Design, synthesis and biological activity of 6-substituted carbamoyl benzimidazoles as new nonpeptidic angiotensin II AT1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.05.056

A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic angiotensin II AT₁ receptor antagonists. The preliminary pharmacological evaluation revealed a nanomolar AT₁ receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6g is an orally active AT₁ receptor antagonist with low toxicity.

Full text of DOI:10.1016/j.bmc.2012.05.056

Bioorganic & Medicinal Chemistry 20 (2012) 4208–4216
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activity of 6-substituted carbamoyl
benzimidazoles as new nonpeptidic angiotensin II AT1 receptor antagonists
Jun Zhang a, Jin-Liang Wang a, Zhi-Ming Zhou a, , Zhi-Huai Li 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:
A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic
angiotensin II AT1 receptor antagonists. The preliminary pharmacological evaluation revealed a nanomo-
lar AT1 receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an
isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Fur-
thermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that
compound 6g is an orally active AT1 receptor antagonist with low toxicity.
Received 4 April 2012
Revised 28 May 2012
Accepted 29 May 2012
Available online 5 June 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Angiotensin II AT1 receptor antagonists
carbamoyl benzimidazole
Hypertension
1. Introduction
the overall interaction between the antagonist and the receptor.
Interestingly, it has been shown in the literature13 that phenyleth-
Antagonism of octapeptide angiotension II (Ang II) at the AT1
receptor offers real promise as a therapy for hypertension and
heart failure,1,2 although the functional role of the AT2 receptor
has yet to be clearly demonstrated.3 The discovery of the AT1-
selective AII antagonist Losartan has stimulated extensive research
on other nonpeptide AII antagonists bearing novel heterocyclic
moieties.4,5 Among the large variety of the heterocyclic systems
developed, the ortho-fused bicyclic moiety benzimidazole appears
to be, from the point of view of the interaction with the AT1 recep-
tor, a particularly effective heterocyclic system.6,7 This compound
is commercially available as Candesartan8 and is the most potent
AT1 antagonist so far. Meanwhile, modification of the benzimid-
azole nucleus also demonstrated that the presence of an acylureas
lipophilic group at the 6-position,9 a carboxylic group at the 7-po-
sition,10 and a nitro group at the 5-position11 were favourable for
AII antagonism. However, benzimidazole-6 carboxylic acid deriva-
tives have been scarcely exploited in treatments for hypertension
and heart failure. The presence of the L3 lipophilic pockets
proposed in the literature12 suggested that a carboxamide with
an alkyl-chain-bearing phenyl group may provide additional inter-
ylamino can effectively lower hypertensive pressure. Therefore,
N-substituted alkyl 1-[20-(1H-tetrazol-5-yl)-1,10-biphenyl-4-yl]-
methyl-4-methyl-2-n-propyl-1H-benzimidazole-6-carboxamide
compounds (Fig. 1) were designed with a carbamoyl group at the
6-position, which are expected to have higher activity than Losar-
tan. A previous study in our laboratory14,15 regarding the electro-
static potential of the benzimidazole nucleus showed that an
electron-donating group at the phenyl ring of the phenylethyl-
amino fragment facilitates the activity. Therefore, in the present
study, we designed, synthesised and evaluated 6-substituted
carbamoyl benzimidazoles bearing methoxy on the phenyl side
and the effect of the length of the bridge chain using Losartan as
reference compound.
N
H
R
N
N
N
N
n
N
NH
O
actions (Van der Waals force, e.g., p–p) that consequently enhance
⇑
Corresponding author. Tel./fax: +86 10 68918982.
E-mail address: zzm@bit.edu.cn (Z.-M. Zhou).
Figure 1. The structure of the designed compounds.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.056

J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4209
N
N
N
H
N
n
R
a
b
N
H
Cl
N
H
N
H
HOOC
O
O
4a-4m
3
2
N
N
N
H
N
n
R
H
N
n
R
N
N
N
N
d
N
N
N
NH
N
c
O
CPh3
O
4
6a-6m
5a-5m
Scheme 1. Reagents and conditions: (a) thionyl dichloride, reflux, 3 h; (b) various substituted amines, Et3N, DCM, rt, 6 h; (c) 5-(40-bromomethyl-1,10-biphenyl-2-yl)-1-
triphenylmethyl-1H tetrazole, t-BuOK, DMF, rt, 12 h; (d) 10%HCl, THF, MeOH; NaOH; HCl, overnight.
addition, compounds 6a, 6c, 6e, 6i, 6j and 6l showed the same
activity level as losartan (10 nM < AT1 IC50 <100 nM). These results
suggested that both the length of the aliphatic chains (which con-
nect the acylamino moiety and the phenyl ring) and the substitu-
tion pattern on the aromatics played key roles in the binding
affinity.
Based on the results of in vitro Ang II-binding assays and func-
tional antagonism studies, 6g was selected for further evaluation
using in vivo models. When evaluated orally in conscious, sponta-
neously hypertensive rats (SHR), at doses of 10 mg/kg, compound
6g significantly decreased blood pressure by more than 30 mmHg
(Fig. 2), suggesting that it is more efficacious than losartan.
2. Results and discussion
2.1. Chemistry
The target compounds 6a–6m were synthesised 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 4a–4m with an 82–
90% yield. The acylamides were then alkylated with 5-(40-bromo-
methyl-1,10-biphenyl-2-yl)-1-triphenylmethyl-1H-tetrazole using
potassium tert-butoxide in DMF to give the corresponding prod-
ucts in yields of 60–70%. The last step, deprotection of 5a–5m
using diluted hydrochloride acid followed by sodium hydroxide,
was accomplished with a greater than 90% yield. All target com-
pounds were identified by IR, 1H NMR, 13C NMR and HRMS.
2.3. Molecular modelling
In the molecular modelling simulation study, six clinically used
sartans (Fig. 3) were selected as the training set, and the pharma-
cophore hypothesis generation was performed using the HipHop
module of the Discovery Studio software.21 Although the 3D struc-
ture of the AT1 receptor is unknown, an ideal pharmacophore
hypothesis can be identified.22 Accordingly, we used the method
as a template representing the geometry of the receptor sites as
a collection of functional groups in space. In this method, the fit va-
lue indicates how well the features in the pharmacophore map the
chemical features in the molecule.23 The higher the fit value, the
more active a compound is. The fit values of the synthesised com-
pounds were determined based on the hypothesis of AT1 receptor
antagonism using the best-fit algorithm as described in the
literature.24
In this study, there are five features in the selected pharmaco-
phore hypothesis, so the highest fit value is 5. Accordingly, the esti-
mated activity could be classified according to the fit value as
highly active (fit value >4, +++), moderately active (2< fit value
<4, ++) and low activity (fit value <4, +). All synthesised compounds
in this study were also classified according to the literature23 by
their activity as highly active (<100 nM, +++), moderately active
(100–10000 nM, ++) and low activity (>10000 nM, +). All highly ac-
tive compounds were predicted correctly, only three moderately
active compounds were predicted to be highly active. The results
showed that despite these disparities, the pharmacophore hypoth-
esis is reasonable. Furthermore, the molecular modelling simula-
tion 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 fea-
ture. The same binding modality was also observed in the
mapping of telmisartan with the pharmacophore (Fig. 4), in which
2.2. Biological evaluation
The prepared compounds were evaluated for their in vitro Ang
II receptor binding affinity based on the competitive inhibition of
[
125I] Ang II binding to the AT1 receptors by a conventional li-
gand-binding assay as described previously.16 The results are ex-
pressed as IC50 values, which is the concentration of a compound
that inhibits [125I] Ang II binding to the receptor by 50%. The antag-
onistic activity of the more potent compounds (6f, 6g, 6h and 6k)
were also investigated in vitro using the contractile response of
isolated rabbit aortic strips in a functional assay.17 Losartan was ta-
ken as a positive control drug in the assays. At last, the most potent
compound 6g was evaluated in an in vivo model.
As summarised in Table 1, the binding assay and the antagonis-
tic activity results showed that all compounds displayed a certain
extent of inhibition activity, as expected. Most of the compounds
showed high activities for the binding affinities in the submicrom-
olar range. Of all the synthesised compounds, compound 6g was
the most potent compound (IC50 = 0.9 nM), exhibiting almost the
same binding affinity as the marketed AT1 receptor antagonist tel-
misartan (IC50 = 1.0, 0.33 nM18), and about 12-fold the affinity of
losartan (IC50 = 16.2, 150,19 6.7 nM20). The pA2 8.77 also shows that
this compound contains more potent antagonist activity than the
positive control losartan (pA2 = 7.9). Compounds 6f (IC50 = 1.3 nM),
6h (IC50 = 2.6 nM) and 6k (IC50 = 3.7 nM) were also much more ac-
tive than losartan. The pA2 of compounds 6f and 6k are 8.32 and
8.74, respectively, which are also higher than that of losartan. In

4210
J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
Table 1
Fit values on the selected pharmacophore and experimental data of the target compounds
N
N
H
N
n
R
N
N
N
NH
O
6a-6m
Compod No.
n
R
Fit value
IC50 (nM)
Est. act. scaleb
Exp. act. scalec
pA2
LC50 (mg Lꢀ1
)
6a
6b
6c
6d
6e
6f
6g
6h
6i
1
1
1
1
1
2
2a
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
4.82
4.93
4.76
4.91
410
19.7
78.3
118.5
96.8
1.3
0.9
2.6
22.5
78
3.7
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
64
66
2-OMe
3-OMe
4-OMe
3,4-Di-OMe
H
+++
+++
++
>100d
>100
>100
>100
>100
>100
>100
>100
>100
17
+++
+++
+++
+++
+++
+++
+++
+++
++
8.32
8.77
7.83
H
3-OMe
4-OMe
2,5-Di-OMe
3,4-Di-OMe
2-F
4-F
Losartan
Telmisartan
6j
6k
6l
8.74
7.90
37.6
209
16.2
1.0
6m
60
210
>100
+++
+++
a
b
c
The group between the amide group and the phenyl group is 2-methylethyl.
Estimated activity scale: highly active (fit value >4, +++), moderately active (2< fit value <4, ++) and low activity (fit value <4, +).
Activity scale: highly active (<100 nM, +++), moderately active (100–10,000 nM, ++) and low activity (>10,000 nM, +).22
d
No LC50 value could be determined because this compound did not produce more than 50% inhibition of the luminescence of P. phosphoreum at the saturated concen-
tration level.
according to the standard Microtox test using luminescent bacte-
ria. This assay is considered to be a useful tool and provides quick
and reliable data.26
In the toxicity assay, the results of the Microtox test are ex-
pressed in terms of LC50 values. LC50 is the concentration in water
that inhibits 50% of a test batch of Photobacterium phosphoreum as
exemplified by compound 6l and 6m in Figure 6. The lower the
LC50 value, the greater the toxicity of the sample is.27 The results
revealed that the LC50 values of most our synthetic compounds
and telmisartan are higher than 100 mg Lꢀ1, as they did not pro-
duce more than 50% inhibition of bacterial luminescence at the sat-
urated concentration level. It is 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 algae were used.28
This method provided a choice to evaluate the preliminary toxicity
of these compounds, and the results showed that the toxicity of
most compounds of the compounds is as low as that of telmisartan.
Figure 2. Effects of 6g and losartan (10 mg/Kg po) on mean arterial pressure in
conscious SHR after oral administration.
3. Conclusions
it was the methyl in the second benzimidazole ring and not the
propyl that mapped onto the hydrophobic aliphatic feature. How-
ever, in the case of losartan, it was the alkyl substituent that
mapped onto the hydrophobic aliphatic feature (Fig. 5). This differ-
ence implied that the interaction modality of 6g, like that of telmi-
sartan, 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 literature25 and were ob-
tained through the docking of ligands with the homology-mod-
elled AT1 receptor.
In summary, a series of 6-substituted carbamoyl benzimidaz-
oles were designed and synthesised as new nonpeptidic AT1
receptor antagonists. Compounds 6g was found to be the most po-
tent 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.
4. Experimental
4.1. Chemistry
2.4. Toxicity assay
The melting points were determined on an XT-4A melting point
apparatus and are uncorrected. All the target compounds were
Furthermore, a preliminary evaluation of toxicity against lumi-
nescent bacteria was performed on the prepared compounds

J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4211
Cl
N
N
N
N
CH3
N
N
OH
O
N
N
NH
N
N
NH
N
N
N
HOOC
N
COOH
Losartan
Candesartan
Telmisartan
HO
N
N
N
HOOC
N
N
N
O
N
N
N
N
NH
N
O
N N
N
N
NH
N
NH
Tasosartan
Irbesartan
Olmesartan
Figure 3. Structures of several AT1 receptor antagonists.
Figure 4. (a) The selected pharmacophore hypothesis; (b) mapping of 6g with the selected hypothesis; (c) mapping of telmisartan with the selected hypothesis.
characterised by IR, 1H NMR, 13C NMR and HRMS. Infrared (IR)
spectra were recorded on an FTIR spectrometer (Nicolet Magna
IR 560) with KBr pellets. 1H NMR and 13C NMR spectra were mea-
sured on a Bruker Avance III 400 NMR spectrometer using TMS as
an internal standard (chemical shift in ppm). High-resolution mass
spectra were recorded on a Bruker Apex IV FTMS spectrometer.
Column chromatography was performed with silica gel (200–300
mesh). Thin layer chromatography was performed on high-silica-
gel GF254 pre-coated plates.
4.1.1. 4-Methyl-2-n-propyl-1H-benzimidazole-6-carboxylic chlo
ride (3)
Figure 5. Mapping of losartan with the selected hypothesis.
A suspension of 2 (2.18 g, 10 mmol) in thionyl chloride (20 mL,
276 mmol) was refluxed for 2 h, and then the excess thionyl chlo-
ride was removed under a vacuum to provide the crude acid chlo-
ride (3) as an off-white solid. The crude product 3 was used in the
next step without further purification.
110
100
90
80
70
60
50
40
30
20
10
0
4.1.2. General synthetic procedure for 4a–4k
To a stirred suspension of the resulting acid chloride in 60 mL of
chloroform at 0 °C was added dropwise to triethylamine (1.52 g,
15 mmol), followed by a solution of one of the substituted amines
(10 mmol) in 10 mL of chloroform. The resulting mixture was stir-
red at 25 °C for 8 h. The reaction mixture was filtered, and the fil-
trate was washed with brine, saturated sodium bicarbonate
solution, brine and water, dried over anhydrous sodium sulphate,
filtered, and concentrated under a vacuum. The residue was puri-
fied by recrystallisation in ethanol to provide pure product as
white solids.
Telmisartan
6g
Losartan
6m
0
10
20
30
40
50
C(%)
60
70
80
90
100 110
Figure 6. The inhibition tendency of some compounds.

4212
J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4.1.2.1.
carboxamide (4a).
2), mp 134–136 °C. IR(KBr),
N-Benzyl-4-methyl-2-n-propyl-1H-benzimidazole-6-
White solid (2.22 g, 72.3% overall from
max/cmꢀ1: 3254, 3183, 2955, 2929,
3114, 3024, 2962, 2930, 2873, 1608, 1550, 1453, 1315, 1017,
761, 700; 1H NMR (400 MHz, DMSO) d: 0.81 (t, J = 7.2 Hz, 3H),
1.71 (m, 2H), 2.49 (s, 3H), 2.79 (t, J = 7.2 Hz, 2H), 2.89 (t,
J = 7.2 Hz, 2H), 3.65 (m, 2H), 3.77 (s, 3H), 6.77–7.20 (m, 4H), 7.67
(s, 1H), 7.78 (s, 1H); ESIMS(m/z): 352.2 (M+H)+.
m
2870, 1633, 1604, 1548, 1423, 1328, 1233, 1078, 893, 691; 1H
NMR (400 MHz, DMSO) d: 0.96 (t, J = 7.2 Hz, 3H), 1.76 (m, 2H),
2.57 (s, 3H), 2.84 (t, J = 7.1 Hz, 2H), 4.32 (s, 2H), 7.06–7.21 (m,
5H), 7.61 (s, 1H), 7.82 (s, 1H), 8.45 (s, 1H); ESIMS(m/z): 308.2
(M+H)+.
4.1.2.9. N-[b-(4-Methoxy)phenethyl]-4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (4i).
76.6% overall from 2), mp 149–151 °C. IR(KBr),
White solid (2.69 g,
m
max/cmꢀ1: 3337,
4.1.2.2.
imidazole-6-carboxamide (4b).
overall from 2), mp 129–131 °C. IR(KBr),
2961, 2931, 2871, 1701, 1598, 1533, 1432, 1320, 1209, 1040,
874, 764, 690; 1H NMR (400 MHz, DMSO) d: 0.94 (t, J = 7.3 Hz,
3H), 1.76 (m, 2H), 2.56 (s, 3H), 2.83 (t, J = 7.1 Hz, 2H), 3.47 (s,
3H), 4.45 (s, 2H), 7.09–7.31 (m, 4H), 7.57 (s, 1H), 7.88 (s, 1H),
8.48 (s, 1H); ESIMS(m/z): 338.2 (M+H)+.
N-(2-Methoxy)benzyl-4-methyl-2-n-propyl-1H-benz-
White solid (2.63 g, 77.9%
max/cmꢀ1: 3187, 3124,
2960, 2931, 2873, 1633, 1606, 1547, 1509, 1417, 1315, 1230,
1098, 1016, 761, 700; 1H NMR (400 MHz, DMSO) d: 0.98 (t,
J = 7.3 Hz, 3H), 1.81 (m, 2H), 2.57 (s, 3H), 2.77 (t, J = 7.2 Hz, 2H),
2.88 (t, J = 7.1 Hz, 2H), 3.40 (m, 2H), 3.67 (s, 3H), 6.84–7.32 (m,
4H), 7.45 (s, 1H), 7.84 (s, 1H); ESIMS(m/z): 352.2 (M+H)+.
m
4.1.2.10. N-[b-(2,5-Dimethoxy)phenethyl]-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxa-mide (4j).
72.4% overall from 2), mp 145–147 °C. IR(KBr),
White solid (2.75 g,
m
max/cmꢀ1: 3330,
4.1.2.3.
imidazole-6-carboxamide (4c).
overall from 2), mp 132–134 °C. IR(KBr),
2927, 2871, 1632, 1604, 1533, 1425, 1311, 1232, 1040, 898, 692;
1H NMR (400 MHz, DMSO) d: 0.95 (t, J = 7.2 Hz, 3H), 1.77 (m, 2H),
2.55 (s, 3H), 2.86 (t, J = 7.1 Hz, 2H), 3.72 (s, 3H), 4.42 (s, 2H),
6.78–7.24 (m, 4H), 7.58 (s, 1H), 7.87 (s, 1H), 8.47 (s, 1H);
ESIMS(m/z): 338.2 (M+H)+.
N-(3-Methoxy)benzyl-4-methyl-2-n-propyl-1H-benz-
White solid (2.52 g, 74.8%
max/cmꢀ1: 3198, 2957,
3026, 2961, 2932, 2833, 1627, 1595, 1555, 1505, 1419, 1319,
1227, 1045, 890, 807, 703; 1H NMR (400 MHz, DMSO) d: 0.95 (t,
J = 7.2 Hz, 3H), 1.76 (m, 2H), 2.55 (s, 3H), 2.78 (t, J = 7.2 Hz, 2H),
2.86 (t, J = 7.2 Hz, 2H), 3.48 (m, 2H), 3.70 (s, 3H), 3.76 (s, 3H),
6.94–7.12 (m, 3H), 7.70 (s, 1H), 7.86 (s, 1H); ESIMS(m/z): 382.2
(M+H)+.
m
4.1.2.11. N-[b-(3,4-Dimethoxy)phenethyl]-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxamide (4k).
78.7% overall from 2), mp 147–149 °C. IR(KBr),m
White solid (3.00 g,
4.1.2.4.
imidazole-6-carboxamide (4d).
overall from 2), mp 133–135 °C. IR(KBr),
2931, 2872, 1628, 1603, 1536, 1510, 1430, 1330, 1245, 1179,
1097, 1036, 890, 817, 766; 1H NMR (400 MHz, DMSO) d: 0.94 (t,
J = 7.2 Hz, 3H), 1.79 (m, 2H), 2.56 (s, 3H), 2.87 (t, J = 7.2 Hz, 2H),
3.78 (s, 3H), 4.47 (s, 2H), 6.82–7.29 (m, 4H), 7.55 (s, 1H), 7.82 (s,
1H), 8.46 (s, 1H); ESIMS(m/z): 338.2 (M+H)+.
N-(4-Methoxy)benzyl-4-methyl-2-n-propyl-1H-benz-
White solid (2.41 g, 71.4%
max/cmꢀ1: 3231, 2959,
max/cmꢀ1: 3247,
3080, 2933, 2871, 1636, 1607, 1595, 1549, 1515, 1441, 1323,
1231, 1141, 1027, 765; 1H NMR (400 MHz, DMSO) d: 0.96 (t,
J = 7.2 Hz, 3H), 1.78 (m, 2H), 2.56 (s, 3H), 2.77 (t, J = 7.2 Hz, 2H),
2.87 (t, J = 7.2 Hz, 2H), 3.47 (m, 2H), 3.72 (s, 3H), 3.78 (s, 3H),
6.98–7.15 (m, 3H), 7.72 (s, 1H), 7.89 (s, 1H); ESIMS(m/z): 382.2
(M+H)+.
m
4.1.2.12.
benzimidazole-6-carboxamide (4l).
76.3% overall from 2), mp 133–135 °C. IR(KBr),
3026, 2959, 2931, 2873, 1701, 1607, 1544, 1429, 1322, 1229,
1200, 1097, 896, 756, 697; 1H NMR (400 MHz, DMSO) d: 0.95 (t,
J = 7.2 Hz, 3H), 1.67 (m, 2H), 2.55 (s, 3H), 2.75 (t, J = 7.2 Hz, 2H),
2.86 (t, J = 7.2 Hz, 2H), 3.53 (m, 2H), 6.93–7.25 (m, 4H), 7.47 (s,
1H), 7.82 (s, 1H); ESIMS(m/z): 340.2 (M+H)+.
N-[b-(2-Fluoro)phenethyl]-4-methyl-2-n-propyl-1H-
White solid (2.59 g,
max/cmꢀ1: 3341,
4.1.2.5.
benzimidazole-6-carboxamide (4e).
75.0% overall from 2), mp 128–130 °C. IR(KBr),
3197, 2958, 2932, 2872, 1629, 1605, 1539, 1516, 1439, 1314,
1264, 1235, 1138, 1026, 894, 806, 764; 1H NMR (400 MHz, DMSO)
d: 0.81 (t, J = 7.3 Hz, 3H), 1.72 (m, 2H), 2.50 (s, 3H), 2.81 (t,
J = 7.2 Hz, 2H), 3.71 (s, 3H), 3.79 (s, 3H), 4.43 (s, 2H), 6.99–7.01
(m, 3H), 7.68 (s, 1H), 7.81 (s, 1H); ESIMS(m/z): 368.2 (M+H)+.
N-(3,4-Dimethoxy)benzyl-4-methyl-2-n-propyl-1H-
White solid (2.75 g,
max/cmꢀ1: 3307,
m
m
4.1.2.13.
benzimidazole-6-carboxamide (4m).
74.4% overall from 2), mp 137–139 °C. IR(KBr),
3026, 2959, 2931, 2873, 1701, 1607, 1544, 1429, 1322, 1229,
1200, 1097, 896, 756, 697; 1H NMR (400 MHz, DMSO) d: 0.96 (t,
J = 7.2 Hz, 3H), 1.69 (m, 2H), 2.56 (s, 3H), 2.78 (t, J = 7.3 Hz, 2H),
2.87 (t, J = 7.2 Hz, 2H), 3.56 (m, 2H), 6.97–7.23 (m, 4H), 7.46 (s,
1H), 7.84 (s, 1H); ESIMS(m/z): 340.2 (M+H)+.
N-[b-(4-Fluoro)phenethyl]-4-methyl-2-n-propyl-1H-
White solid (2.52 g,
max/cmꢀ1: 3341,
4.1.2.6.
azole-6-carboxamide (4f).
from 2), mp 152–154 °C. IR(KBr),
3025, 2958, 2930, 2872, 1631, 1606, 1545, 1417, 1321, 1233,
1087, 894, 741, 697; 1H NMR (400 MHz, DMSO) d: 0.94 (t,
J = 7.2 Hz, 3H), 1.78 (m, 2H), 2.55 (s, 3H), 2.79 (t, J = 7.2 Hz, 2H),
2.85 (t, J = 7.1 Hz, 2H), 3.49 (m, 2H), 7.20–7.31 (m, 5H), 7.47 (s,
1H), 7.87 (s, 1H), 8.42 (s, 1H); ESIMS(m/z): 322.2 (M+H)+.
N-(b-Phenethyl)-4-methyl-2-n-propyl-1H-benzimid-
White solid (2.41 g, 75.1% overall
max/cmꢀ1: 3341, 3193, 3060,
m
m
4.1.3. General synthetic procedure for 5a–5k
4.1.2.7. N-(2-Methyl-2-phenyl)ethyl-4-methyl-2-n-propyl-1H-
To a stirred solution of compound 4 (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-bromomethyl-2-(1-triphenylmethyltetrazole-5-yl)-1,10-biphe-
nyl (2.1 mmol) was added. After stirring at 25 °C for 12 h, the mix-
ture was poured into water (80 mL) and extracted with ethyl
acetate (40 mL ꢁ 3). The combined ethyl acetate layers were
washed with brine (50 mL ꢁ 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 the pure
product in the form of a white crystalline powder.
benzimidazole-6-carboxamide (4g).
79.7% overall from 2), mp 140–142 °C. IR(KBr),
White solid (2.67 g,
m
max/cmꢀ1: 3337,
2960, 2931, 2873, 1633, 1606, 1547, 1509, 1417, 1315, 1230,
1098, 1016, 761, 700; 1H NMR (400 MHz, DMSO) d: 0.94 (t,
J = 7.2 Hz, 3H), 1.58 (d, J = 7.2 Hz, 3H), 1.78 (m, 2H), 2.56 (s, 3H),
2.84 (t, J = 7.2 Hz, 2H), 4.97 (s, 1H), 7.15–7.31 (m, 5H), 7.46 (s,
1H), 7.89 (s, 1H), 8.44 (s, 1H); ESIMS(m/z): 336.2 (M+H)+.
4.1.2.8. N-[b-(3-Methoxy)phenethyl]-4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (4h).
75.7% overall from 2), mp 148–150 °C. IR(KBr),
White solid (2.66 g,
m
max/cmꢀ1: 3342,

J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4213
4.1.3.1. N-Benzyl-1-[20-(1-triphenylmethyltetrazole-5-yl)-1,10-
biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-
(400 MHz, CDCl3) d: 0.94(t, J = 7.4 Hz, 3H), 1.32(d, J = 6.8 Hz, 1H),
1.74–1.81(m, 2H), 2.66 (s, 3H), 2.72(t, J = 9.2 Hz, 2H), 3.02–
3.08(m, 1H), 3.32–3.39 (m, 1H), 5.20(s, 2H), 5.95(s, 1H), 6.75–
7.51(m, 29H), 7.90–7.93(m, 1H); ESIMS(m/z): 812.4(M+H)+.
6-carboxamide (5a).
89.7%), mp 156–157 °C. IR(KBr),
White crystalline powder (1405 mg,
m
max/cmꢀ1: 3299, 1650, 1596,
1541, 1469, 1378, 1284, 1217, 1029; 1H NMR (400 MHz, CDCl3)
d: 0.95(t, J = 7.40 Hz, 3H), 1.73–1.83(m, 2H), 2.70(s, 3H), 2.74(t,
J = 8.00 Hz, 2H), 4.578 (d, J = 5.60 Hz, 2H), 5.22(s, 2H), 6.35(t,
J = 5.4 Hz, 1H), 6.70–7.60(m, 29H), 7.90–7.93(m, 1H); ESIMS(m/z):
784.4(M+H)+.
4.1.3.8. N -[b-(3-Methoxy)phenethyl]-1-[20-(1-triphenylmethyl-
tetrazole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxamide (5h).
powder (1210 mg, 73.1%), mp 94–97 °C. IR(KBr),
White crystalline
m
max/cmꢀ1: 3388,
1652, 1596, 1538, 1488, 1352, 1272, 1215, 1190, 1033; 1H NMR
(400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.75–1. 81(m, 2H),
2.69(s, 3H), 2.75(t, J = 8.0 Hz, 2H), 2.87(t, J = 7.0 Hz, 2H), 3.61–
3.66(m, 2H), 3.76(s, 3H), 5.22(s, 2H), 6.12(d, J = 4.8 Hz, 1H), 6.76–
7.52(m, 28H), 7.90–7.92(m, 1H); ESIMS(m/z): 828.4(M+H)+.
4.1.3.2.
ole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (5b). White crystalline pow-
der (1404 mg, 86.3%), mp 107–109 °C. IR(KBr),
max/cmꢀ1: 3427,
N-(2-Methoxy)benzyl-1-[20-(1-triphenylmethyltetraz-
m
1646, 1599, 1513, 1492, 1448, 1355, 1247, 1120, 1028; 1H NMR
(400 MHz, CDCl3) d: 0.94(t, J = 7.4 Hz, 3H), 1.72–1.80(m, 2H),
2.70(s, 3H), 2.74(t, J = 7.8 Hz, 2H), 3.84(s, 3H), 4.60(d, J = 5.6 Hz,
2H), 5.22(s, 2H), 6.58(t, J = 5.6 Hz, 1H), 6.75–7.61(m, 28H), 7.91–
7.93(m, 1H); ESIMS(m/z): 814.4(M+H)+.
4.1.3.9. N-[b-(4-Methoxy)phenethyl]-1-[20-(1-triphenylmethyl-
tetrazole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxamide (5i).
White crystalline
powder (1174 mg, 70.9%), mp 105–107 °C. IR(KBr),
m
max/cmꢀ1
:
3327, 1653, 1595, 1539, 1470, 1357, 1240, 1215, 1033; 1H NMR
(400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.75–1.81(m, 2H),
2.69(s, 3H), 2.75(t, J = 7.8 Hz, 2H), 2.83(t, J = 7.0 Hz, 2H), 3.58–
3.63(m, 2H), 3.77(s, 3H), 5.22(s, 2H), 6.08(t, J = 5.60 Hz, 1H),
6.77–7.52(m, 28H), 7.90–7.92(m, 1H); ESIMS(m/z): 828.4(M+H)+.
4.1.3.3.
ole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (5c). White crystalline pow-
der (1467 mg, 90.2%), mp 94–96 °C. IR(KBr),
max/cmꢀ1: 3395,
N-(3-Methoxy)benzyl-1-[20-(1-triphenylmethyltetraz-
m
1655, 1598, 1531, 1490, 1357, 1268, 1214, 1186, 1036; 1H NMR
(400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.73–1.81(m, 2H),
2.70(s, 3H), 2.74(t, J = 8. Hz, 2H), 3.77(s, 3H), 4.55(d, J = 5.6 Hz,
2H), 5.22(s, 2H), 6.36(t, J = 5.6 Hz, 1H), 6.76–7.59(m, 28H), 7.90–
7.92(m, 1H); ESIMS(m/z): 814.4(M+H)+.
4.1.3.10. N-[b-(2,5-Dimethoxy)phenethyl]-1-[20-(1-triphenylm-
ethyltetrazole-5-yl)-1,10-biphen-yl-4-yl]methyl-4-methyl-2-n-
propyl-1H-benzimidazole-6-carboxamide (5j).
talline powder (1497 mg, 87.3%), mp 176–178 °C. IR(KBr),
White crys-
max/cm
m
ꢀ1: 3293, 1646, 1596, 1540, 1497, 1444, 1348, 1281, 1223, 1032;
1H NMR (400 MHz, CDCl3) d: 0.94(t, J = 7.4 Hz, 3H), 1.70(s, 3H),
1.72–1.82(m, 2H), 2.70–2.75(m, 5H), 2.91(t, J = 6.4 Hz, 2H), 3.59–
3.64(m, 2H), 3.72(s, 3H), 3.77(s, 3H), 5.22(s, 2H), 6.58(t, J = 4.8 Hz,
1H), 6.71–7.58(m, 27H), 7.90–7.92(m, 1H); ESIMS(m/z):
858.4(M+H)+.
4.1.3.4.
ole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-
benzimidazole-6-carboxamide (5d). White crystalline pow-
der (1427 mg, 87.7%), mp 104–107 °C. IR(KBr),
max/cmꢀ1: 3299,
N-(4-Methoxy)benzyl-1-[20-(1-triphenylmethyltetraz-
m
1650, 1600, 1511, 1468, 1380, 1287, 1187, 1036; 1H NMR
(400 MHz, CDCl3) d: 0.94(t, J = 7.4 Hz, 3H), 1.73–1.80(m, 2H),
2.67(s, 3H), 2.74(t, J = 8.0 Hz, 2H), 3.78(s, 3H), 4.51(d, J = 5.6 Hz,
2H), 5.21(s, 2H), 6.32(t, J = 5.6 Hz, 1H), 6.75–7.60(m, 28H), 7.90–
7.93(m, 1H); ESIMS(m/z): 814.4(M+H)+.
4.1.3.11. N-[b-(3,4-Dimethoxy)phenethyl]-1-[20-(1-triphenylm-
ethyltetrazole-5-yl)-1,10-biphen-yl-4-yl]methyl-4-methyl-2-n-
propyl-1H-benzimidazole-6-carboxamide (5k).
talline powder (1337 mg, 78.0%), mp 127–128 °C. IR(KBr),
White crys-
max/cm
m
4.1.3.5. N -(3,4-Dimethoxy)benzyl-1-[20-(1-triphenylmethyltet-
razole-5-yl)-1,10- biphenyl-4-yl]-methyl-4-methyl-2-n-propyl-
ꢀ1: 3356, 1652, 1594, 1540, 1490, 1357, 1262, 1236, 1155, 1028;
1H NMR (400 MHz, CDCl3) d: 0.94(t, J = 7.4 Hz, 3H), 1.73–1.88(m,
3H), 2.68(s, 3H), 2.74(t, J = 7.8 Hz, 2H), 2.83(t, J = 7.0 Hz, 2H),
3.59–3.64(m, 2H), 3.811(s, 3H), 3.843(s, 3H), 5.22(s, 2H), 6.10–
6.25(br, 1H), 6.73–7.55(m, 27H), 7.89–7.91(m, 1H); ESIMS(m/z):
858.4(M+H)+.
1H-benzimidazole-6-carboxamide (5e).
powder (1167 mg, 69.2%), mp 109–112 °C. IR(KBr),
White crystalline
m
max/cmꢀ1
:
3297, 1641, 1594, 1510, 1447, 1357, 1261, 1188, 1028, 1006,
875, 750, 698; 1H NMR (400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz,
3H), 1.75–1.81(m, 2H), 2.70(s, 3H), 2.75(t, J = 7.8 Hz, 2H), 3.83(s,
3H), 3.85(s, 3H), 4.51(d, J = 5.6 Hz, 2H), 5.23(s, 2H), 6.32(t,
J = 5.6 Hz, 1H), 6.76–7.59(m, 27H), 7.90–7.92(m, 1H); ESIMS(m/z):
844.4(M+H)+.
4.1.3.12. N-[b-(2-Fluoro)phenethyl]-1-[20-(1-triphenylmethyl-
tetrazole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxamide (5l).
White crystalline
powder (1076 mg, 66.0%), mp 184–186 °C. IR(KBr),
m
max/cmꢀ1
:
4.1.3.6.
yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimid-
azole-6-carboxamide (5f). White crystalline powder
(1351 mg, 84.7%), mp 161–162 °C. IR(KBr),
max/cmꢀ1: 3329, 1653,
N-(b-Phenethyl)-1-[20-(1-triphenylmethyltetrazole-5-
3378, 1655, 1597, 1536, 1491, 1452, 1355, 1272, 1227, 1034; 1H
NMR (400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.73–1.83(m,
2H), 2.70(s, 3H), 2.75(t, J = 8.0 Hz, 2H), 2. 94(t, J = 6.8 Hz, 2H),
3.61–3.66(m, 2H), 5.22(s, 2H), 6.17(s, 1H), 6.77–7.52(m, 28H),
7.90–7.92(m, 1H); ESIMS(m/z): 816.4(M+H)+.
m
1595, 1541, 1470, 1358, 1290, 1216, 1032; 1H NMR (400 MHz,
CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.64–1.83(m, 2H), 2.69(s, 3H),
2.75(t, J = 7.8 Hz, 2H), 2.89(t, J = 7.0 Hz, 2H), 3.67(m, 2H), 5.22(s,
2H), 6.09(t, J = 5.8 Hz, 1H), 6.76–7.51(m, 29H), 7.90–7.92(m, 1H);
ESIMS(m/z): 798.4(M+H)+.
4.1.3.13. N-[b-(4-Fluoro)phenethyl]-1-[20-(1-triphenylmethyl-
tetrazole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-
1H-benzimidazole-6-carboxamide (5m).
White crystalline
powder (1290 mg, 79.1%), mp 181–183 °C. IR(KBr),
m
max/cmꢀ1
:
4.1.3.7. N-(2-Methyl-2-phenyl)ethyl-1-[20-(1-triphenylmethyl-
tetrazole-5-yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-
3323, 1654, 1597, 1541, 1509, 1470, 1445, 1357, 1289, 1218; 1H
NMR (400 MHz, CDCl3) d: 0.95(t, J = 7.4 Hz, 3H), 1.73–1.81(m,
2H), 2.69(s, 3H), 2.75(t, J = 7.8 Hz, 2H), 2.85(t, J = 7.2 Hz, 2H),
3.56–3.62(m, 2H), 5.23(s, 2H), 6.09(t, J = 5.4 Hz, 1H), 6.77–7.51(m,
28H), 7.89–7.91(m, 1H); ESIMS(m/z): 816.4(M+H)+.
1H-benzimidazole-6-carboxamide (5g).
powder (1229 mg, 75.7%), mp 102–104 °C. IR(KBr),
3378, 1653, 1596, 1540, 1469, 1356, 1286, 1215, 1033; 1H NMR
White crystalline
m
max/cmꢀ1
:

4214
J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4.1.4. General Synthetic Procedure for 6a–6k
4.1.4.5. N-(3,4-Dimethoxy)benzyl-1-[20-(1H-tetrazol-5-yl)-1,10-
biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-
A solution of one of 5a–5k (1 mmol), 40 mL of tetrahydrofuran,
40 mL of methanol, and 9 mL of 3 N aqueous hydrochloric acid
were stirred at 25 °C for 12 h. The solution was adjusted to pH
12 with 1 N aqueous sodium hydroxide, and the organic solvents
were removed under a vacuum. The residual liquid was filtered,
and the filtrate was adjusted to pH 6–7 with 3 N aqueous hydro-
chloric acid. The resulting precipitate was filtered, washed with
water, dried, and then recrystallised in ethyl acetate/ethanol
(4:1) to afford the target product as a white powder.
6-carboxamide (6e).
194–196 °C, IR(KBr),
White powder (433 mg, 72.0%), mp
m
max/cmꢀ1: 3239, 1368, 1594, 1541, 1515,
1459, 1357, 1265, 1232, 1139, 1025, 853, 803, 761; 1H NMR
(400 MHz, DMSO-d6) d: 0.94(t, J = 7.4 Hz, 3H), 1.70–1.77(m, 2H),
2.55(s, 3H), 2.79(t, J = 7.4 Hz, 2H), 3.70(s, 6H), 4.40(d, J = 5.6 Hz,
2H), 5.46(s, 2H), 6.82–7.06(m, 7H), 7.29–7.59(m, 5H), 7.91(s, 1H),
8.82(t, J = 5.2 Hz, 1H); HRMS calcd for
C
35H36N7O3[M+H]+
602.2874, found 602.2863.
4.1.4.1.
yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-6-carbox-
amide (6a). White powder (447 mg, 82.6%), mp 185–187 °C,
IR(KBr),
N-Benzyl-1-[20-(1H-tetrazol-5-yl)-1,10-biphenyl-4-
4.1.4.6. N-(b-Phenethyl)-1-[20-(1H -tetrazol-5-yl)-1,10-biphenyl-
4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-6-carbox-
amide (6f).
White powder (459 mg, 82.6%), mp 176–178 °C,
m
max/cmꢀ1: 3286, 1638, 1590, 1542, 1457, 1350, 1276,
IR(KBr),
m
max/cmꢀ1: 3296, 1639, 1591, 1542, 1456, 1404, 1351,
1209, 1080, 1004, 875, 758; 1H NMR (400 MHz, DMSO-d6) d:
0.95(t, J = 7.4 Hz, 3H), 1.73–1.78(m, 2H), 2.56(s, 3H), 2.80(t,
J = 7.6 Hz, 2H), 4.48 (d, J = 6.0 Hz, 2H), 5.47(s, 2H), 6.90–7.61(m,
14H), 7.93(s, 1H), 8.93(t, J = 5.6 Hz, 1H); HRMS calcd for
1275, 1215, 1086, 1006, 839, 758; 1H NMR (400 MHz, DMSO-d6)
d: 0.94(t, J = 7.40 Hz, 3H), 1.67–1.77(m, 2H), 2.56(s, 3H), 2.79 (t,
J = 7.60 Hz, 2H), 2.85(t, J = 7.60 Hz, 2H), 3.46–3.51(m, 2H), 5.50(s,
2H), 7.0–7.30(m, 9H), 7.49–7.67(m, 5H), 7.84(s, 1H), 8.41–8.44(t,
J = 5.60 Hz, 1H); HRMS calcd for C34H34N7O[M+H]+ 556.2819,
found 556.2808.
C
33H32N7O[M+H]+ 542.2663, found 542.2656.
4.1.4.2.
biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-
N-(2-Methoxy)benzyl-1-[20-(1H-tetrazol-5-yl)-1,10-
4.1.4.7. N-(2-Phenyl)propyl-1-[20-(1H -tetrazol-5-yl)-1,10-biphe-
nyl-4-yl]methyl-4-methyl-2-n-propyl-1H -benzimidazole-6-car-
6-carboxamide (6b).
192–193 °C, IR(KBr),
White powder (507 mg, 88.7%), mp
m
max/cmꢀ1: 3263, 1635, 1591, 1536, 1492,
boxamide (6g).
180 °C, IR(KBr),
White powder (501 mg, 88.0%), mp 178–
m
max/cmꢀ1: 3285, 1640, 1593, 1539, 1456, 1404,
1458, 1392, 1275, 1240, 1119, 1026, 877, 754; 1H NMR
(400 MHz, DMSO-d6) d: 0.95(t, J = 7.4 Hz, 3H), 1.71–1.80(m, 2H),
2.57(s, 3H), 2.80(t, J = 7.4 Hz, 2H), 3.81(s, 3H), 4.45 (d, J = 5.6 Hz,
2H), 5.47(s, 2H), 6.87–7.52(m, 12H), 7.62(s, 1H), 7.95(s, 1H),
8.71(t, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d: 13.99,
14.27, 16.68, 20.69, 20.95, 28.90, 46.02, 55.49, 59.94, 107.36,
110.57, 120.29, 121.49, 125.60, 127.01, 127.32, 127.53, 127.62,
127.97, 128.07, 129.63, 130.29, 130.67, 134.84, 135.08, 140.19,
140.93, 143.98, 156.72, 156.78, 166.93; HRMS calcd for
1349, 1270, 1212, 1119, 1021, 875, 757; 1H NMR (400 MHz,
DMSO-d6) d: 0.93(t, J = 7.4 Hz, 3H), 1.23 (d, J = 6.8 Hz, 3H), 1.67–
1.77(m, 2H), 2.55 (s, 3H), 2.79(t, J = 7.6 Hz, 2H), 3.07–3.13(m, 1H),
3.36–3.47 (m, 2H), 5.50(s, 2H), 7.0–7.30 (m, 9H), 7.50–7.68(m,
5H), 7.82(s, 1H), 8.33–8.36(t, J = 5.6 Hz, 1H); HRMS calcd for
C
35H36N7O[M+H]+ 570.2976, found 570.2968.
4.1.4.8. N-[b-(3-Methoxy)phenethyl]-1-[20-(1H -tetrazol-5-yl)-
1,10-biphenyl-4-yl] methyl-4-methyl-2-n-propyl-1H -benzimid-
C
34H34N7O2[M+H]+ 572.2769, found 572.2757.
azole-6-carboxamide (6h).
White powder (469 mg, 80.1%),
3427, 1637, 1593, 1541,
N-(3-Methoxy)benzyl-1-[20-(1H-tetrazol-5-yl)-1,10-
mp 164–166 °C, IR(KBr),
m
max/cmꢀ1
:
4.1.4.3.
biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-
1457, 1352, 1262, 1215, 1149, 1093, 875, 757; 1H NMR
(400 MHz, DMSO-d6) d: 0.95(t, J = 7.4 Hz, 3H), 1.71–1.78(m, 2H),
2.55(s, 3H), 2.78–2.84(m, 4H), 3.45–3.52(m, 2H), 3.70(s, 3H),
5.47(s, 2H), 6.74–7.56(m, 13H), 7.84(s, 1H), 8.44(t, J = 5.6 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) d: 13.99, 14.27, 16.67, 20.70,
20.95, 28.89, 35.43, 46.00, 55.03, 59.94, 107.21, 111.76, 114.38,
121.09, 121.34, 125.66, 127.14, 127.55, 128.35, 129.49, 129.62,
130.34, 130.69, 134.77, 135.17, 140.26, 140.75, 141.43, 143.87,
156.72, 159.44, 159.53, 166.77; HRMS calcd for C35H36N7O2[M+H]+
586.2925, found 586.2915.
6-carboxamide (6c).
181–183 °C, IR(KBr),
White powder (431 mg, 75.4%), mp
m
max/cmꢀ1: 3274, 1635, 1595, 1542, 1458,
1360, 1263, 1212, 1149, 1040, 1009, 869, 759; 1H NMR
(400 MHz, DMSO-d6) d: 0.95(t, J = 7.4 Hz, 3H), 1.71–1.80(m, 2H),
2.56(s, 3H), 2.80(t, J = 7.6 Hz, 2H), 3.71(s, 3H), 4.45(d, J = 5.6 Hz,
2H), 5.47(s, 2H), 6.78–7.60(m, 13H), 7.93(s, 1H), 8.90(t, J = 5.8 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d: 14.00, 14.27, 16.67, 20.69,
20.95, 28.89, 42.81, 46.03, 55.13, 59.94, 107.34, 112.17, 113.19,
119.64, 121.46, 125.62, 127.07, 127.65, 128.01, 128.10, 129.50,
129.63, 130.31, 130.67, 131.11, 134.83, 135.09, 140.19, 140.90,
141.74, 144.00, 156.81, 159.45, 159.81, 166.80, 170.53; HRMS
calcd for C34H34N7O2[M+H]+ 572.2769, found 572.2757.
4.1.4.9.
1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H -benzimid-
azole-6-carboxamide (6i). White powder (447 mg, 76.4%),
mp 180–182 °C, IR(KBr), 3274, 1637, 1593, 1544,
max/cmꢀ1
N-[b-(4-Methoxy)phenethyl]-1-[20-(1H-tetrazol-5-yl)-
m
:
N-(4-Methoxy)benzyl-1-[20-(1H-tetrazol-5-yl)-1,10-
1511, 1459, 1352, 1297, 1245, 1176, 1034, 877, 820, 757; 1H
NMR (400 MHz, DMSO-d6) d: 0.91(t, J = 7.4 Hz, 3H), 1.68–1.74(m,
2H), 2.54(s, 3H), 2.75–2.80(m, 4H), 3.40–3.45 (m, 2H), 3.68(s,
3H), 5.49(s, 2H), 6.81–7.15(m, 8H), 7.48–7.66(m, 5H), 7.83 (s,
4.1.4.4.
biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimidazole-
6-carboxamide (6d).
197–198 °C, IR(KBr),
White powder (472 mg, 82.6%), mp
m
max/cmꢀ1: 3285, 1636, 1585, 1536, 1512,
1458, 1347, 1294, 1246, 1174, 1031, 1006, 817, 759; 1H NMR
(400 MHz, DMSO-d6) d: 0.93(t, J = 7.4 Hz, 3H), 1.67–1.76(m, 2H),
2.56(s, 3H), 2.79(t, J = 7.6 Hz, 2H), 3.72(s, 3H), 4.41(d, J = 5.6 Hz,
2H), 5.51(s, 2H), 6.87–7.67(m, 13H), 7.91(s, 1H), 8.82(t, J = 5.8 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d: 13.97, 14.27, 16.66, 20.67,
20.95, 28.87, 42.33, 45.91, 55.22, 59.94, 170.50, 113.84, 121.33,
123.64, 126.39, 127.59, 128.00, 128.18, 128.82, 129.41, 130.79,
131.23, 132.04, 134.83, 136.44, 138.65, 141.13, 143.85, 155.10,
1H), 8.38(t, J = 5.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d
6) d:
13.97, 16.67, 20.69, 28.87, 34.56, 45.92, 55.13, 59.94, 107.37,
113.94, 121.22, 123.68, 126.39, 127.54, 128.00, 128.42, 129.41,
129.77, 130.77, 131.21, 131.67, 134.78, 136.42, 138.68, 141.12,
143.78,
C
156.71,
157.83,
166.72;
HRMS
calcd
for
35H36N7O2[M+H]+586.2925, found 586.2913.
4.1.4.10. N-[b-(2,5-Dimethoxy)phenethyl]-1-[20-(1H-tetrazol-5-
yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benz-
156.78, 158.34, 166.65; HRMS calcd for
572.2769, found 572.2757.
C
34H34N7O2[M+H]+
imidazole-6-carboxamide (6j).
White powder (489 mg,

J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
4215
79.5%), mp 129–131 °C, IR(KBr),
m
max/cmꢀ1: 3373, 1626, 1588,
receptor (25 lg) and standard or test compounds. The binding
1541, 1501, 1459, 1347, 1273, 1224, 1152, 1039, 877, 762; 1H
NMR (400 MHz, DMSO-d6) d: 0.93(t, J = 7.4 Hz, 3H), 1.69–1.75(m,
2H), 2.54(s, 3H), 2.76–2.81(m, 4H), 3.41–3.46(m, 2H), 3.63(s, 3H),
3.70(s, 3H), 5.47(s, 2H), 6.70–7.06(m, 7H), 7.40–7.60(m, 5H),
7.84(s, 1H), 8.36(t, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 13.98, 14.27, 16.67, 20.68, 20.95, 28.88, 30.11, 45.92, 55.39,
55.97, 59.94, 107.26, 111.74, 111.80, 116.40, 121.29, 126.08,
126.56, 127.51, 127.63, 128.42, 128.75, 129.49, 130.01, 130.60,
130.73, 134.79, 135.92, 139.54, 140.77, 143.82, 151.57, 153.16,
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. The IC50 values were estimated from the lin-
ear portion of the competition curves.
4.3. Procedure for angiotensin II receptor functional
antagonism in rabbit aorta strips
156.69, 157.00, 166.72; HRMS calcd for
616.3031, found 616.3021.
C
36H38N7O3[M+H]+
Japanese White Rabbits (2–3 kg body weight, Vitalriver Com-
pany, China) were killed by cervical dislocation after a slight anaes-
thesia with a 20% Urethane solution. The thoracic aorta was
carefully dissected out and placed in ice-cold Krebs-Henseleit solu-
tion of the following composition (mM): NaCl 118; KCl 4.7; KH2PO4
1.2; MgSO4ꢂ7H2O 1.17; CaCl2ꢂ2H2O 2.5; NaHCO3 25; glucose 11.1.29
It was cut into helical strips that were 3–4 mm wide and 15–
20 mm long. These strips were mounted in 10-mL tissue baths,
the tissue baths were kept at 37 °C and aerated with 95% O2 and
5% CO2. Each strip was connected to a force transducer, and the
changes in the 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 begin-
ning of the experiment, a 67 mM KCl solution was administered
to check the sensitivity of the preparations as well as to determine
their maximal contractile response. After a 30 min washout, test
substances or their respective vehicles were added. Sixty minutes
later, cumulative concentration–response curves of angiotensin II
were obtained. Only one curve was obtained from each strip, and
the contractile response was expressed as a percentage (%) of the
maximal contraction achieved with KCl. The pA2 values were cal-
culated using Schild’s plot.30 The antagonist potency was evaluated
by the estimation of the pA2 values.
4.1.4.11. N-[b-(3,4-Dimethoxy)phenethyl]-1-[20-(1H-tetrazol-5-
yl)-1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benz-
imidazole-6-carboxamide (6k).
73.6%), mp 185–186 °C, IR(KBr),
White powder (453 mg,
m
max/cmꢀ1: 3235, 1640, 1593,
1548, 1514, 1458, 1351, 1261, 1139, 1025, 877, 760; 1H NMR
(400 MHz, DMSO-d6) d: 0.95(t, J = 7.4 Hz, 3H), 1.69–1.79(m, 2H),
2.55(s, 3H), 2.76–2.81(m, 4H), 3.40–3.51 (m,2H), 3.69(d,
J = 2.0 Hz, 6H), 5.48(s, 2H), 6.72–7.08(m, 7H), 7.37–7.59(m, 5H),
7.84(s, 1H), 8.40(t, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 13.98, 14.27, 16.67, 20.69, 20.95, 28.88, 34.95, 41.34, 45.97,
55.48, 107.25, 112.08, 112.72, 120.63, 121.29, 125.90, 127.44,
127.54, 128.23, 128.40, 129.3155.48, 55.68, 59.95, 129.54,
130.49, 130.71, 132.31, 134.78, 135.60, 140.04, 140.55, 143.84,
147.36, 148.76, 156.72, 156.72, 158.04, 166.75; HRMS calcd for
C
36H38N7O3[M+H]+ 616.3031, found 616.3021.
4.1.4.12.
1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimid-
N-[b-(2-Fluoro)phenethyl]-1-[20-(1H-tetrazol-5-yl)-
azole-6-carboxamide (6l).
mp 183–184 °C, IR(KBr),
White powder (458 mg, 79.9%),
m
max/cmꢀ1: 3286, 1632, 1593, 1544,
1486, 1454, 1349, 1278, 1229, 1108, 1009, 757; 1H NMR
(400 MHz, DMSO-d6) d: 0.94(t, J = 7.4 Hz, 3H), 1.67–1.77(m, 2H),
2.55(s, 3H), 2.79(t, J = 7.6 Hz, 2H), 2.89(t, J = 7.4 Hz, 2H), 3.47–
3.52(m, 2H), 5.50(s, 2H), 7.08–7.33(m, 8H), 7.50–7.67(m, 5H),
7.84(s, 1H), 8.46(t, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 13.97, 14.27, 16.67, 20.69, 20.95, 28.87, 45.91, 59.94, 107.38,
115.18, 115.40, 121.22, 123.71, 124.51, 124.54, 126.26, 126.40,
127.55, 128.00, 128.33, 128.40, 128.48, 129.41, 130.78, 131.21,
131.30, 131.44, 134.77, 137.41, 138.69, 141.12, 143.82, 156.73,
4.4. Procedure for oral activity in the spontaneously
hypertensive rats (SHR)
Male SHR aged 19–21 weeks were used in this study. Six ani-
mals served as controls and received the vehicle (10 mL/Kg). The
hypertensive animals were divided into two groups (n = 6). Group
1 received losartan (standard drug: 10 mg/kg), and group 2 was gi-
ven the same doses of the tested compound 6i. Both the vehicle
and test compound were given by oral administration.31 Blood
pressure and heart rate were measured by tail plethysmography
(BP-98A, Softron, Japan) after a warming period in unanesthetised
rats. The BP measurements required only a few minutes per indi-
vidual rat. All data were expressed as means SEM.32
159.72, 162.14, 166.80; HRMS calcd for
574.2725, found 574.2714.
C
34H33FN7O[M+H]+
4.1.4.13.
1,10-biphenyl-4-yl]methyl-4-methyl-2-n-propyl-1H-benzimid-
azole-6-carboxamide (6m). White powder (388 mg, 67.7%),
mp 188–190 °C, IR(KBr), 3230, 1637, 1593, 1544,
max/cmꢀ1
N-[b-(4-Fluoro)phenethyl]-1-[20-(1H-tetrazol-5-yl)-
m
:
1508, 1456, 1404, 1349, 1275, 1219, 1155, 1006, 817, 759; 1H
NMR (400 MHz, DMSO-d6) d: 0.96(t, J = 7.2 Hz, 3H), 1.71–1.79(m,
2H), 2.55(s, 3H), 2.79–2.85(m, 4H),3.44–3.49(m, 2H), 5.47(s, 2H),
6.90–7.56(m, 13H), 7.83(s, 1H), 8.45(t, J = 5.6 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 13.99, 14.27, 16.68, 20.71, 20.95, 28.88,
34.52, 41.15, 46.02, 59.94, 107.21, 115.04, 115.25, 121.34, 125.58,
127.06, 127.56, 128.06, 128.29, 129.64, 130.30, 130.56, 130.64,
130.67, 131.23, 134.67, 135.06, 135.97, 136.00, 140.17, 140.94,
143.89, 156.73, 159.77, 159.90, 162.18, 166.79, 170.53; HRMS
calcd for C34H33FN7O[M+H]+ 574.2725, found 574.2714.
4.5. Procedure for preliminary toxicity evaluation
The preliminary toxicity evaluation was performed on a water
toxicity analyser (BHP 9511, Beijing Hamamatsu Inc.) based on
the inhibition of P. phosphoreum (T3 mutation), which was sup-
plied by the Institute of Soil Science, Academia Sinica, Nanjing,
PR China in the form of a freeze-dried powder. For each test, a se-
rial 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 P. 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 trea-
ted 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 representing the concentration in water that
inhibits 50% of a test batch of P. phosphoreum.
4.2. Procedure for the receptor binding assay
The AT1 receptor binding assay was carried out by competitive
displacement of the binding of [125I] Sar1 Ile8-Ang II with the angio-
tensin AT1 receptor as described previously.18 Each 180-
lL incuba-
tion contained the following: [125I] Sar1 Ile8-Ang II (25 pM), AT1

4216
J. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 4208–4216
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Molecular modelling studies were performed using a Silicon
Graphics desktop (SGI) Fuel work station. The training set was se-
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AT1 receptor antagonists was generated using the HipHop module
in Discovery Studio, version 2.0, from Accelrys Inc. The molecules
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Acknowledgements
Financial support of this work was provided by the National
Technological Project of the Manufacture and Innovation of Key
New Drugs(2009ZX09103-143). We also thank Beijing Hamamatsu
Photon Technique Inc. for the providing of the toxicity test
instrument.
21. Discovery Studio, 2.0 Version. Accelrys, 2005.
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