
using a vacuum device; dried filters disks were punched out and
counted in a gamma counter. IC50 values were estimated from the
linear portion of the competition curves.
7.6. Molecular modelling experiments
7.3. Procedure for angiotensin II receptor functional antagonism in
rabbit aorta strips
Molecular modelling studies were performed using a Silicon
Graphics desktop (SGI) Fuel work station. The training set was
selected as described above, and the pharmacophore model for
AT1 receptor antagonists was generated using the HipHop module
in Discovery Studio, version 2.0, from Accelrys Inc. Molecules were
built in a 3D window, and conformational models for each
molecule were generated using the diverse conformation module.
Then the resulting sd files were used for common features
hypothesis generation using the HipHop module by default.
Through these experiments, we specified the features that are
crucial for binding with Ang II receptor, in agreement with the
literature [20,21].
Japanese White Rabbits (2e3 kg body weight, Vitalriver
Company, China) were killed by cervical dislocation, after a slight
anesthesia with 20% Urethane solution. The thoracic aorta was
carefully dissected out, placed in ice-cold Krebs-Henseleit solu-
tion of the following composition (mM): NaCl 118; KCl 4.7;
KH2PO4 1.2; MgSO4$7H2O 1.17; CaCl2$2H2O 2.5; NaHCO3 25;
glucose 11.1 [28]. It was cut into helical strips 3e4 mm wide and
15e20 mm long. These strips were mounted in 10-mL tissue
baths, the tissue baths were kept at 37 ꢁC and aerated with 95%

190
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Acknowledgments
Financial support of this work by the Fundamental Research
Foundation of Beijing Institute of Technology (1050050320704)
and the National Technological Project of the Manufacture and
Innovation of Key New Drugs (2009ZX09103-143) was appreciated.
We also thank Beijing Harmamatsu Photon Technique Inc. for the
providing of the toxicity test instrument.
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