Nonpeptidic angiotensin II AT1 receptor antagonists derived from 6-substituted aminocarbonyl and acylamino benzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2013.08.014

Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12 showed nanomolar AT₁ receptor binding affinity and high AT₁ receptor selectivity over AT₂ receptor in a preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT₁ IC ₅₀ = 3 nM, AT₂ IC₅₀ > 10,000 nM, PA ₂ = 8.51) and 11g (AT₁ IC₅₀ = 0.1 nM, AT ₂ IC₅₀ = 149 nM, PA₂ = 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addition, they were orally active AT₁ receptor antagonists in spontaneous hypertensive rats.

Full text of DOI:10.1016/j.ejmech.2013.08.014

European Journal of Medicinal Chemistry 69 (2013) 44e54
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Nonpeptidic angiotensin II AT1 receptor antagonists derived from
6-substituted aminocarbonyl and acylamino benzimidazoles
,
a
a
,
a
Jun Zhang a, Jin-Liang Wang a , Wei-Fa Yu , Zhi-Ming Zhou , Wen-Chang Tao ,
Yi-Cheng Wang a, Wei-Zhe Xue a, Di Xu a, Li-Ping Hao a, 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, 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:
Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and syn-
thesized as nonpeptidic angiotensin II AT1 receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12
showed nanomolar AT1 receptor binding affinity and high AT1 receptor selectivity over AT2 receptor in a
preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT1
IC50 ¼ 3 nM, AT2 IC50 > 10,000 nM, PA2 ¼ 8.51) and 11g (AT1 IC50 ¼ 0.1 nM, AT2 IC50 ¼ 149 nM,
PA2 ¼ 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addi-
tion, they were orally active AT1 receptor antagonists in spontaneous hypertensive rats.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 29 December 2012
Received in revised form
6 August 2013
Accepted 8 August 2013
Available online 19 August 2013
Keywords:
Angiotensin II AT1 receptor antagonists
Aminoacyl benzimidazole
Acylamino benzimidazole
Hypertension
1. Introduction
heterocycles by means of a methylene group. Among the large
variety of heterocyclic systems developed, the ortho-fused bicyclic
The renineangiotensin aldostenone system (RAAS) is a highly
complex system that is essential in fluid/electrolyte homeostasis
and blood pressure regulation [1,2]. Angiotensin II (Ang II) is an
active octapeptide and a potent vasoconstrictor in the RAAS, which
is produced in vivo from angiotensin I by the angiotensin-
converting enzyme. In humans, Ang II interacts with two main
receptor subtypes, namely, AT1 and AT2. The AT1 receptor subtype
mediates virtually all the known physiological actions of Ang II in
cardiovascular, neuronal, endocrine, and hepatic cells, among
others [3]. AT1 receptor antagonists are clinically used as therapy
for hypertension and heart failure [4], moreover, they show po-
tentials for the prevention of target-organ damage and anti-tumor
effects [5]. Seven of these drugs, including one recently listed drug
azilsartan [6], have already been licensed for the treatment of high
blood pressure (Fig. 1). All these drugs contain common structural
features represented by a biphenyl fragment bearing an acidic
moiety (i.e., tetrazole, carboxylic group) that is linked to
moiety benzimidazole appears to be a particularly effective het-
erocyclic system considering its interaction with the AT1 receptor
[7]. Such effectiveness is exemplified by the commercially available
candesartan [8], the most potent AT1 antagonist to date. It’s
demonstrated that substitutions at the benzimidazole ring [8e12],
such as a nitro group at the 5-position, an acylureas lipophilic group
at the 6-position, and a carboxylic group at the 7-position, can
promote selective AT1 antagonism. Notably, 6-carbamoyl benz-
imidazole has been rarely used to evaluate the corresponding effect
for AT1 antagonism. Bansal’s proposal [11] of the presence of L3
lipophilic pockets at the AT1 receptor encouraged us to postulate
that a carbamoyl group at the 6-position of benzimidazole with an
alkyl-chain-bearing phenyl group may provide additional in-
teractions (e.g.,
pep interaction) that consequently enhance the
overall interaction between the antagonist and the receptor. The
literature shows [13] that the phenylethylamino fragment pos-
sesses
a biological effect that lowers hypertensive pressure.
Furthermore, Rapposell [14] reported that the simple reversion of
the amido-function of the model compound induces a dramatic
change of affinity toward the AT1 receptor. Thus, new 6-
aminocarbonyl benzimidazoles A, which are expected to have
higher activity than losartan, were designed. For comparison, the
* Corresponding authors. Tel./fax: þ86 10 68918982.
E-mail addresses: jl.wang@hotmail.com (J.-L. Wang), zzm@bit.edu.cn
(Z.-M. Zhou).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.014

J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
45
Cl
N
O
i-Pr
N
H
OH
N
N
N
N
N
COOH
N
NH
NH
N
Losartan
Valsartan
N
N
N
N
N
N
N
O
n-Bu
O
N
O
N
N
N
N
NH
N
NH
N
COOH
O
O
O
O
Candesartan
Telmisartan
Irbesartan
OH
O
O
N
O
N
N
O
O
O
N
O
N
N
N
NH
O
N
NH
O
O
O
O
O
Olmesartan
Azilsartan
Fig. 1. Representive AT1 receptor antagonists.
congeners 6-acylamino benzimidazoles B were also designed and
synthesized to examine their effect as nonpeptidic selective AT1
receptor antagonists (Fig. 2).
give
40-[(6-amino-4-methyl-2-n-propyl-1H-benzimidazol-1-yl)
methyl]biphenyl-2-carboxylic acid (10). This compound was
coupled with different acyl chlorides to give the target compounds
11ae11g. The target compound 12 was obtained by direct hydro-
lysis of compound 9 using aqueous sodium hydroxide in boiling
methanol.
2. Results and discussion
All target compounds were identified by IR, 1H NMR, 13C NMR
and HRMS. As confirmatory evidence of the regiochemistry of the
final products, a Nuclear Overhauser Effect (NOE) experiment was
conducted on one of the alkylated products, namely, compound 5h
(Fig. 3). In DMSO-d6, 5h showed an NOE between the hydrogen of
2.1. Chemistry
The target compounds were prepared following the route in
Schemes 1 and 2. The starting material 4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxylic acid (1) was prepared from 3-methyl-
4-nitrobenzoic acid following the previously reported method [15].
This compound was transformed to acyl chloride and then coupled
with various amines to give acylamide compounds 3ae3i with
yield ranging from 72% to 85%. The acylamides were then alkylated
with methyl 40-(bromomethyl)biphenyl-2-carboxylate (4) using
potassium tert-butoxide in DMF to furnish the corresponding
products in yields of 57% to 72%. Finally, compounds 5ae5i were
hydrolyzed using aqueous sodium hydroxide in boiling methanol
with a yield of over 70%. In Scheme 2, compound 2 was coupled
with ammonium carbonate to give 4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (7). This compound was alkylated
with compound 4 first, and then was converted to the corre-
sponding carbamate (9) by a refined Hofmann rearrangement [16].
Compound 9 was refluxed with sodium hydroxide in dioxane to
the methyl at C-4 (
between the protons on the methylene adjacent to N-1 (
the proton at C-7 ( 7.95), as well as an NOE between the protons on
the methylene adjacent to N-1 ( 5.60) and the proton at the
methylene adjacent to C-2. Furthermore, the structures of com-
pounds 5a, 5f, and 6a were confirmed by X-ray crystallography [17]
(Figs. 4e6). Taken together, these structures indicate that N1
alkylation and not N3 alkylation occurred.
d
2.59) and the proton at C-5 (
d
7.60), an NOE
d
5.60) and
d
d
2.2. 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-
N
O
N
H
N
N
R
N
H
N
R
O
COOH
COOH
A
B
Fig. 2. The structure of the designed compounds.

46
J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
N
N
N
H
N
ii
i
N
H
R
Cl
HO
N
H
N
H
O
O
O
3a-3i
2
1
N
N
H
N
H
N
N
R
N
iv
4, iii
R
O
O
COOH
COOMe
6a-6i
5a-5i
a: R= n-propyl; b: R= n-butyl; c: R= 4-morpholinyl; d: R= 1-piperidinyl;
e: R= 4-methylphenyl; f: R= n-propyl; g: R= 4-morpholinyl;
h: R= phenyl; i: R= 4-morpholinyl.
Reagents and conditions: (i) SOCl2, refluxed; (ii) RNH2, DCM, Et3N; (iii) KOtBu, DMF; (iv) NaOH, MeOH, reflux,
then HCl, 0°C.
Scheme 1. Synthesis of compounds 6ae6i.
N
N
N
O
H2N
N
N
N
O
N
H
iii
i
4, ii
Cl
H2N
O
N
H
N
H
O
O
8
9
7
2
MeOOC
MeOOC
vi
iv
N
N
N
N
O
N
N
v
O
H2N
R
N
H
O
N
H
10
12
11a-11g
HOOC
HOOC
HOOC
a: R= methyl; b: R= ethyl; c: R= n-propyl; d: R= phenyl;
e: R= 4-methylphenyl;f: R= phenyl; g: R= phenyl.
Reagents and conditions: (i) (NH4)2CO3, DCM, Et3N; (ii) KOtBu, DMF; (iii) NaOCl, KF/Al2O3, MeOH, refluxed; (iv)
NaOH, Dioxane, reflux; (v) RCOCl, DCM, Et3N; (vi) NaOH, MeOH, refluxed, then HCl, 0°C.
Scheme 2. Synthesis of compounds 11ae11g and 12.
binding assay as described previously [18]. The results are
expressed as IC50 values, which are the concentrations of a com-
pound that inhibit [125I] Ang II binding to the receptor by 50%. The
antagonistic activity of the more potent compounds (6f, 6g, 11e, 11f,
11g, and 12) was also investigated in vitro using the contractile
response of isolated rabbit aortic strips in a functional assay.
Losartan was taken as a positive control drug in the assays.
Finally, the most potent compounds 6f and 11g were evaluated in
an in vivo model [19]. As a rational design for this work, pharma-
cophore hypothesis generation was performed using the HipHop
module of the Discovery Studio software [20] using the training set
consisting of six clinical sartans [21,22]. The fit values of the syn-
thesized compounds were determined on the selected hypothesis
of the AT1 receptor antagonist using the best fit algorithm as
described in the literature [23].
As summarized in Table 1, the binding assay and the antago-
nistic activity results showed that as expected, all compounds
displayed a certain degree of inhibition activity. Several compounds
showed high selectivity for the AT1 receptor over the AT2 receptor,
given that the binding affinities were in the submicromolar range
for the AT1 receptor and in the micromolar range for the AT2 re-
ceptor. Of all the synthesized compounds, compounds 6f and 11g
were the most potent (6f: AT1 IC50 ¼ 3 nM, PA2 ¼ 8.51; 11g: AT1
IC50 ¼ 0.1 nM, PA2 ¼ 8.43), exhibiting almost the same binding
affinity as the marketed AT1 receptor antagonist telmisartan
(IC50 ¼ 1.0 nM, 0.33 nM [24]) and approximately 10 times the
4
N
2
H
N
7
N
1
O
O
O
Fig. 3. NOESY of compound 5h.

J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
47
to the fit value as highly active (fit value > 4, þþþ), moderately
active (2 < fit value < 4, þþ), and with low activity (fit value < 4, þ).
All synthesized compounds of this study were also classified ac-
cording to the literature [28] by their activity as highly active
(<100 nM, þþþ), moderately active (100 nm to 10,000 nM, þþ)
and low activity (>10,000 nM, þ). All but two highly active com-
pounds were predicted correctly, and only seven moderately active
compounds were predicted to be highly active. Despite these dis-
parities, the pharmacophore hypothesis could still be considered
relatively reasonable. Furthermore, the molecular modeling simu-
lation performed above reflected the interaction of ligands with the
AT1 receptor. Unexpectedly, the benzene ring in the phenylethyl,
and not the propyl at the 2-position of the benzimidazole ring,
mapped onto the hydrophobic aliphatic feature (Fig. 8). The same
binding modality was also observed in the mapping of our previ-
ously published analogues [21,22] with the pharmacophore, in
which the methyl in the second benzimidazole ring, and not the
propyl, mapped onto the hydrophobic aliphatic feature. Notably,
the different interaction modalities of telmisartan and losartan
have been reported in the literature [29] and were obtained
through the docking of ligands with the homology-modeled AT1
receptor.
Fig. 4. Crystal structure of compound 5a.
3. Conclusions
affinity of losartan (IC50 ¼ 16.2 nM, 150 nM [25], 6.7 nM [26]).
Compounds 6g, 11e, 11f, and 12 were also much more active than
losartan. The pA2 of these compounds were 8.20, 8.25, 8.21, and
8.15, respectively, which are higher than that of losartan
(pA2 ¼ 7.92). In addition, compounds 6b, 6i, 11b, 11c, 11d, and 11e
Two families of 6-substituted aminocarbonyl and acylamino
benzimidazole derivatives were designed and synthesized as
nonpeptidic Ang II AT1 receptor antagonists. Compounds 6f and 11g
were found to be the two most potent AT1-selective Ang II receptor
antagonists. These compounds would be valuable as lead com-
pounds for further rational design of more potent Ang II AT1 re-
ceptor antagonists.
showed the same activity level as losartan (10 nM
< AT1
IC50 < 100 nM). These results suggest that both 6-substituted
aminocarbonyl benzimidazole and acylamino benzimidazoles are
good angiotensin II AT1 receptor antagonists.
Based on the results of in vitro Ang II-binding assay and func-
tional antagonism, 6f and 11g were selected for further evaluation
of in vivo models. When evaluated orally in conscious spontane-
ously hypertensive rats (SHR), both compounds at doses of 10 mg/
kg significantly decreased blood pressure by more than 30 mmHg
(Fig. 7), which is more efficacious than losartan.
In the molecular modeling simulation section, fit value indicates
how well the features in the pharmacophore map the chemical
features in the molecule [27]. The higher the fit value, the more
active the compound. In this study, five features were included in
the selected pharmacophore hypothesis; the highest fit value was
5. Accordingly, the estimated activity could be classified according
4. Experimental section
4.1. Chemistry
The melting points were determined on an XT e 4A melting
point apparatus and are uncorrected. All the target compounds
were characterized by IR, 1H NMR, 13C NMR and HRMS. Infrared (IR)
spectra were recorded on a Bruker Alpha Spectrometer, Nuclear
magnetic resonance (1H NMR and 13C NMR) spectra were recorded
Fig. 5. Crystal structure of compound 5f.
Fig. 6. Crystal structure of compound 6a.

48
J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
Table 1
Fit values on the selected pharmacophore and experimental data of the target compounds.
N
N
N
O
H
R
N
R
N
N
H
n
n
O
COOH
COOH
6a-6i
11a-11g,12
No.
n
R
Fit value
IC50 (nM)
Est. act. scalea
Act. (AT1R) scaleb
pA2
AT1
AT2
6a
6b
6c
6d
6e
6f
6g
6h
6i
11a
11b
11c
11d
11e
11f
11g
12
0
0
0
0
0
2
2
3
3
0
0
0
0
0
1
2
0
n-Propyl
n-Butyl
4-Morpholinyl
1-Piperidinyl
4-Methylphenyl
Phenyl
4-Morpholinyl
Phenyl
4-Morpholinyl
Methyl
Ethyl
n-Propyl
Phenyl
4-Methylphenyl
Phenyl
Phenyl
Methoxy
Losartan
Telmisartan
4.78
4.70
4.77
4.87
4.90
4.84
3.14
4.93
3.24
4.84
4.88
4.48
4.40
4.44
4.36
4.79
4.49
4.76
4.91
1345
13
1008
20
39
3
11
115
10
1430
0.4
0.3
3.9
4.9
1.2
0.1
0.7
16.2
1.0
e
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþ
þþ
164
e
þþþ
þþ
42
61
>10,000
>10,000
e
þþþ
þþþ
þþþ
þþþ
þþ
8.51
8.20
þþþ
þþ
718
e
þþþ
þþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
78
53
6
>10,000
77
149
4366
>10,000
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
8.25
8.21
8.43
8.15
7.92
a
Estimated activity scale: highly active (fit value > 4, þþþ), moderately active (2 < fit value < 4, þþ), and with low activity (fit value < 4, þ).
Activity scale: highly active (<100 nM, þþþ), moderately active (100 nM to 10,000 nM, þþ), and with low activity (>10,000 nM, þ) [22].
b
on a Bruker NMR spectrometer using TMS as internal standard
280 mmol) was refluxed for 3 h, then the excess thionyl chloride
was removed under reduced pressure to obtain the crude acyl
chloride (2) as an off-white solid. It was used for the next step
directly.
(chemical shift in ppm). High-resolution mass spectra were recor-
ded on a Fourier Transform Ion Cyclotron Resonance Mass Spec-
trometer. Silica gel column chromatography was performed with
silica gel (200e300 mesh) and F254 pre-coated silica gel TLC plate.
4.1.2. General synthetic procedure for 3ae3i
4.1.1. 4-Methyl-2-n-propyl-1H-benzimidazole-6-carboxylic
chloride (2)
To a stirred suspension of acyl chloride (2) in 60 mL of
dichloromethane at 0 ꢀC was added dropwise triethylamine (1.52 g,
15 mmol), followed by a solution of one of the selected amines
(10 mmol) in 10 mL of dichloromethane. The resulting mixture was
stirred at room temperature for 8 h. The reaction mixture was
filtered, and the filtrate was washed with 1N hydrochloric acid,
saturated sodium bicarbonate solution and saturated sodium
chloride solution, dried over anhydrous sodium sulphate, filtered,
and concentrated under vacuum. The residue was purified by
recrystallization in ethanol to provide pure 3ae3i as white solids in
yield of 72e85%.
A
mixture of 4-methyl-2-n-propyl-1H-benzimidazole-6-
carboxylic (1, 2.18 g, 10 mmol) and thionyl chloride (20 mL,
4.1.2.1. N-n-Propyl-4-methyl-2-n-propyl-1H-benzimidazole-6-
carboxamide (3a). White solid, yield 83%. m.p.:124e125 ꢀC. IR (ATR,
cmꢁ1): 3290 (NH), 1605 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
0.91 (t, J ¼ 7.3 Hz, 3H, 300-Me), 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.59 (m,
2H, 20-CH2), 1.76 (m, 2H, 200-CH2), 2.55 (s, 3H, 4-Me), 2.78 (t,
J ¼ 7.2 Hz, 2H, 10-CH2), 3.21 (m, 2H, 100-CH2), 7.47 (s, 1H, 5-H), 7.81 (s,
1H, 7-H); ESIMS (m/z): 260.3 [M þ H]þ.
4.1.2.2. N-n-Butyl-4-methyl-2-n-propyl-1H-benzimidazole-6-
carboxamide (3b). White solid, yield 80%. m.p.:119e121 ꢀC. IR (ATR,
cmꢁ1): 3123 (NH), 1624 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
0.91 (t, J ¼ 7.3 Hz, 3H, 400-Me), 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.32 (m,
Fig. 7. Effects of 6f, 11g and losartan (10 mg/kg po) on mean arterial pressure in
conscious SHR after oral administration.
2H, 300-CH2), 1.59 (m, 2H, 200-CH2), 1.79 (m, 2H, 20-CH2), 2.55 (s, 3H,

J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
49
Fig. 8. (a) The selected pharmacophore hypothesis; (b) mapping of 6f and 11g with the selected hypothesis.
4-Me), 2.81 (t, J ¼ 7.3 Hz, 2H, 10-CH2), 3.53 (m, 2H, 100-CH2), 7.49 (s,
1H, 5-H), 7.85 (s, 1H, 7-H); ESIMS (m/z): 274.3 [M þ H]þ.
4.1.2.9. N-[3-(4-Morpholinyl)propyl]-4-methyl-2-n-propyl-1H-benz-
imidazole-6-carboxamide (3i). Light yellow solid, yield 82%.
m.p.:99e101 ꢀC. IR (ATR, cmꢁ1): 3177 (NH), 1633 (C]O); 1H NMR
4.1.2.3. N-(4-Morpholinyl)-4-methyl-2-n-propyl-1H-benzimidazole-
6-carboxamide (3c). White solid, yield 78%. m.p.:179e181 ꢀC. IR
(ATR, cmꢁ1): 3107 (NH), 1629 (C]O); 1H NMR (400 MHz, DMSO-d6)
(400 MHz, DMSO-d6)
d
: 0.95 (t, J ¼ 7.3 Hz, 3H, 30-Me), 1.76 (m, 2H,
20-CH2), 2.12 (m, 2H, 200-CH2), 2.37 (t, J ¼ 7.2 Hz, 4H, 300-CH2), 2.52 (s,
3H, 4-Me), 2.78 (t, J ¼ 7.2 Hz, 2H, 400-CH2), 2.83 (t, J ¼ 7.2 Hz, 2H, 10-
CH2), 3.62 (m, 2H, 100-CH2), 3.82 (t, J ¼ 7.2 Hz, 4H, 500-CH2), 7.47 (s,
1H, 5-H), 7.84 (s, 1H, 7-H); ESIMS (m/z): 345.4 [M þ H]þ.
d
: 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.74 (m, 2H, 20-CH2), 2.37 (s, 3H, 4-
Me), 2.56 (t, J ¼ 7.2 Hz,2H, 10-CH2), 3.46 (t, J ¼ 7.2 Hz, 4H, 100-CH2),
3.72 (t, J ¼ 7.2 Hz, 4H, 200-CH2), 7.52 (s, 1H, 5-H), 7.86 (s, 1H, 7-H);
ESIMS (m/z): 288.3 [M þ H]þ.
4.1.3. General synthetic procedure for 5ae5i
To a stirred solution of compound 3 (2 mmol) in 15 mL of
dimethylformamide at 0 ꢀC, potassium tret-butoxide (2.2 mmol)
was added. The mixture was stirred for 30 min at 0 ꢀC, and then
methyl 40-bromomethylbiphenyl-2-carboxylate (4, 2.1 mmol)
was added. After stirring at room temperature for 12 h, the
mixture was poured into ice water (80 mL) and extracted with
ethyl acetate (40 mL ꢂ 3). The combined ethyl acetate layers
were washed with brine (50 mL ꢂ 3), dried over anhydrous so-
dium sulfate, and concentrated under a reduced pressure to
provide an off-white solid. The solid was purified by column
chromatography [elution: petroleum ether/ethyl acetate (1:1, v/
v)] to provide the pure product as white solids in yield of 57e
72%.
4.1.2.4. N-(1-Piperidinyl)-4-methyl-2-n-propyl-1H-benzimidazole-6-
carboxamide (3d). White solid, yield 72%. m.p.:174e176 ꢀC. IR (ATR,
cmꢁ1): 3119 (NH), 1590 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.54 (m, 2H, 300-CH2), 1.62 (m, 4H, 200-
CH2),1.74 (m, 2H, 20-CH2), 2.31 (s, 3H, 4-Me), 2.57 (t, J ¼ 7.2 Hz, 2H,
10-CH2), 3.46 (m, 4H, 100-CH2), 7.49 (s, 1H, 5-H), 7.94 (s, 1H, 7-H);
ESIMS (m/z): 286.3 [M þ H]þ.
4.1.2.5. N-(4-Methylphenyl)-4-methyl-2-n-propyl-1H-benzimid-
azole-6-carboxamide (3e). White solid, yield 73%. m.p.:215e217 ꢀC.
IR (ATR, cmꢁ1): 3212 (NH), 1642(C]O); 1H NMR (400 MHz, DMSO-
d6)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.74 (m, 2H, 20-CH2), 2.31 (s, 3H,
400-Me), 2.36 (s, 3H, 4-Me), 2.57 (t, J ¼ 7.2 Hz, 2H, 10-CH2), 7.15e8.18
(m, 6H, Ar-H); ESIMS (m/z): 308.3 [M þ H]þ.
4.1.3.1. Methyl 40-[(6-n-propylaminocarbonyl-4-methyl-2-n-propyl-
1H-benzimidazol-1-yl)
White solid, yield 57%. m.p.: 175e177 ꢀC. IR (ATR, cmꢁ1): 1720 (C]
O),1627 (C]O); 1H NMR (400 MHz, DMSO-d6)
methyl]biphenyl-2-carboxy-late
(5a).
4.1.2.6. N-(2-Phenylethyl)-4-methyl-2-n-propyl-1H-benzimidazole-
6-carboxamide (3f). White solid, yield 85%. m.p.:152e154 ꢀC. IR
(ATR, cmꢁ1): 3193 (NH), 1631 (C]O); 1H NMR (400 MHz, DMSO-d6)
d: 0.91 (t, J ¼ 7.23 Hz,
3H, 300-Me), 0.96 (t, J ¼ 7.23 Hz, 3H, 30-Me), 1.58 (m, 2H, 200-CH2),
1.75 (m, 2H, 20-CH2), 2.55 (s, 3H, 4-Me), 2.77 (t, J ¼ 7.16 Hz, 2H, 10-
CH2), 3.20 (t, J ¼ 7.24 Hz, 2H, 100-CH2), 3.63 (s, 3H, COOMe), 5.56 (s,
2H, N-CH2-Ar), 7.07e7.89 (m, 10H, Ar-H), 8.45 (s, 1H, CONH); ESIMS
(m/z): 484.0 [M þ H]þ.
d
: 0.94 (t, J ¼ 7.3 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.55 (s, 3H, 4-
Me), 2.78 (t, J ¼ 7.2 Hz, 2H, 10-CH2), 2.95 (t, J ¼ 7.2 Hz, 2H, 200-CH2),
3.82 (t, J ¼ 7.2 Hz, 2H,100-CH2), 7.06e7.18(m, 5H, Ar-H), 7.47 (s,1H, 5-
H), 7.84 (s, 1H, 7-H); ESIMS (m/z): 322.2 [M þ H]þ.
4.1.2.7. N-[2-(4-Morpholinyl)ethyl]-4-methyl-2-n-propyl-1H-benz-
imidazole-6-carboxamide (3g). Light yellow solid, yield 76%.
m.p.:149e151 ꢀC. IR (ATR, cmꢁ1): 3188 (NH), 1635 (C]O); 1H NMR
4.1.3.2. Methyl 40-[(6-n-butylaminocarbonyl-4-methyl-2-n-propyl-
1H-benzimidazol-1-yl)methyl]biphenyl-2-carboxylate
White solid, yield 64%. m.p.: 196e198 ꢀC. IR (ATR, cmꢁ1): 1721 (C]
O),1629 (C]O); 1H NMR (400 MHz, DMSO-d6)
(5b).
(400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.3 Hz, 3H, 30-Me), 1.76 (m, 2H,
d: 0.91 (t, J ¼ 7.24 Hz,
20-CH2), 2.37 (t, J ¼ 7.2 Hz, 4H, 300-CH2), 2.52 (s, 3H, 4-Me), 2.78 (t,
J ¼ 7.2 Hz, 2H, 10-CH2), 2.83 (t, J ¼ 7.2 Hz, 2H, 200-CH2), 3.62 (m,
2H,100-CH2), 3.82 (t, J ¼ 7.2 Hz, 4H,400-CH2), 7.47 (s, 1H, 5-H), 7.84 (s,
1H, 7-H); ESIMS (m/z): 331.3 [M þ H]þ.
3H, 400-Me), 0.95 (t, J ¼ 7.25 Hz, 3H, 30-Me), 1.32 (m, 2H, 300-CH2),
1.59 (m, 2H, 200-CH2),1.75 (m, 2H, 20-CH2), 2.56 (s, 3H, 4-Me), 2.81 (t,
J ¼ 7.16 Hz, 2H, 10-CH2), 3.53 (m, 2H, 100-CH2), 3.60 (s, 3H, COOMe),
5.56 (s, 2H, N-CH2-Ar), 7.11e7.92 (m, 10H, Ar-H), 8.45 (s, 1H, CONH);
ESIMS (m/z): 498.1 [M þ H]þ.
4.1.2.8. N-(3-Phenylpropyl)-4-methyl-2-n-propyl-1H-benzimidazole-
6-carboxamide (3h). White solid, yield 79%. m.p.:189e191 ꢀC. IR
(ATR, cmꢁ1): 3181 (NH), 1633 (C]O); 1H NMR (400 MHz, DMSO-d6)
4.1.3.3. Methyl
pyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carbo-xylate
White solid, yield 61%. m.p.: 124e126 ꢀC. IR (ATR, cmꢁ1): 1725 (C]
40-[[6-(4-morpholinyl)carbonyl-4-methyl-2-n-pro-
(5c).
d
: 0.95(t, J ¼ 7.2 Hz, 3H, 30-Me), 1.77(m, 2H, 20-CH2), 2.08(m, 2H, 200-
CH2), 2.49(s, 3H, 4-Me), 2.58(t, J ¼ 6.0 Hz, 2H, 300-CH2), 2.76(t,
J ¼ 7.2 Hz, 2H, 10-CH2), 3.45(t, J ¼ 6.0 Hz, 2H, 100-CH2), 7.07e7.88(m,
7H, Ar-H), 8.41(s, 1H, CONH); ESIMS (m/z): 336.4 [M þ H]þ.
O), 1627 (C]O);1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz,
3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.58 (s, 3H, 4-Me), 2.81 (t,
J ¼ 7.6 Hz, 2H, 10-CH2), 3.52 (m, 4H, 100-CH2), 3.61 (s, 3H, COOMe),

50
J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
3.74 (m, 4H, 200-CH2), 5.57 (s, 2H, N-CH2-Ar), 7.19e7.92 (m, 10H, Ar-
H); ESIMS (m/z): 512.5 [M þ H]þ.
the filtrate was concentrated under vacuum to remove methanol.
The residue was adjusted to pH 6.5 with 3N aqueous hydrochloric
acid. The resulting precipitate was filtered, dried, and then recrys-
tallized in methanolechloroform (1:1) to provide pure 6ae6i as
white solids in yield of 71e83%.
4.1.3.4. Methyl 40-[[6-(1-piperidinyl)carbonyl-4-methyl-2-n-propyl-
1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxy-late
White solid, yield 68%. m.p.: 159e161 ꢀC. IR (ATR, cmꢁ1): 1718 (C]
O), 1618 (C]O); 1H NMR (400 MHz, DMSO-d6)
(5d).
d: 0.96 (t, J ¼ 7.2 Hz,
4.1.4.1. 40-[(6-n-Propylaminocarbonyl-4-methyl-2-n-propyl-1H-ben-
zimidazol-1-yl)methyl]biphenyl-2-carboxylic acid (6a). White solid,
yield 75%. m.p.: 285e286 ꢀC. IR (ATR, cmꢁ1): 3340 (OH), 1710 (C]
3H, 30-Me), 1.53 (m, 2H, 300-CH2), 1.62 (m, 4H, 200-CH2),1.76 (m, 2H,
20-CH2), 2.55 (s, 3H, 4-Me), 2.75 (t, J ¼ 7.2 Hz, 2H, 10-CH2), 3.48 (m,
4H, 100-CH2), 3.63 (s, 3H, COOMe), 5.55 (s, 2H, N-CH2-Ar), 7.09e
7.86(m, 10H, Ar-H); ESIMS (m/z): 510.4 [M þ H]þ.
O), 1644 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.91 (t, J ¼ 7.2 Hz,
3H, 300-Me), 0.96 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.59 (m, 2H, 200-CH2), 1.74
(m, 2H, 20-CH2), 2.56 (s, 3H, 4-Me), 2.78 (t, J ¼ 7.2 Hz, 2H, 10-CH2),
3.21 (t, J ¼ 7.2 Hz, 2H, 100-CH2), 5.55 (s, 2H, N-CH2-Ar), 7.07e7.89 (m,
10H, Ar-H), 8.45 (s, 1H, CONH), 12.72 (br, 1H, COOH); 13C NMR
4.1.3.5. Methyl 40-[[6-(4-methylphenyl)aminocarbonyl-4-methyl-2-
n-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylate (5e).
White solid, yield 65%. m.p.: 177e179 ꢀC. IR (ATR, cmꢁ1): 1723
(100 MHz, DMSO-d6) d: 11.88, 14.20, 16.86, 20.89, 22.86, 29.06,
(C]¼O), 1641 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t,
41.41, 46.15, 99.86, 107.59, 121.33, 126.33, 127.67, 128.66, 129.08,
129.44, 130.80, 131.22, 132.52, 134.99, 136.29, 140.37, 140.75, 143.99,
156.84, 166.85, 169.85; HRMS (ESI) Calcd for C29H32N3O3 [M þ H]þ:
470.2444, Found 470.2435.
J ¼ 7.2 Hz, 3H, 30-Me), 1.75 (m, 2H, 20-CH2), 2.38 (s, 3H, 400-Me), 2.56
(s, 3H, 4-Me), 2.71 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 3.76 (s, 3H, COOMe),
5.59 (s, 2H, N-CH2-Ar), 7.12e7.88 (m, 14H, Ar-H), 8.42 (d, J ¼ 8.0 Hz,
1H, CONH); ESIMS (m/z): 532.4 [M þ H]þ.
4.1.4.2. 40-[(6-n-Butylaminocarbonyl-4-methyl-2-n-propyl-1H-ben-
zimidazol-1-yl)methyl]biphenyl-2-carboxylic acid (6b). White solid,
yield 79%. m.p.: 253e254 ꢀC. IR (ATR, cmꢁ1): 3360 (OH), 1705 (C]
4.1.3.6. Methyl 40-[[6-(2-phenylethyl)aminocarbonyl-4-methyl-2-n-
propyl-1H-benzimidazol-1-yl] methyl]biphenyl-2-carboxylate (5f).
White solid, yield 72%. m.p.: 191e193 ꢀC. IR (ATR, cmꢁ1): 1731 (C]
O), 1638 (C]O);1H NMR (400 MHz, DMSO-d6)
d
: 0.91 (t, J ¼ 7.2 Hz,
O), 1629 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz,
3H, 400-Me), 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.34 (m, 2H, 300-CH2), 1.59
(m, 2H, 200-CH2), 1.78 (m, 2H, 20-CH2), 2.58 (s, 3H, 4-Me), 2.82 (t,
J ¼ 7.2 Hz, 2H, 10-CH2), 3.56 (m, 2H, 100-CH2), 5.55 (s, 2H, N-CH2-Ar),
7.09e7.92 (m, 10H, Ar-H), 8.44 (s, 1H, CONH), 12.71 (br, 1H, COOH);
3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.57 (s, 3H, 4-Me), 2.71 (t,
J ¼ 7.6 Hz, 2H, 10-CH2), 2.92 (t, J ¼ 7.2 Hz, 2H, 200-CH2), 3.81 (t,
J ¼ 7.2 Hz, 2H, 100-CH2), 3.78 (s, 3H, COOMe), 5.58 (s, 2H, N-CH2-Ar),
7.14e7.85 (m, 14H, Ar-H), 8.45 (d, J ¼ 8.0 Hz, 1H, CONH); ESIMS (m/
z): 546.6 [M þ H]þ.
13C NMR (100 MHz, DMSO-d6)
d: 13.70, 13.78, 16.44, 19.64, 20.47,
28.64, 31.32, 40.06, 45.73, 107.17, 120.90, 125.91, 127.24, 128.66,
129.02, 130.38, 130.79, 134.57, 135.87, 139.96, 140.33, 143.56, 156.42,
166.39, 169.43; HRMS (ESI) Calcd for C30H34N3O3 [M þ H]þ:
484.2600, Found 484.2594.
4.1.3.7. Methyl 40-[[6-[2-(4-morpholinyl)ethyl]aminocarbonyl-4-
methyl-2-n-propyl-1H-benzimidazol-1-yl]methyl]bi-phenyl-2-
carboxylate (5g). White solid, yield 63%. m.p.: 67e68 ꢀC. IR (ATR,
cmꢁ1): 1720 (C]O), 1635 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
4.1.4.3. 6-(4-Morpholinyl)carbonyl-4-methyl-2-n-propyl-1H-benzi-
midazol-1-yl]methyl]biphenyl-2-carboxylic acid (6c). White solid,
yield 83%. m.p.: 159e161 ꢀC. IR (ATR, cmꢁ1): 3375 (OH), 1708 (C]
0.95 (t, J ¼ 7.18 Hz, 3H, 30-Me), 1.74 (m, 2H, 20-CH2), 2.39 (m, 4H, 300-
CH2), 2.55 (s, 3H, 4-Me), 2.79 (t, J ¼ 7.22 Hz, 2H,10-CH2), 2.88 (m, 2H,
200-CH2), 3.49 (m, 4H, 100-CH2), 3.62 (s, 3H, COOMe), 3.72 (m, 4H, 400-
CH2), 5.55 (s, 2H, N-CH2-Ar), 7.09e7.93 (m, 10H, Ar-H); ESIMS (m/z):
555.5 [M þ H]þ.
O), 1597 (C]O); 1H NMR (400MHz, DMSO-d6)
d
: 0.96 (t, J ¼ 7.2 Hz,
3H, 30-Me), 1.74 (m, 2H, 20-CH2), 2.53 (s, 3H, 4-Me), 2.71 (t,
J ¼ 7.2 Hz, 2H, 10-CH2), 3.52 (m, 4H, 100-CH2), 3.77 (m, 4H, 200-CH2),
5.55 (s, 2H, N-CH2-Ar), 7.07e7.89 (m, 10H, Ar-H), 12.72 (br, 1H,
4.1.3.8. Methyl 40-[[6-(3-phenylpropyl)aminocarbonyl-4-methyl-2-n-
propyl-1H-benzimidazol-1-yl] methyl]biphenyl-2-carboxylate (5h).
White solid, yield 66%. m.p.: 137e139 ꢀC. IR (ATR, cmꢁ1): 1730 (C]
COOH);; 13C NMR (100 MHz, DMSO-d6)
d: 13.87, 16.42, 20.75, 28.67,
46.01, 54.97, 66.13, 107.39, 109.32, 121.22, 126.29, 127.37, 128.04,
128.73, 129.16, 130.46, 130.92, 131.57, 132.21, 134.17, 135.96, 140.08,
140.44, 156.12, 169.54, 169.90; HRMS (ESI) Calcd for C30H32N3O4
[M þ H]þ: 498.2393, Found 498.2387.
O), 1624 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94(t, J ¼ 7.2 Hz,
3H, 30-Me), 1.75(m, 2H, 20-CH2), 2.12(m, 2H, 200-CH2), 2.55(s, 3H, 4-
Me), 2.62(t, J ¼ 6.0 Hz, 2H, 300-CH2), 2.81(t, J ¼ 7.2 Hz, 2H, 10-CH2),
3.48(t, J ¼ 6.0 Hz, 2H, 100-CH2), 3.65(s, 3H, COOMe), 5.57(s, 2H, N-
CH2-Ar), 7.09e7.89(m, 15H, Ar-H), 8.42(s, 1H, CONH); ESIMS (m/z):
560.6 [M þ H]þ.
4.1.4.4. 40-[[6-(1-Piperidinyl)carbonyl-4-methyl-2-n-propyl-1H-ben-
zimidazol-1-yl]methyl]biphenyl-2-carboxylic acid (6d). White solid,
yield 82%. m.p.: 199e201 ꢀC. IR (ATR, cmꢁ1): 3370 (OH), 1709 (C]
O), 1594 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.95 (t, J ¼ 7.2 Hz,
4.1.3.9. Methyl 40-[[6-[3-(4-morpholinyl)propyl]aminocarbonyl-4-
methyl-2-n-propyl-1H-benzimidazol-1-yl] methyl]biphenyl-2-
carboxylate (5i). White solid, yield 69%. m.p.: 58e60 ꢀC. IR (ATR,
3H, 30-Me), 1.54(m, 2H, 300-CH2), 1.63(m, 4H, 200-CH2), 1.76 (m, 2H,
20-CH2), 2.63 (s, 3H, 4-Me), 2.89 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 3.48(m,
4H, 100-CH2), 5.75 (s, 2H, 2H, N-CH2-Ar), 7.20e8.62 (m, 10H, Ar-H),
cmꢁ1): 1720 (C]O), 1635 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
12.70 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6)
d: 13.78,
0.94 (t, J ¼ 7.3 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 1.82 (m, 2H, 200-
CH2), 2.39 (t, J ¼ 7.2 Hz, 4H, 400-CH2), 2.48 (m, 2H, 300-CH2), 2.52 (s,
3H, 4-Me), 2.78 (t, J ¼ 7.2 Hz, 2H, 10-CH2), 3.42 (m, 2H, 100-CH2), 3.67
(s, 3H, COOMe), 3.81 (m, 4H, 500-CH2), 5.59 (s, 1H, N-CH2-Ar),7.09e
7.96 (m,10H, ArH), 8.52 (s,1H, CONH); ESIMS (m/z): 569.5 [M þ H]þ.
16.32, 20.63, 24.04, 25.49, 28.61, 45.83, 106.68, 120.64, 126.14,
127.25, 128.59, 129.62, 130.34, 130.77, 132.15, 134.16, 135.93, 139.93,
140.32, 142.17, 155.78, 169.43, 169.59; HRMS (ESI) Calcd for
C
31H34N3O3 [M þ H]þ: 496.2600, Found 496.2589.
4.1.4.5. 40-[[6-(4-Methylphenyl)aminocarbonyl-4-methyl-2-n-pro-
pyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carbo-xylic acid (6e).
White solid, yield 75%. m.p.: 292e294 ꢀC. IR (ATR, cmꢁ1): 3360
4.1.4. General synthetic procedure for compounds 6ae6i
A mixture of compound 5 (0.4 mmol), methanol (10 mL) and 2N
aqueous sodium hydroxide (5 mL) was refluxed for 2 h. After
cooling to room temperature, the reaction mixture was filtered, and
(OH), 1718 (C]O), 1654 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.45 (s, 3H, 400-

J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
51
Me), 2.63 (s, 3H, 4-Me),3.09 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 5.75 (s, 2H, N-
CH2-Ar), 7.06w8.62 (m, 15H, Ar-H), 12.70 (s, 1H, COOH); 13C NMR
(100 MHz, DMSO-d6) d: 13.78, 16.46, 20.43, 20.46, 28.68, 45.78,
107.72, 120.36, 121.33, 125.96, 127.26, 127.49, 128.68, 128.87, 130.40,
130.80, 132.10, 132.26, 134.64, 135.85, 136.76, 139.99, 140.35, 143.95,
156.82, 165.62, 169.42; HRMS (ESI) Calcd for C33H32N3O3 [M þ H]þ:
518.2444, Found 518.2428.
15 mmol), followed by ammonium carbonate (0.96 g,10 mmol). The
resulting mixture was stirred at room temperature for 8 h and then
was filtered, the filtrate was washed with 1N hydrochloric acid,
saturated sodium bicarbonate solution and saturated sodium
chloride solution, dried over anhydrous sodium sulphate, filtered
and concentrated under vacuum. The residue was purified by
recrystallization in ethanol to provide pure product as a white solid,
yield 86%. m.p.: 284e286 ꢀC. IR (ATR, cmꢁ1): 3161 (NH), 1657 (C]
4.1.4.6. 40-[[6-(2-Phenylethyl)aminocarbonyl-4-methyl-2-n-propyl-
O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.95 (t, J ¼ 7.2 Hz, 3H, 30-Me),
1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxy-lic
acid
(6f).
1.74 (m, 2H, 20-CH2), 2.35 (s, 3H, 4-Me), 2.55 (t, J ¼ 7.2 Hz, 2H, 10-
CH2), 7.49 (s, 1H, 5-H), 7.88 (s, 1H, 7-H); ESIMS (m/z): 218.2
[M þ H]þ.
White solid, yield 79%. m.p.: 284e286 ꢀC. IR (ATR, cmꢁ1): 3288 (OH),
1739 (C]O), 1631 (C]O); 1H NMR (400 MHz, DMSO-d6)
d: 0.94 (t,
J ¼ 7.3 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.56 (s, 3H, 4-Me),
2.78w2.84 (m, 4H, 10-CH2, 200-CH2), 3.47 (t, J ¼ 6.5 Hz, 2H, 100-CH2),
5.56(s, 2H, N-CH2-Ar), 7.09e7.88 (m,15H, Ar-H), 8.48(d, J ¼ 5.6Hz,1H,
4.1.6. Methyl 40-[(6-aminocarbonyl-4-methyl-2-n-propyl-1H-
benzimidazol-1-yl)methyl]biphenyl-2-carboxylate (8)
CONH), 12.70 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6)
d
: 13.93,
To a stirred solution of compound 7 (2 mmol) in 15 mL of
dimethylformamide at 0 ꢀC, potassium tert-butoxide (2.2 mmol)
was added. The mixture was stirred for 30 min at 0 ꢀC, and then 40-
bromomethylbiphenyl-2-carboxylate (4, 2.1 mmol) was added.
After stirring at room temperature for 12 h, the mixture was poured
into water (80 mL) and extracted with ethyl acetate (40 mL ꢂ 3).
The combined ethyl acetate layers were washed with brine
(50 mL ꢂ 3), dried over anhydrous magnesium sulphate, and
concentrated under a reduced pressure to provide an off-white
solid. The solid was purified by column chromatography (elution:
1:1 petroleum ether/ethyl acetate) to provide a white crystalline
solid, yield 74%. m.p.: 162e164 ꢀC. IR (ATR, cmꢁ1): 1732 (C]O),
16.58, 20.64, 28.80, 35.34, 41.06, 45.90, 107.36, 109.36, 121.10, 126.11,
127.41, 128.39, 128.73, 128.80, 129.18, 130.52, 130.93, 132.44, 134.67,
135.87,13970,140.10,140.45,156.64,166.64,169.53; HRMS (ESI) Calcd
for C34H33N3O3 [M þ H]þ: 532.2600, Found 532.2589.
4.1.4.7. 40-[[6-[2-(4-Morpholinyl)ethyl]aminocarbonyl-4-methyl-2-
n-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic
(6g). White solid, yield 71%. m.p.: 165e167 ꢀC. IR (ATR, cmꢁ1): 3370
(OH), 1691 (C]O),1598 (C]O); 1H NMR (400 MHz, DMSO-d6)
: 0.94
acid
d
(t, J¼ 7.2 Hz, 3H, 30-Me),1.76(m, 2H, 20-CH2), 2.42(m, 4H, 300-CH2), 2.67
(s, 3H, 4-Me), 2.76 (t, J ¼ 7.2 Hz, 2H, 10-CH2), 2.93 (m, 2H, 200-CH2), 3.45
(m, 4H, 100-CH2), 3.77 (m, 4H, 400-CH2), 5.82 (s, 2H, N-CH2-Ar), 7.29e
8.22 (m, 10H, Ar-H), 8.78 (s,1H, CONH), 12.65 (s, 1H, COOH); 13C NMR
1620 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.95 (t, J ¼ 7.18 Hz,
3H, 30-Me), 1.74 (m, 2H, 20-CH2), 2.55 (s, 3H, 4-Me), 2.79 (t,
J ¼ 7.22 Hz, 2H, 10-CH2), 3.65 (s, 3H, COOMe), 5.55 (s, 2H, N-CH2-Ar),
7.09e7.93 (m, 10H, Ar-H); ESIMS (m/z): 442.3 [M þ H]þ.
(100 MHz, DMSO-d6) d: 14.51, 17.16, 21.19, 29.37, 37.25, 37.25, 46.52,
53.98, 58.17, 66.83,107.91,121.69,126.19,127.10,127.23,128.00,128.07,
129.38, 135.23, 135.39, 137.60, 142.10, 144.35, 157.18, 167.21, 173.51;
HRMS (ESI) Calcd for C32H37N4O4 [MþH]þ: 541.2815, Found 541.2801.
4.1.7. Methyl 40-[(6-methoxycarbonylamino-4-methyl-2-n-propyl-
1H-benzimidazol-1-yl)methyl]biphenyl-2-carboxylate (9)
4.1.4.8. 40-[[6-(3-Phenylpropyl)aminocarbonyl-4-methyl-2-n-propyl-
To a stirred mixture of compound 8 (1.32 g, 3 mmol) and KF/
Al2O3 (6 g) was added 10% sodium hypochlorite solution, the re-
action mixture was refluxed for 30 min and then cooled, filtered,
the filtrate was concentrated and the residue was purified by col-
umn chromatography, 0.82 g white solid was obtained. Yield 79%.
m.p.: 154e156 ꢀC. IR (ATR, cmꢁ1): 1730 (C]O), 1632 (C]O); 1H
1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxy-lic
acid
(6h).
White solid, yield 76%. m.p.: 239e241 ꢀC. IR (ATR, cmꢁ1): 3380
(OH), 1693 (C]O), 1598 (C]O); 1H NMR (400 MHz, DMSO-d6)
d:
0.94 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 1.96 (m, 2H, 200-
CH2), 2.58 (s, 3H, 4-Me), 2.68 (t, J ¼ 7.6 Hz, 2H, 300-CH2), 2.82 (t,
J ¼ 7.6 Hz, 2H, 10-CH2), 3.51 (m, 2H, 100-CH2), 5.57 (s, 2H, N-CH2-Ar),
7.19e7.92 (m, 15H, Ar-H), 8.45 (s, 1H, CONH), 12.66(s, 1H, COOH);
NMR (400 MHz, DMSO-d6)
d
: 1.01 (t, J ¼ 7.2 Hz, 3H, 30-Me), 1.82 (m,
2H, 20-CH2), 2.65 (s, 3H, 4-Me), 2.87 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 3.64
(s, 3H, NHCOOMe), 3.76 (s, 3H,COOMe),5.36 (s, 2H, N-CH2-Ar),
6.79e7.85 (m, 10H, Ar-H), 8.42 (s, 1H, CONH); ESIMS (m/z): 472.2
[M þ H]þ.
13C NMR (100MHz, DMSO-d6)
d: 14.31, 16.99, 19.04, 21.03, 29.19,
31.46, 33.17, 46.29, 107.77, 121.51, 126.18, 126.47, 127.79, 127.83,
128.74, 128.78, 129.21, 129.58, 130.92, 131.32, 132.70, 135.18, 136.39,
140.53, 140.87, 142.26, 144.14, 157.01, 167.09, 170.01; HRMS (ESI)
Calcd for C35H36N3O3 [M þ H]þ: 546.2757, Found 546.2745.
4.1.8. 40-[(6-Amino-4-methyl-2-n-propyl-1H-benzimidazol-1-yl)
methyl]biphenyl-2-carboxylic acid (10)
4.1.4.9. 40-[[6-[3-(4-Morpholinyl)propyl]aminocarbonyl-4-methyl-2-
The mixture of compound 9 (0.7 g, 2.1 mmol), sodium hydroxide
(0.4 g, 10 mmol) and dioxane (20 mL) was heated at 100 ꢀC for 3 h,
then was concentrated, ethanol was added and the solution was
acidified with conc. hydrochloride acid to pH ¼ 6, the mixture was
filtered and the filtrate was concentrated to obtain 0.44 g light
brown solid, yield 53%. m.p.: 195e197 ꢀC. IR (ATR, cmꢁ1): 3300
n-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic
acid
(6i). White solid, yield 72%. m.p.: 205e206 ꢀC. IR (ATR, cmꢁ1): 3370
(OH), 1690 (C]O), 1628 (C]O);1H NMR (400MHz, DMSO-d6)
d: 0.95
(t, J ¼ 7.2 Hz, 3H, 30-Me),1.76 (m, 2H, 20-CH2),1.85 (m, 2H, 200-CH2), 2.38
(m, 4H, 400-CH2), 2.46 (m, 2H, 300-CH2), 2.57 (s, 3H, 4-Me), 2.62 (t,
J ¼ 7.6 Hz, 2H, 10-CH2), 3.27 (m, 2H, 100-CH2), 3.77 (m, 4H, 500-CH2),5.57
(s, 2H, N-CH2-Ar), 7.12e8.22(m,10H, Ar-H), 8.31(s,1H, CONH),12.72(s,
(OH), 1710 (C]O); 1H NMR (400 MHz, DMSO-d6)
d: 0.94 (t,
J ¼ 7.2 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.58 (s, 3H, 4-Me), 2.81
(t, J ¼ 7.6 Hz, 2H, 10-CH2), 5.57 (s, 2H, N-CH2-Ar), 7.19e7.92 (m, 10H,
Ar-H); HRMS (ESI) Calcd for C25H26N3O2 [M þ H]þ: 400.2025, Found
400.2033.
1H, COOH); 13CNMR (100 MHz, DMSO-d6)
d:13.93,16.59, 20.62, 26.09,
28.77, 30.82, 37.92, 45.91, 53.39, 56.21, 66.24, 107.37, 1210.7, 125.66,
126.59, 127.01, 127.38, 127.62, 128.26, 128.79, 129.28, 134.67, 134.94,
137.29,141.38,143.74,156.56,162.39,166.58,172.86; HRMS (ESI) Calcd
for C33H39N4O4 [M þ H]þ: 555.2971, Found 555.2966.
4.1.9. General procedure for compounds 11ae11g
To a stirred mixture of compound 10 (0.40 g,1 mmol) in 20 mL of
dichloromethane at 0 ꢀC was added triethylamine (1.5 mmol), and
then added dropwise different acyl chloride (1 mmol) in 5 mL of
dichloromethane. The resulting mixture was stirred at room
4.1.5. 4-Methyl-2-n-propyl-1H-benzimidazole-6-carboxamide (7)
To a stirred suspension of acyl chloride (2) in 60 mL of
dichloromethane at 0 ꢀC was added dropwise triethylamine (1.52 g,

52
J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
temperature for 5 h, then 10 mL of water was added. The organic
phase was separated, washed with diluted hydrochloride acid and
water, dried over anhydrous sodium sulphate, filtered, and the
filtrate was concentrated under reduced pressure. The residue was
recrystallized in ethanol to provide pure 11ae11g as light yellow or
white solids in yield of 68e80%.
134.21, 135.11, 135.26, 137.13, 138.86, 141.71, 141.99, 144.07, 154.70,
165.49, 173.43; HRMS (ESI) Calcd for C33H32N3O3 [M þ H]þ:
518.2444, Found 518.2442.
4.1.9.6. 40-[(6-Phenylacetylamino-4-methyl-2-n-propyl-1H-benzimi-
dazol-1-yl)methyl]biphenyl-2-carboxylic acid (11f). White solid,
yield 78%, m.p.: 186e188 ꢀC. IR (ATR, cmꢁ1): 3280 (OH), 1717 (C]
4.1.9.1. 40-[(6-Acetylamino-4-methyl-2-n-propyl-1H-benzimidazol-
1-yl)methyl]biphenyl-2-carboxylic acid (11a). Light yellow solid,
yield 68%, m.p.: 215e217 ꢀC. IR (ATR, cmꢁ1): 3420 (OH), 1716 (C]
O), 1653 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz,
3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.58 (s, 3H, 4-Me), 2.81 (t,
J ¼ 7.6 Hz, 2H, 10-CH2), 3.56 (s, 2H, 100-CH2), 5.57 (s, 2H, N-CH2-Ar),
O), 1682 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz,
7.19e7.92 (m, 15H, Ar-H); 13C NMR (100 MHz, DMSO-d6)
d: 14.34,
3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.12 (s, 3H, 100-Me), 2.58 (s, 3H, 4-
17.00, 21.14, 29.12, 29.49, 43.75, 46.32, 98.95, 115.14, 126.20, 126.88,
127.19, 128.21, 128.46, 128.68, 129.18, 129.61, 130.00, 134.36, 135.13,
135.60, 136.73, 138.50, 141.41, 154.57, 169.22, 172.26; HRMS (ESI)
Calcd for C33H32N3O3 [M þ H]þ: 518.2444, Found 518.2441.
Me), 2.81 (t, J ¼ 7.6 Hz, 2H,10-CH2), 5.57 (s, 2H, N-CH2-Ar), 7.19e7.92
(m, 10H, Ar-H); 13C NMR (100 MHz, DMSO-d6)
d: 14.42, 16.99, 21.16,
24.44, 29.48, 46.19, 98.69, 115.06, 126.33, 127.59, 128.17, 129.13,
129.21, 129.33, 130.72, 132.00, 134.59, 135.14, 136.22, 138.31, 140.32,
140.74, 154.43, 168.43, 171.42; HRMS (ESI) Calcd for C27H28N3O3
[M þ H]þ: 442.2131, Found 442.2128.
4.1.9.7. 40-[[6-(3-Phenylpropionyl)amino-4-methyl-2-n-propyl-1H-
benzimidazol-1-yl]methyl]biphenyl-2-carboxylic
acid
(11g).
White solid, yield 74%, m.p.: 168e170 ꢀC. IR (ATR, cmꢁ1): 3420 (OH),
4.1.9.2. 40-[(6-n-Propionylamino-4-methyl-2-n-propyl-1H-benzimi-
dazol-1-yl)methyl]biphenyl-2-carboxylic acid (11b). White solid,
yield 78%, m.p.: 165e167 ꢀC. IR (ATR, cmꢁ1): 3340 (OH), 1717 (C]O),
1683 (C]O), 1650 (C]O); 1H NMR (400 MHz, DMSO-d6)
d: 0.94 (t,
J ¼ 7.2 Hz, 3H, 30-Me), 1.81 (m, 2H, 20-CH2), 2.62 (s, 3H, 4-Me), 2.77
(m, 2H,100-CH2), 2.81 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 2.96 (m, 2H, 200-CH2),
5.67 (s, 2H, N-CH2-Ar), 7.19e7.92 (m, 15H, Ar-H), 12.56 (s, 1H,
1658 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz, 3H,
30-Me), 1.25 (t, J ¼ 7.2 Hz, 3H, 200-Me), 1.76 (m, 2H, 20-CH2), 2.26 (m,
2H, 100-CH2), 2.58 (s, 3H, 4-Me), 2.81 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 5.57 (s,
2H, N-CH2-Ar), 7.19e7.92 (m, 10H, Ar-H); 13C NMR (100 MHz, DMSO-
COOH); 13C NMR (100 MHz, DMSO-d6)
d: 14.24, 17.02, 21.17, 29.12,
31.39, 38.38, 46.29, 98.88, 115.15, 126.17, 126.35, 127.19, 128.17,
128.44, 128.70, 128.76, 129.17, 130.01, 134.39, 135.13, 135.66, 138.43,
141.36, 141.76, 154.48, 170.48, 172.25; HRMS (ESI) Calcd for
d6) d: 10.23, 14.34, 16.99, 21.16, 29.10, 29.93, 46.16, 98.70, 115.09,
126.34, 127.71, 128.22, 129.17, 129.45, 130.84, 131.03, 134.55, 135.18,
136.40, 138.30, 140.53, 140.64, 154.45, 170.46, 172.08; HRMS (ESI)
Calcd for C28H30N3O3 [M þ H]þ: 456.2287, Found 456.2276.
C
34H34N3O3 [M þ H]þ: 532.2600, Found 532.2594.
4.1.10. 40-[(6-Methoxycarbonylamino-4-methyl-2-n-propyl-1H-
benzimidazol-1-yl)methyl]biphenyl-2-carboxylic acid (12)
4.1.9.3. 40-[(6-n-Butyrylamino-4-methyl-2-n-propyl-1H-benzimida-
zol-1-yl)methyl]biphenyl-2-carboxylic acid (11c). White solid, yield
81%, m.p.: 178e180 ꢀC. IR (ATR, cmꢁ1): 3336 (OH),1717 (C]O),1653
The mixture of compound 9 (0.5 g, 1.1 mmol), 1N aqueous so-
dium (5.5 mL, 5.5 mmol) and methanol (20 mL) was heated at 60 ꢀC
for 3 h, then was concentrated, 10 mL water was added, cooled, and
the solution was acidified with 1N hydrochloride acid to pH ¼ 6, the
precipitate formed was filtered to obtain 0.39 g white solid, yield
77%. m.p. 198e200 ꢀC; IR (ATR, cmꢁ1): 3331 (OH), 1717 (C]O), 1610
(C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.92 (t, J ¼ 7.2 Hz, 3H, 30-
Me), 0.98 (t, J ¼ 7.2 Hz, 3H, 300-Me), 1.78 (m, 2H, 20-CH2), 1.86 (m, 2H,
200-CH2), 2.53 (s, 3H, 4-Me), 2.62 (t, J ¼ 7.2 Hz, 2H, 100-CH2), 2.82 (t,
J ¼ 7.2 Hz, 2H, 10-CH2), 5.45 (s, 2H, N-CH2-Ar), 7.06e7.96 (m, 10H,
(C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.94 (t, J ¼ 7.2 Hz, 3H, 30-
Ar-H), 12.45 (s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6)
d
: 14.15,
Me), 1.76 (m, 2H, 20-CH2), 2.58 (s, 3H, 4-Me), 2.81 (t, J ¼ 7.6 Hz, 2H,
14.33, 16.99, 19.08, 21.15, 29.10, 38.77, 46.15, 98.72, 115.14, 126.33,
127.71, 128.21, 129.19, 129.46, 130.85, 131.03, 134.54, 135.18, 136.41,
138.33, 140.53, 140.66, 154.45, 170.48, 171.24; HRMS (ESI) Calcd for
10-CH2), 3.76 (s, 3H, COOMe), 5.57 (s, 2H, N-CH2-Ar), 7.19e7.92 (m,
10H, Ar-H); 13C NMR (100 MHz, DMSO-d6)
d: 14.33, 16.99, 21.18,
29.10, 46.20, 51.93, 97.81, 114.56, 126.44, 127.74, 128.35, 129.17,
129.50, 130.87, 131.17, 133.03, 134.24, 135.36, 136.44, 138.07, 140.47,
140.75, 154.31, 154.61, 170.14; HRMS (ESI) Calcd for C27H28N3O4
[M þ H]þ: 458.2080, Found 458.2082.
C
29H32N3O3 [M þ H]þ: 470.2444, Found 470.2437.
4.1.9.4. 40-[(6-Benzoylamino-4-methyl-2-n-propyl-1H-benzimidazol-
1-yl)methyl]biphenyl-2-carboxylic acid (11d). White solid, yield
79%, m.p.: 195e197 ꢀC. IR (ATR, cmꢁ1): 3270 (OH), 1699 (C]O),
4.2. Biological evaluation
1653 (C]O); 1H NMR (400 MHz, DMSO-d6)
d
: 0.93 (t, J ¼ 7.2 Hz, 3H,
30-Me), 1.78 (m, 2H, 20-CH2), 2.68 (s, 3H, 4-Me), 2.85 (t, J ¼ 7.6 Hz,
4.2.1. Procedure for receptor binding assay
2H, 10-CH2), 5.67 (s, 2H, N-CH2-Ar), 7.19e7.92 (m, 15H, Ar-H), 12.45
The AT1 receptor binding assay was carried out by the
competitive displacement of the binding of [125I] Sar1 Ile8-Ang II
with angiotensin AT1 receptor as described previously [18]. Each
(s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6)
d: 14.34, 17.03, 21.15,
29.13, 46.22, 100.23, 116.49, 126.42, 127.76, 128.02, 128.15, 128.79,
129.03, 129.19, 129.75, 130.90, 131.24, 131.83, 132.00, 133.28, 134.17,
135.15, 135.56, 136.48, 138.90, 140.47, 154.80, 165.66, 170.13; HRMS
(ESI) Calcd for C32H30N3O3 [M þ H]þ: 504.2287, Found 504.2279.
180-
pM), AT1(AT2) receptor (25
m
L incubate contained the following: [125I] Sar1 Ile8-Ang II (25
mg), and standard (losartan) or test
compounds (6aw6i, 11aw11g, 12). The 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
filter disks were punched out and counted in a gamma counter. IC50
values were estimated from the linear portion of the competition
curves.
4.1.9.5. 40-[[6-(4-Methylbenzoyl)amino-4-methyl-2-n-propyl-1H-
benzimidazol-1-yl]methyl]biphenyl-2-carboxylic
acid
(11e).
White solid, yield 75%, m.p.: 175e177 ꢀC. IR (ATR, cmꢁ1): 3300 (OH),
1699 (C]O), 1653 (C]O); 1H NMR (400 MHz, DMSO-d6)
d: 0.94 (t,
J ¼ 7.2 Hz, 3H, 30-Me), 1.76 (m, 2H, 20-CH2), 2.38 (s, 3H, 400-Me), 2.58
(s, 3H, 4-Me),2.81 (t, J ¼ 7.6 Hz, 2H, 10-CH2), 5.57 (s, 2H, N-CH2-Ar),
4.2.2. Procedure for Angiotensin II receptor functional antagonism
in rabbit aorta strips
7.19e7.92 (m, 14H, Ar-H); 13C NMR (100 MHz, DMSO-d6)
d: 14.37,
17.05, 21.16, 21.45, 29.15, 46.31, 100.41, 116.62, 126.00, 126.68,
126.85, 127.70, 128.00, 128.11, 129.18, 129.28, 129.45, 132.01, 132.68,
Japanese white rabbits (2 kg to 3 kg body weight, Vitalriver
Company, China) were killed by cervical dislocation after slight

J. Zhang et al. / European Journal of Medicinal Chemistry 69 (2013) 44e54
53
anesthesia with 20% urethane solution. The thoracic aorta was
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Financial support of this work by the Fundamental Research
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and the National Technological Project of the Manufacture and
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