Design, synthesis and biological evaluation of new 5-nitro benzimidazole derivatives as AT1 antagonists with anti-hypertension activities

Article abstract of DOI:10.1016/j.bmc.2014.02.008

The design, synthesis, in vitro and in vivo evaluation of 5-nitro benzimidazole with 1,4-disubsituted or 1,5-disubsituted indole derivatives as novel angiotensin II receptor antagonist is outlined. Radioligand binding assays showed that 2-(4-((2-butyl-5-n

Full text of DOI:10.1016/j.bmc.2014.02.008

Accepted Manuscript
Design, Synthesis and Biological Evaluation of New 5-Nitro Benzimidazole
Derivatives as AT1 Antagonists with Anti-Hypertension Activities
Weibo Zhu, Yajing Da, Dan Wu, Huiling Zheng, Linfeng Zhu, Li Wang, Yijia
Yan, Zhilong Chen
PII:
S0968-0896(14)00103-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.02.008
BMC 11397
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2014
4 February 2014
7 February 2014
Please cite this article as: Zhu, W., Da, Y., Wu, D., Zheng, H., Zhu, L., Wang, L., Yan, Y., Chen, Z., Design,
Synthesis and Biological Evaluation of New 5-Nitro Benzimidazole Derivatives as AT1 Antagonists with Anti-
Hypertension Activities, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.02.008
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Design, Synthesis and Biological Evaluation of New 5-Nitro Benzimidazole
Derivatives as AT1 Antagonists with Anti-Hypertension Activities
Weibo Zhu, Yajing Da, Dan Wu, Huiling Zheng, Linfeng Zhu, Li Wang, Yijia Yan,
Zhilong Chen*
Department of Pharmaceutical Science and Technology, College of Chemistry and Biology,
Donghua University, Shanghai 201620, China
* Corresponding author: Zhilong Chen, Department of Pharmaceutical Science and Technology,
College of Chemistry and Biology, Donghua University, 2999 North Renmin Road, Shanghai
201620, China. Tel (Fax): 86-21-67792654, Email: zlchen1967@yahoo.com
1

ABSTRACT: The design, synthesis, in vitro and in vivo evaluation of 5-nitro benzimidazole
with 1, 4-disubsituted or 1, 5-disubsituted indole derivatives as novel angiotensin II receptor
antagonist is outlined. Radioligand binding assays showed that 2-(4-((2-butyl-5-nitro-
1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl)benzoic acid, compound 3, displayed a high
affinity for the angiotensin II type 1 receptor with IC50 value of 1.03±0.26 nM. The biological
evaluation on spontaneously hypertensive rats and renal hypertensive rats showed that 3 could
cause significant decrease on MBP in a dose dependent manner, whose maximal response
lowered 30 mmHg of MBP at 5 mg/kg and 41 mmHg of MBP at 10 mg/kg after oral
administration, and the significant antihypertensive effect lasted beyond 24 h, which is better
than Losartan. Taken together 3 could be considered as an effective and durable
anti-hypertension drug candidate. These encouraging results are deserved of further investigation
towards its use for therapeutic benefit.
Keywords: 5-Nitro benzimidazole derivatives; AT1 antagonist; Synthesis; Anti-hypertension
2

1. Introduction
The pivotal role of the rennin-angiotensin system (RAS) in the regulation of blood pressure
and fluid volume homeostasis has been well established. This system consists of a cascade of
1
enzymatic reactions, leading to the production of angiotensin II (AII) which is the final site of
action in the RAS, elicits multiple biological effects such as increase in blood pressure, vascular
2
contraction and modulation of central drinking behavior . Angiotensin II has become the most
common target in the treatment of cardiovascular disease, such as diabetic nephropathy, cardiac
hypertrophy, arrhythmia and heart failure. Losartan is the first non-peptide orally active
3
angiotensin II receptor antagonist . Since then, a series of angiotensin II receptor blockers
(ARBs) were used in clinics for the treatment of hypertension. These ARBs share a common
mechanism of action, which block the effects of angiotensin II by selectively antagonizing the
angiotensin II type 1 receptor 4. Recent researches suggest that ARBs are also useful for treating
congestive heart failure and reducing the cardiac and vascular remodeling associated with
cardiovascular disease.
As the active AII receptor antagonists were widely developed, a lot of receptor binding
5
models were proposed based on the structure-activity relationships. Alka Bali proposed a
drug–receptor interaction model that 5-substituted benzimidazole nucleus coupled through a
methylene linker to pendent biphenyl system bearing a carboxyl group, and nitro group at
5-position, which had been found to be favorable for AII antagonism. Among this series
compound 7 displayed higher activities than Losartan. Its binding profile proposed in literature
(Fig.1a) depicted that n-butyl chain and biphenyl system respectively interacted with lipophilic
pockets L1 and L2 through van der Waal interactions. N-3 of benzimidazole nucleus and
terminal carboxyl group interacted with receptor through H-bonding interactions, while nitro
group at 5-position interacted with pocket L3 through van der Waal and/or H-bonding
interactions.
We designed a series of compounds based on 5-nitrobenzimidazole as nucleus and
N-phenylindole groups as rigid structure bearing a carboxyl group. 1, 4- and 1, 5- disubstituted
3

indoles were introduced to show which one could display a higher affinity. In addition, the
appropriate alkyl groups were optimized through altering the length of alkyl chain. The binding
profiles of 3 (Fig.1b) and 6 (Fig.1c) are similar to that of 7. The dominant conformation of 7, 3
and 6 were shown as Fig.2 using Spartan 8. The energy-minimized stereo-conformation of 3, 6
showed a high degree of fit with 7 and as such warranted biological evaluation.
a.
b.
c.
L3
L3
L3
O
H
O
H
H
O
O
N
O N
O
N
L1
L1
L1
N
N
N
N
N
N
L2
L2
L2
N
N
O
O
O
O
O
O
H
H
H
Fig.1 (a) Binding profile of 7 where L1–L2 are lipophilic pockets, L3 is a lipophilic or H-bond donor
pocket, and H is H-bond donor site. (b) Binding profile of 3 where L1–L2 are lipophilic pockets, L3 is a
lipophilic or H-bond donor pocket, and H is H-bond donor site. (c) Binding profile of 6 where L1–L2 are
lipophilic pockets, L3 is a lipophilic or H-bond donor pocket, and H is H-bond donor site.
Fig.2 Energy-minimized conformation of 7, 3, 6 and their overlay conformation
Six new designed compounds were synthesized, which were measured for their affinity to
the AT1 receptor to displace 125I-AII from its specific binding sites in rat vascular smooth muscle
cells (VSMC). The in vivo anti-hypertension properties were evaluated in spontaneously
hypertensive rats and renal hypertensive rats.
2. Results and discussion
4

2.1 Chemistry
The preparation of indole derivatives 1-6 was performed by means of a multistep procedure
described in Scheme 1-2.
The synthesis of 1-4 was accomplished starting from the suitable commercially available
4-Nitro-benzene-1, 2-diamine (8), which reacted with different acyl chlorides in THF using Et3N
as base to give amide 9a-d. They were cyclized to produce benzoimidazoles 10a-d with con. HCl
in EtOH. 4-Methylindole 11a was N-protected by acylation with benzoyl chloride in the presence
of Et3N and 4-(dimethylamino)pyridine (DMAP) to give (4-methyl-1H-indol-1-yl) (phenyl)
methanone 12a, which was brominated with NBS in the presence of AIBN to produce
(4-bromomethyl-1H-indol-1-yl) (phenyl)methanone 13a. 14a-d was obtained after reaction of
13a with 10a-d in the presence of K2CO3, and then hydrolyzed with 2M aqueous NaOH. 14a-d
reacted with 2-fluoro-benzonitrile using K2CO3 as base in DMF yielded 15a-d. The final
products 1-4 were acquired after hydrolysis of 15a-d. At each stage of the synthetic sequence the
product was isolated, purified by column chromatography and characterized by NMR and mass
spec techniques.
5

H
O2N
O2N
NH2
NH2
O2N
N
R
N
a
b
R
O
NH2
N
H
9a: R=Et
10a-d
9a-d
8
9b: R=n-pr
9c: R=n-Bu
9d: R=n-amy
e,f
Br
d
c
N
N
O
O
N
H
11a
12a
13a
O2N
O2N
N
R
N
N
O2N
R
N
N
N
R
g
h
N
N
N
H
CN
COOH
1: R=Et
2: R=n-pr
14a-d
15a-d
1-4
3: R=n-Bu
4: R=n-amy
Scheme 1. Reagents and conditions: (a) RCOCl, Et3N, THF; (b) HCl, C2H5OH, reflux; (c) benzoyl chloride,
Et3N, DMAP, CH2Cl2; (d) NBS, AIBN, CCl4, reflux; (e) K2CO3, CH3COCH3, reflux; (f) NaOH, H2O, CH3OH,
reflux; (g) 2-fluorobenzonitrile, K2CO3, DMF, reflux; (h) NaOH, H2O, CH3OH, reflux.
The preparation of 5 and 6 were similar to 1-4.
6

O2N
Br
N
R
10b: R=n-pr
10c: R=n-Bu
N
H
b
a
N
N
O
c,d
O
N
H
11b
12b
13b
O2N
O2N
N
N
N
N
O2N
R
R
N
N
R
e
f
N
N
CN
COOH
N
H
14e-f
15e-f
5: R=n-pr
6: R=n-Bu
5-6
Scheme 2. Reagents and conditions: (a) benzoyl chloride, Et3N, DMAP, CH2Cl2; (b) NBS, AIBN, CCl4, reflux;
(c)K2CO3, CH3COCH3, reflux; (d) NaOH, H2O, CH3OH, reflux; (e) 2-fluorobenzonitrile, K2CO3, DMF, reflux;
(f) NaOH, H2O, CH3OH, reflux.
One of the committed steps was the preparation of 14a-f. In this step two compounds were
obtained, namely, target compound and its regioisomer caused by the tautomeric forms of 10a-d
(Fig.3) under refluxing of acetone.
H
H
O2N
N
O2N
N
R
R
N
N
H
Fig.3 Tautomeric forms of 10a-d
14b was selected as an example to identify the position of nitro group. 14b and its
regioisomer 16 were separated by silica gel column chromatography. Nuclear Overhauser Effect
Spectroscopy (NOESY) was used to confirm the structure of 14b and 16. Irradiation of the
benzylic CH2 gave enhancement of the aromatic protons at position 7 of the benzimidazole
7

moiety (Fig.4). The difference between proton at position 7 in 14b and 16 was that chemical
shift of the former (at δ 7.26ppm, red circle in Fig.5) was lower than the latter (at δ 8.21ppm, red
circle in Fig.5). This indicates that the nitro group is at position 5 of benzimidazole in 14b while
at position 6 in 16. The structures of 14a, c-f could also be confirmed as the identification of 14b.
4
4
5
O2N
N
N
5
N
N
2
2
6
O2N
6
7
7
N
H
N
H
16
14b
Fig.4 compounds 14b and 16
Fig.5 The NOESY of 14b (left) and 16(right).
2.2 Biological evaluation
2.2.1 Radioligand binding assays
The prepared compounds were evaluated for their activities to competitively inhibit 125I-AII
8

binding to the AT1 receptor by a conventional ligand-binding assay with VSMC. The specific
binding of 125I-AII was inhibited in a concentration-dependent manner by compounds 1, 2, 3, 4,
5, 6 and Losartan, as exemplified by 3 and 4 in Fig.6. The IC50 and Ki values were shown in
Table 1. Compound 3 and 5 exhibited more affinity to AT1 receptors than other compounds. 4
didn’t compete well in the receptor assay. The result indicated that 3 and 5 might have better in
vivo antihypertensive activity.
4
125
losartan
3
100
75
50
25
0
-13 -12 -11 -10 -9
-8
-7
-6
-5
compound, log(M)
Fig.6 Inhibitory effects of 3, 4 and Losartan (10-6-10-12M) on the specific binding of 125I-AII to AT1
receptors in VSMCs
Table 1. IC50 and Ki value of the tested compounds
O2N
O2N
N
N
N
N
R
R
N
N
COOH
COOH
1-4
5-6
9

IC50 SEM
(nM)
Compound
R
Mp (oC)
Formula
Ki (nM)
Et
191-194
196-199
200-203
205-208
197-200
201-204
C25H20N4O4
C26H22N4O4
C27H24N4O4
C28H26N4O4
C26H22N4O4
C27H24N4O4
4.12 1.15
5.43 0.27
1.03 0.26
3.04 1.17
4.23 0.47
0.97 0.43
1
n-pr
2
n-Bu
n-amyl
n-pr
3
15.74 0.32 13.42 2.78
4
3.32 0.78
5.11 0.89
3.54 0.34
2.22 1.21
4.89 0.34
2.53 1.12
5
6
n-Bu
Losartan
2.2.2 In vivo antihypertensive activities
In spontaneously hypertensive rats (SHR), the effects of compounds 1, 2, 3, 4, 5, 6 (5, 10
mg/kg) and Losartan (10 mg/kg) on the mean blood pressure (MBP) after oral administration are
shown in Fig.7. It displayed that under the dose of 5 mg/kg or 10 mg/kg, 4 could not decrease
blood pressure significantly compared with the negative control group. 2 could decrease blood
pressure but not obviously enough. 1, 5, 6 had similar anti-hypertensive effects with Losartan at
the dose of 10 mg/kg. The maximal reduction of MBP of 1, 5, 6 and Losartan were 30 mmHg, 28
mmHg, 32 mmHg and 31 mmHg, respectively. 3 decreased the MBP with the maximal reduction
reached 4 to 5 h following administration. A dose related response was observed for 3(5, 10
mg/kg) with giving a MBP of 30 mmHg and 41 mmHg respectively. In addition, the duration of
antihypertensive action of 3 was seen to last for at least 24 h. Compared with Losartan, 3 has a
higher and longer antihypertensive effect at the same dose.
In renal hypertensive rats (RHR), the effects of compounds 1, 2, 3, 4, 5, 6 (5, 10 mg/kg) and
Losartan (10 mg/kg) on the mean blood pressure (MBP) after oral administration are shown in
Fig.8. In RHR, the same effects were observed as in SHR. 2, 4 had no significant
antihypertensive effect. 1, 5, 6 at 10 mg/kg showed the similar effects to Losartan at the same
dose. 3 at 5 mg/kg showed the equal effect to that of Losartan at 10 mg/kg. At 10 mg/kg, 3 had a
more effective and durable antihypertensive effect.
10

compound 2 5mg/kg
compound 2 10mg/kg
control
losartan 10mg
10
0
-10
-20
-30
-40
-50
-60
*
-1 0
1 2 3 4 5 6 7 8 9 10 1112 24
Time(h)
Fig.7 Effects of compounds 1, 2, 3, 4, 5, 6, and Losartan on mean blood pressure (MBP) in spontaneously
hypertensive rats. *, ** Significant difference from the control, p＜0.05 and p＜0.01, respectively.
11

compound 2 5mg/kg
compound 2 10mg/kg
control
losartan 10mg
10
0
-10
-20
-30
-40
-50
-60
-1 0 1 2 3 4 5 6 7 8 9 10 1112 24
Time(h)
Fig.8 Effects of compounds 1, 2, 3, 4, 5, 6, and Losartan on mean blood pressure (MBP) in renal hypertensive
rats. *, ** Significant difference from the control, p＜0.05 and p＜0.01, respectively.
12

3. Conclusions
A series of 5-nitro benzimidazole with 1, 4-disubsituted or 1, 5-disubsituted indole
derivatives were designed and synthesized. Their in vitro AII antagonistic activities as well as in
vivo antihypertensive activities were evaluated. Among them, 3 with n-butyl chain at 2-position
of benzimidazole and 1, 4-disubstituted indole group was found to be more potent than Losartan.
A binding profile for target compounds was proposed where 1, 4-disubstituted or 1,
5-disubstituted indole group could interact with L2 lipophilic pocket, and 5-nitro benzimidazole
could interact with the receptor through binding sites. The length of alkyl chain on 2-position of
benzimidazole is very important, and the compound with butyl chain has the highest activity. The
persuasive explanation is that alkyl chain with suitable length could interact more quickly and
bind more tightly with L1 lipophilic pocket. 1, 4-disubstituted and 1, 5-disubstituted indole
group were introduced to form different spatial conformations, and the former showed better
activity. In addition, a simple method for synthesis of 1, 4-disubstituted and 1, 5-disubstituted
indole compounds had been developed. In summary, 3 could be considered as a novel effective
anti-hypertension drug candidate with long-lasting blood pressure-lowering effect, and deserved
for further investigation.
4. Experimental section
4.1 Chemistry
All chemical reagents were of highest commercially available quality and applied without
further purification. Yields refer to purified products were not optimized. Melting points (m.p.)
were measured on an electro thermal melting point apparatus and were uncorrected. 1H NMR
spectra were measured on a Bruker 400 MHz spectrometer using TMS (Me4Si) as internal
standard. ESI-MS spectra were recorded on a Micromass triple quadrupole mass spectrometer.
Column chromatography was performed using silica gel H (300-400).
4.2 Preparations
4.2.1 N-(2-amino-5-nitrophenyl) butyramide(9b)
9b was prepared by the general procedure described by Katritzky 6.
4.2.2 N-(2-amino-5-nitrophenyl)propionamide(9a)
9a was prepared by the general procedure described by Katritzky 6.
13

4.2.3 N-(2-amino-5-nitrophenyl) valeramide(9c)
9c was prepared by the general procedure described by Katritzky 6.
4.2.4 N-(2-amino-5-nitrophenyl)hexanamide (9d)
9d was prepared by the general procedure described by Katritzky 6.
4.2.5 2-propyl-5-nitro-1H-benzo[d]imidazole (10b)
10b was prepared by the general procedure described by Olguin 7.
4.2.6 2-ethyl-5-nitro-1H-benzo[d]imidazole (10a)
10a was prepared by the general procedure described by Olguin 7.
4.2.7 2-butyl -5-nitro-1H-benzo[d]imidazole(10c)
10c was prepared by the general procedure described by Olguin 7.
4.2.8 2-pentyl-5-nitro-1H-benzo[d]imidazole（10d)
10d was prepared by the general procedure described by Olguin 7.
4.2.9 (4-methyl-1H-indol-1-yl) (phenyl) methanone (12a)
12a was prepared by the general procedure described by Dhanoa 8.
4.2.10 (5-methyl-1H-indol-1-yl) (phenyl) methanone (12b)
12b was prepared by the general procedure described by Dhanoa 8.
4.2.11 ((4-bromomethyl)-1H-indol-1-yl)(phenyl) methanone (13a)
13a was prepared by the general procedure described by Dhanoa 8.
4.2.12 ((5-bromomethyl)-1H-indol-1-yl)(phenyl) methanone (13b)
13b was prepared by the general procedure described by Dhanoa 8.
4.2.13 1-((1H-indol-4-yl)methyl)-2-propyl-5-nitro-1H-benzo[d]imidazole (14b)
A solution of 10b (500 mg, 2.44 mmol) in acetone (50 ml) was treated with K2CO3(435 mg,
3.15 mmol) and 13a（765 mg，2.44 mmol), and the mixture stirred at 60oC for 6h. The resulting
mixture was diluted with water and extracted with ethyl acetate (50 ml×3). The organic layer was
washed with saturated salt water (75 ml) and dried over MgSO4. After filtration, the solvent was
removed under reduced pressure. The residue was treated with the solution of 2M NaOH (12 ml)
in 12ml MeOH, the mixture stirred and heated under reflux for 3h. After completed, the solvent
was removed in vacuo. The resulting mixture was diluted with water and extracted with DCM
(15 ml×4). The organic layer was washed with saturated salt water (20 ml) and dried over
MgSO4. After filtration, the solvent was removed under reduced pressure. The residue was
purified by CC to give 14b (249 mg) as a yellow solid. Yield: 30.6%. m.p.: 172-174 oC. 1H NMR
(400MHz, CDCl3) δ: 8.72 (1H, d, J=1.9Hz, Ph-H), 8.44 (1H, d, N-H), 8.13 (1H, dd, J1=2.0Hz,
J2=8.8Hz, Ph-H), 7.41 ( 1H, d, J=8.1Hz, Ph-H), 7.32 (1H, t, J=2.8,Hz, Ph-H), 7.26 ( 1H, s, Ph-H),
7.10 (1H, t, J=7.0Hz, indo-H), 6.47 (1H, d, J=7.2Hz, Ph-H), 6.45 (1H, s, indo-H), 5.70 ( 2H, s,
14

Ph-CH2), 2.93 ( 2H, t, J=7.5Hz, -CH2CH2CH3), 1.92( 2H, m, J=7.4Hz, -CH2CH2CH3), 1.03 (3H,
t, J=7.3Hz, -CH2CH2 CH3). MS(ESI): [M+H]+ calcd 335.1; found 335.0.
4.2.14 1-((1H-indol-4-yl)methyl)-2-ethyl-5-nitro-1H-benzo[d]imidazole(14a)
14a was prepared according to the procedure described for the preparation of 14b. Yield:
37.0%. m.p.: 168-171 oC. 1H NMR (400 MHz, CDCl3) δ: 8.73 (d, 1H, J=1.81Hz, Ph-H), 8.47 (s,
1H, N-H), 8.14 (dd, 1H, J1=1.74Hz, J2=8.92Hz, Ph-H), 7.41 (d, 1H, J=8.22Hz, Ph-H), 7.32 (d,1H,
J=2.78Hz, Ph-H), 7.27 (d, 1H, J=6.45Hz, indo-H), 7.10 (t,1H, J=7.86Hz, Ph-H), 6.49 (d, 1H,
J=7.32, Ph-H), 6.44 (s,1H, indo-H), 5.32 (s, 2H, PhCH2), 2.96 (q, 2H, J=7.48Hz, -CH2CH3), 1.47
(t, 3H, J=7.59Hz, -CH2CH3). MS(ESI): [M+H]+ calcd 321.1; found 321.0.
4.2.15 1-((1H-indol-4-yl)methyl)-2-butyl-5-nitro-1H-benzo[d]imidazole (14c)
14c was prepared according to the procedure described for the preparation of 14b. Yield:
32.7%. m.p.: 173-175 oC. 1H NMR (400MHz, CDCl3) δ: 8.67（d, 1H, J=2.10Hz, Ph-H）, 8.46 (s,
1H, N-H), 8.09 (dd, 1H, J1=2.15Hz, J2=8.90Hz, Ph-H), 7.38 (d, 1H, J=8.12Hz, Ph-H), 7.29 (t, 1H,
J=2.96Hz, Ph-H), 7.22 (d, 1H, J=8.90Hz, indo-H), 7.07 (t, 1H, J=7.76Hz, Ph-H), 6.44 (m, 2H,
Ph-H, indo-H), 5.67 (s, 2H, Ph-CH2-), 2.89 (t, 2H, J=7.72Hz, -CH2CH2CH2CH3), 1.83 (quin, 2H,
J=7.56Hz, -CH2CH2CH2CH3), 1.40 (sext, 2H, J=7.56Hz, -CH2CH2CH2CH3), 0.90 (t, 3H,
J=7.32Hz, -CH2CH2CH2CH3). MS(ESI): [M+H]+ calcd 349.2; found 349.3.
4.2.16 1-((1H-indol-4-yl)methyl)-2-pentyl-5-nitro-1H-benzo[d]imidazole (14d)
14d was prepared according to the procedure described for the preparation of 14b. Yield:
o
1
18.6%. m.p.: 175-178 C. H NMR (400MHz,CDCl3) δ: 8.68 (d, 1H, J=2.16Hz, Ph-H), 8.41 (s,
1H, N-H), 8.09 (dd, 1H, J1=2.10Hz, J2=8.84Hz, Ph-H), 7.38 (d, 1H, J=8.16Hz, Ph-H), 7.29 (t, 1H,
J=2.64Hz, Ph-H), 7.23 (d, 1H, J=8.76Hz, indo-H), 7.08 (t, 1H, J=7.48Hz, Ph-H), 6.45 (m, 2H,
Ph-H, indo-H), 5.67 (s, 2H, Ph-CH2), 2.89 (t, 2H, J=7.72Hz, -CH2CH2CH2CH2CH3), 1.83 (sext,
2H, J=7.76Hz, -CH2CH2CH2CH2CH3), 1.32 (m, 4H, -CH2CH2CH2CH2CH3), 0.85 (t, 3H,
J=7.12Hz, -CH2CH2CH2CH2CH3). MS(ESI): [M+H]+ calcd 363.2; found 363.4.
4.2.17 1-((1H-indol-5-yl)methyl)-2-propyl-5-nitro-1H-benzo[d]imidazole(14e)
14e was prepared according to the procedure described for the preparation of 14b. Yield:
1
30.3%. m.p.: 171-173 oC. H NMR (400MHz, CDCl3) δ: 8.67 (d, 1H, J=2.10Hz, Ph-H), 8.40 (s,
1H, N-H), 8.13 (dd, 1H, J1=2.15Hz, J2=8.88Hz, Ph-H), 7.36 (d, 1H, J=8.40Hz, Ph-H), 7.30 (m,
2H, Ph-H, indo-H), 7.25 (d, 1H, J=2.87Hz, Ph-H), 6.90 (d, 1H, J=8.40Hz, Ph-H) , 6.49 (s, 1H,
indo-H), 5.49 (s, 2H, Ph-CH2), 2.92 (t, 2H, J=7.51Hz, -CH2CH2CH3), 2.05-1.89(m, 2H,
-CH2CH2CH3), 1.03(t, 3H, -CH2CH2CH3). MS(ESI): [M+H]+ calcd 335.1; found 335.1.
4.2.18 1-((1H-indol-5-yl)methyl)-2-butyl-5-nitro-1H-benzo[d]imidazole (14f)
15

14f was prepared according to the procedure described for the preparation of 14b. Yield:
26.4%. m.p.: 172-174 oC. 1H NMR (400MHz, CDCl3) δ: 8.66（d, 1H, J=1.92Hz, Ph-H）, 8.23 (s,
1H, N-H), 8.12 (dd, 1H, J1=1.98Hz, J2=8.96Hz, Ph-H), 7.35 (d, 1H, J=8.4Hz, Ph-H), 7.29 (m, 2H,
Ph-H, indo-H), 7.24 (t, 1H, J=2.92Hz, Ph-H), 6.89 (d, 1H, J=8.36Hz, Ph-H), 6.48 (t, 1H,
J=2.04Hz, indo-H), 5.48 (s, 2H, Ph-CH2-), 2.92 (t, 2H, J=7.68Hz, -CH2CH2CH2CH3), 1.85 (quin,
2H, J=8.16Hz, -CH2CH2CH2CH3), 1.45-1.37 (m, 2H, -CH2CH2CH2CH3), 0.91 (t, 3H, J=7.36Hz,
-CH2CH2CH2CH3). MS(ESI): [M+H]+ calcd 349.2; found 349.3.
4.2.19 2-(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzonitrile
(15b)
A solution of 14b (158 mg, 0.47 mmol) and K2CO3(135 mg , 0.98 mmol) in DMF (10 ml)
was treated with 2-fluorobenzonitrile (0.08 ml, 0.74 mmol), and the mixture stirred and heated
under reflux for 5h under nitrogen. After completed, the resulting mixture was diluted with water
and extracted with EA (30 ml×4). The organic layer was washed with saturated salt water (40
ml×4) and dried over MgSO4. After filtration, the solvent was removed under reduced pressure.
The residue was purified by CC to give 15b (163 mg) as a yellow solid. Yield: 79.3%. m.p.:
1
183-186 oC. H-NMR (400MHz, CDCl3) δ: 8.67（1H, d, J=2.0Hz, Ph-H）, 8.09（1H, dd, J1=
2.1Hz, J2= 6.7Hz, Ph-H）, 7.87 (1H, d, J=7.8Hz, Ph-H), 7.78 (1H, t, J=7.7Hz, Ph-H), 7.60
(1H, d, J=8.1Hz, Ph-H), 7.56 (1H, t, J=7.6Hz, Ph-H), 7.47 ( 1H, d, J=3.4Hz, Ph-H) , 7.26 ( 1H,
d, J=2.1Hz, Ph-H), 7.24 (1H, s, Ph-H), 7.09 (1H, t, J=7.6Hz, indo-H), 6.98 (1H, d, J=2.1Hz, Ph-
=3.4Hz, Ph-H), 6.46 (1H, d, J=7.3Hz, indo-H), 5.71 ( 2H, s, Ph-CH2), 2.90 (2H, t, J=7.4Hz,
-CH2CH2CH3), 1.91 (2H, q, J=7.4Hz, -CH2CH2CH3), 1.03 (3H, t, J=7.3Hz, -CH2CH2CH3).
MS(ESI): [M+H]+ calcd 436.2; found 436.5.
4.2.20 2-(4-((2-ethyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzonitrile
(15a)
15a was prepared according to the procedure described for the preparation of 15b.Yield:
o
1
74.9%. m.p.: 179-181 C. H-NMR (400MHz, CDCl3) δ: 8.71 ( 1H, s, Ph-H), 8.13 (1H, s,
J=8.8Hz, Ph-H), 7.88 (1H, d,J=7.8Hz, Ph-H), 7.78 (1H, s, J=7.7Hz, Ph-H), 7.61 (1H, q, J=7.9Hz,
Ph-H), 7.56 (1H, d,J=7.6Hz, Ph-H), 7.47 (1H, d, J=3.3Hz, Ph-H), 7.28 (2H, t, J=9.0Hz, Ph-H),
7.11 ( 1H, t, J=7.4Hz, indo-H), 6.68 (1H, d, J=3.3Hz, Ph-H), 6.47 (1H, d, J=7.2Hz, indo-H), 5.71
( 2H, s, Ph-CH2), 2.95 (2H, q, J=7.4Hz, -CH2CH3), 1.47 (3H, t, J=7.4Hz, -CH2CH3). MS(ESI):
[M-H]- calcd 420.2; found 420.6.
4.2.21 2-(4-((2-butyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl)
benzonitrile(15c)
16

15c was prepared according to the procedure described for the preparation of 15b.Yield:
84.8%. m.p.: 184-187 oC. 1H NMR (400MHz, CDCl3) δ: 8.68 (1H, d, J=2.0Hz, Ph-H), 8.12 (1H,
dd, J1=2.0Hz, J2=9.0Hz, Ph-H), 7.88 (1H, d, J=7.7Hz, Ph-H), 7.78 (1H, t, J=8.0Hz, Ph-H), 7.61
(1H, d, J=8.0Hz, Ph-H), 7.56 ( 1H, t, J=7.6Hz, Ph-H), 7.47 ( 1H, d, J=3.4Hz, Ph-H), 7.28 (1H, s,
Ph-H), 7.25 (1H, s, indo-H), 7.10 (1H, t, J=7.4Hz, Ph-H), 6.69 (1H, q, J=3.3Hz, Ph-H), 6.47 ( 1H,
q, J=7.2Hz, indo-H), 5.71 (2H, s, Ph-CH2), 2.92 (2H, t, J=7.6Hz, CH2CH2CH2CH3), 1.90-1.83
(2H, m, J=7.4Hz, CH2CH2CH2CH3), 1.43 (2H, s, J=7.5Hz, CH2CH2CH2CH3), 0.92 (3H, t,
J=7.3Hz, CH2CH2CH2CH3). MS(ESI): [M+H]+ calcd 450.2; found 450.5.
4.2.22 2-(4-((2-pentyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzonitrile
(15d)
15d was prepared according to the procedure described for the preparation of 15b.Yield:
84.3%. m.p.: 186-188oC. 1H-NMR (400MHz, CDCl3) δ: 8.67 ( 1H, d, J=2.04Hz, Ph-H), 8.13 (1H,
dd, J1=2.1Hz, J2=8.7Hz, Ph-H), 7.88 (1H, d, J=7.7Hz, Ph-H), 7.78 ( 1H, t, J=8.0Hz, Ph-H), 7.60
(1H, m, Ph-H), 7.56 (1H, s, Ph-H), 7.46 (1H, d, J=3.4Hz, Ph-H), 7.25 ( 2H, m, Ph-H, indo-H),
7.10 (1H, s, Ph-H), 6.68 ( 1H, d, J=3.3Hz, Ph-H), 6.47 ( 1H, d, J=7.8Hz, indo-H), 5.70 (2H, s,
Ph-CH2), 2.91 (2H, t, J=7.5Hz, -CH2CH2CH2CH2CH3), 1.89-1.85 (2H, m,-CH2CH2CH2CH2CH3),
1.40-1.33 (4H, m,-CH2CH2CH2CH2CH3), 0.86 ( 3H, t, J=7.0Hz, -CH2CH2CH2CH2CH3).
MS(ESI): [M+H]+ calcd 464.2; found 464.5.
4.2.23 2-(5-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl)
benzonitrile(15e)
15e was prepared according to the procedure described for the preparation of 15b. Yield:
79.5%. m.p.: 182-184 oC. 1H-NMR (400MHz, CDCl3) δ: 8.66 ( 1H, d, J=2.0Hz, Ph-H), 8.12 (1H,
d, J1=2.1Hz, J2=8.9Hz, Ph-H),7.84 ( 1H, d, J=0.8Hz, Ph-H), 7.74 ( 1H, d, J=0.7Hz, Ph-H), 7.54
(2H, t, J=8.4Hz, Ph-H), 7.40 ( 1H, d, J=3.3Hz, Ph-H), 7.31 (2H, d, J=2.2Hz, indo-H, Ph-H), 7.26
(1H, m, Ph-H), 6.95 (1H, s, Ph-H), 6.68 (1H, d,J=3.3Hz, indo-H), 5.51 (2H, s, Ph-CH2-), 2.91
(2H, q, J=8.6Hz, -CH2CH2CH3), 1.92 (2H, q, J=7.5Hz, -CH2CH2CH3), 1.06 ( 3H, t, J=7.3Hz,
-CH2CH2CH3). MS(ESI): [M+H]+ calcd 436.2; found 436.2.
4.2.24 2-(5-((2-butyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzonitrile
(15f)
15f was prepared according to the procedure described for the preparation of 15b. Yield:
77.1%. m.p.: 183-185 oC. 1H-NMR (400MHz, CDCl3) δ: 8.66 (1H, d, J=2.0Hz, Ph-H), 8.13 ( 1H,
dd, J1=2.1Hz，J2=8.9Hz, Ph-H),8.02 (1H, d, J=7.7Hz,Ph-H), 7.85 (1H, t, J=7.6Hz, Ph-H), 7.54
( 2H, m, Ph-H), 7.40 (1H, d, J=3.4Hz, Ph-H), 7.32 (1H, d, J=2.6Hz，Ph-H)，7.30-7.25 ( 2H, m,
17

Ph-H, indo-H), 6.95 (1H, d, J=3.5Hz, Ph-H), 6.68 (1H, s, indo-H), 5.51 ( 2H, s, Ph-CH2) , 2.95
(2H, t, J=7.6Hz, -CH2CH2CH2CH3), 1.92-1.84( 2H, m, -CH2CH2CH2CH3), 1.51-1.44 (2H, m,
-CH2CH2CH2CH3), 0.95 (3H, t, J=7.3Hz, -CH2CH2CH2CH3). MS(ESI): [M+H]+ calcd 450.2;
found 450.3.
4.2.25 2-(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic
acid (2)
A solution of 5M NaOH(10 ml) in 10ml methanol was treated with 15b（107 mg，0.25 mmol),
and the mixture stirred and heated under reflux for 34 h. After completed, the solvent was
removed in vacuo. The pH value of the mixture was adjusted to 5-6 by careful addition of 6M
aqueous hydrochloride. The resulting mixture was extracted with DCM (20 ml×5). The organic
layer was washed with saturated salt water (40 ml×1) and dried over MgSO4. After filtration, the
solvent was removed under reduced pressure. The residue was purified by CC to give 2 as a
yellow solid 57 mg. Yield: 51.2%. m.p.: 196-199 oC. 1H-NMR (DMSO，400MHz, ) δ: 12.19 (1H,
s, -COOH), 8.52(1H, d, J=2.15Hz, Ph-H), 8.12-8.09 (1H, dd, J1=2.2Hz, J2=8.9Hz, Ph-H),
7.70-7.68 ( 1H, m，Ph-H) , 7.58(1H, s, Ph-H), 7.52(1H, m, indo-H), 7.37-7.35(2H, m, Ph-H),
7.28-7.27(1H, m, Ph-H), 7.17-7.15 (1H, m, Ph- H), 6.97-6.94 (1H, m, Ph-h), 6.54 (1H, d,
J=3.03Hz, Ph-H), 6.30 (1H, d, J=4.3Hz, indo-H), 5.87 (2H, s，Ph-CH2-), 2.88(2H, t, J=7.4Hz,
-CH2CH2CH3)，1.82(2H, m, -CH2CH2CH3), 0.96 ( 3H, t, J=7.4Hz, -CH2CH2CH3). 13C-NMR
(DMSO, 125 MHz) δ: 170.56, 160.42, 143.08, 143.08, 142.02, 140.68, 140.68, 139.31, 135.67,
131.13, 127.72, 127.48, 127.31, 126.49, 121.85, 118.07, 116.64, 115.08, 111.21, 110.76, 105.08,
99.80, 45.64, 29.22, 20.37, 14.13. HRMS (ESI): m/z calculated for C26H22N4O4 [M+Na]+:
477.15387; found 477.15106. Anal. Calcd for C26H22N4O4: C, 68.71; H, 4.88; N, 12.33. Found:
C, 68.83; H, 4.96; N, 12.29.
4.2.26 2-(4-((2-ethyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic acid
(1)
1 was prepared according to the procedure described for the preparation of 2. Yield: 47.2%.
o
1
m.p.: 191-194 C. H-NMR (400MHz, DMSO) δ: 12.9 (1H, s, -COOH), 8.54 (1H, d, J=2.15Hz,
Ph-H), 8.13(1H, dd, J1=2.2Hz, J2=8.9Hz, Ph-H), 7.94 (1H, d, J=7.9Hz, Ph-H), 7.74 (2H, t,
J=8.8Hz, Ph-H), 7.60 (1H, t, J=7.5Hz, Ph-H), 7.53 -7.51 (2H, m, Ph-H, indo-H), 7.02 (2H, d,
J=8.5, Ph-H), 6.64 ( 1H, d, J=3.2Hz, Ph-H), 6.42 (1H, d, J=4.3Hz, indo-H), 5.91 (2H, s,
Ph-CH2-), 2.91 (2H, q, J1=7.4Hz, J2=14.8Hz, -CH2CH3), 1.30 ( 3H, t, J=7.4Hz, -CH2CH3).
13C-NMR: 167.18, 161.04, 142.59, 141.49, 140.37, 137.18, 136.79,132.58, 130.69, 130.24,
128.46, 128.09, 127.72, 126.14, 122.08, 117.71, 116.76, 114.68, 110.66, 109.53, 100.46, 45.00,
18

40.12, 20.48, 11.04. HRMS (ESI): m/z calculated for C25H20N4O4 [M+H]+: 441.15628; found
441.15573. Anal. Calcd for C25H20N4O4: C, 68.17; H, 4.58; N, 12.72. Found: C, 68.06; H, 4.43;
N, 12.54.
4.2.27 2-(4-((2-butyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic acid
(3)
3 was prepared according to the procedure described for the preparation of 2.Yield: 56.8%.
o
1
m.p.: 200-203 C. H NMR (400MHz, DMSO) δ: 12.8 (1H, s,-COOH), 8.51 (1H, d, J=2.2Hz,
Ph-H), 8.11 (1H, dd, J1=2.2Hz, J2=8.8Hz, Ph-H), 7.94 ( 1H, d, J=7.7Hz, Ph-H), 7.71-7.69 (2H, m,
Ph-H), 7.60 ( 1H, t, J=7.6Hz, Ph-H), 7.51-7.48 ( 2H, m, Ph-H, indo-H), 7.03-7.00（2H, m, Ph-H）,
6.64 ( 1H, d, J=3.3Hz, Ph-H), 6.42 (1H, m, indo-H), 5.91 (2H, s, Ph-CH2-), 2.88 (2H, t, J=7.4Hz,
-CH2CH2CH2CH3), 1.70 (2H, m, -CH2CH2CH2CH3), 1.34 (2H, m, -CH2CH2CH2CH3), 0.83 (3H,
t, J=7.3Hz,-CH2CH2CH2CH3).13C-NMR: 169.81, 162.90, 145.40, 144.31, 143.02, 140.04, 139.58,
135.51, 133.52, 132.99, 132.69, 131.28, 130.90, 130.57, 128.96, 124.86, 120.44, 119.59,
117.41,113.49, 112.29, 103.29, 47.85, 31.76, 29.34, 24.50, 16.38. HRMS (ESI): m/z calculated
for C27H24N4O4 [M+H]+: 469.18758; found 469.18703. Anal. Calcd for C27H24N4O4: C, 69.22; H,
5.16; N, 11.96. Found: C, 69.16; H, 5.03; N, 11.89.
4.2.28 2-(4-((2-pentyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic
acid (4)
4 was prepared according to the procedure described for the preparation of 2. Yield: 47.7%.
m.p.: 205-208 oC. 1H NMR (400MHz, DMSO) δ: 12.85 (1H, s, -COOH), 8.52 (1H, d，J=2.1Hz,
Ph-H), 8.11 (1H, dd，J1=2.1Hz, J2=8.9Hz, Ph-H), 7.93 (1H, d, J=1.4Hz, Ph-H), 7.74-7.69 (2H,
m，Ph-H), 7.60 (1H, m，Ph-H), 7.51-7.48 (2H, m，Ph-H, indo-H), 7.02-7.00 ( 2H, m，Ph-H), 6.63
( 1H, d，J=3.2Hz, Ph-H), 6.31 ( 1H, m，indo-H), 5.90 (2H, s，Ph-CH2-), 2.88 (2H, t，J=6.7Hz,
CH2CH2CH2CH2CH3), 1.73 (2H, m ， CH2CH2CH2CH2CH3), 1.26-1.24 (4H, m ，
CH2CH2CH2CH2CH3), 0.81 (3H, t，J=7.0Hz, CH2CH2CH2CH2CH3).13C-NMR: 162.92, 145.40,
144.30, 143.01, 139.52, 134.51, 133.21, 130.85, 128.92, 124.69, 120.43, 119.45, 117.41, 113.49,
112.48, 103.01, 47.87, 42.89, 42.69, 42.48, 42.06, 41.85, 33.54, 29.59, 28.92, 24.53, 16.54.
HRMS (ESI): m/z calculated for C28H26N4O4 [M+H]+: 483.20323; found 483.20268. Anal. Calcd
for C28H26N4O4: C, 69.70; H, 5.43; N, 11.61. Found: C, 69.78; H, 5.51; N, 11.56.
4.2.29 2-(5-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic
acid (5)
5 was prepared according to the procedure described for the preparation of 2. Yield: 61.2%.
o
1
m.p.: 197-200 C. HNMR (400MHz, DMSO) δ: 12.84(1H, s, -COOH), 8.49 ( 1H, d，J=2.1Hz,
19

Ph-H), 8.12 (1H, dd，J1=2.1Hz, J2=8.9Hz, Ph-H), 7.91 ( 1H, d，J=1.4Hz, Ph-H), 7.77-7.76 (2H,
m，J=8.9Hz, Ph-H), 7.57 ( 1H, d, J=0.8Hz, Ph-H), 7.48 (1H, d，J=7.8Hz, indo-H), 7.44 ( 1H, d，
J=3.2Hz, Ph-H), 7.35 (1H, s, Ph-H), 7.05 (1H, s，Ph-H), 6.98 (1H, d，J=2.8Hz, Ph-H), 6.58 (1H,
d，indo-H), 5.66 ( 2H, s, Ph-CH2), 2.94 (2H, t, J=7.4Hz, -CH2CH2CH3), 1.85-1.79 (2H, m，
-CH2CH2CH3), 0.98 (3H, t，J=7.3Hz, -CH2CH2CH3). 13C-NMR: 167.72, 160.36, 143.21, 142.03,
140.44, 137.83, 136.72, 133.34, 131.28, 131.10, 130.31, 129.13, 128.64, 128.18, 121.45, 119.29,
118.28, 115.09, 111.47, 103.43, 47.52, 40.31, 39.90, 29.34, 20.61, 14.28. HRMS (ESI): m/z
calculated for C26H22N4O4 [M+H]+: 455.17193; found 455.17138. Anal. Calcd for C26H22N4O4:
C, 68.71; H, 4.88; N, 12.33. Found: C, 68.80; H, 4.92; N, 12.30.
4.2.30 2-(5-((2-butyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-indol-1-yl) benzoic acid
(6)
6 was prepared according to the procedure described for the preparation of 2. Yield: 52.5%.
o
1
m.p.: 201-204 C. H NMR (400MHz, DMSO-d6) δ: 12.80 (1H, s，-COOH), 8.48 (1H, d，
J=1.9Hz, Ph-H), 8.12 (1H, dd，J1=2.0 Hz, J2=8.9Hz, Ph-H), 7.91 (1H, d，J=7.7 Hz, Ph-H), 7.77
-7.71 (2H, m, Ph-H), 7.57 (1H, t，J=7.5 Hz, Ph-H), 7.46 ( 1H, d，J=7.8 Hz, indo-H), 7.42(1H, d,
J=3.1 Hz, Ph-H), 7.36 ( 1H, s, Ph-H), 7.05 (1H, d，J=8.4 Hz, Ph-H), 6.97 ( 1H, d，J=8.5 Hz,
Ph-H), 6.57 ( 1H, d, J=3.1 Hz, indo-H), 5.66 (2H, s, -PhCH2-), 2.96 (2H, t, J=7.6 Hz,
CH2CH2CH2CH3), 1.78-1.74 (2H, m, CH2CH2CH2CH3), 1.40-1.37 (2H, m，CH2CH2CH2CH3),
0.88(3H, t, J=7.3 Hz, CH2CH2CH2CH3). 13C-NMR: 159.81, 142.56, 141.57, 139.96, 137.09,
136.07, 130.53, 128.53, 128.21, 127.86, 127.60, 120.77, 118.62, 117.58, 114.54, 110.81, 110.35,
102.66, 46.91, 40.12, 39.91, 39.08, 38.87, 28.67, 26.58, 21.80, 13.68. HRMS (ESI): m/z
calculated for C27H24N4O4 [M+H]+: 469.18758; found 469.18703. Anal. Calcd for C27H24N4O4:
C, 69.22; H, 5.16; N, 11.96. Found: C, 69.11; H, 5.22; N, 11.88.
4.3 Radioligand binding assay
The vascular smooth muscle cells (VSMCs) were gained from thoracic aorta of SD rats
(Second Military Medical University, China) and cultured by the tissue explants methods 9. One
section of aorta was removed and placed in Dulbecco’s Modified Eagle’s Medium (DMEM).
Adherent fat and connective tissue were gently removed with fine sterile forceps. The aorta was
minced into small cube-shaped specimens and digested with collagenase for 1h, at 37oC. The
homogenate was centrifuged at 10000×g for 5 mins. Then they were incubated with 1ml of
DMEM supplemented with 15% fetal bovine serum (FBS) at 37oC in 95% air 5% CO2. Cells at
20

passage 3-7 were used for the experiments.
Each 250µL incubate contained the following: 0.1nM 125I-Angiotensin II (Zhongshan
Hospital, Shanghai, China) and concentration of test compounds. The final concentrations were
1×10-6-1×10-12M. They were incubated in 24-well plates with VSMCs for 60 min at 37oC.
Nonspecific binding was measured in the presence of 1 µM AII and represented 5-10% of total
binding. After the reaction, removed the liquid immediately, washed five times with PBS, and
digested cells with 0.1M NaOH for 10 min. The radioactivity was counted with a γ-counter
(Wallac 1470 Wizard, PerkinElmer, Finland). The IC50 value (concentration for 50%
displacement of the specifically bound 125I-Angiotensin II) was determined by regression
analysis of displacement curves 10. The inhibition constant (Ki value) was calculated from the
formula Ki= IC50/ (1+ [L]/kd) 11, where [L] was the concentration of radioligand present in tubes.
4.4 In vivo study of anti-hypertensive effect
For preparing renal hypertensive rats, the left renal arteries of Sprague-Dawley rats
(250-350 g, Second Military Medical University, China) were completely ligated under sodium
pentobarbital (40 mg/kg) anesthesia. Thereafter，the rats with SBP higher than 160 mmHg were
selected and used as renal hypertensive rats. Spontaneous hypertensive rats (250-300 g) came
from Second Military Medical University, China. Both spontaneous and renal hypertensive rats
were randomly divided into different experimental groups of 10 animals (negative control group,
positive control groups, low-dose groups and high-dose groups). Each compound was suspended
in a 0.5% solution of sodium carboxymethyl cellulose and administered orally at the dose of 5
mg/kg and 10 mg/kg separately. Losartan (10 mg/kg, SanXin Zhujiang Chemical Engineering
Company) was used as positive control group. The negative control group was administered the
same volume of sodium carboxymethyl cellulose solution. To measure the blood pressure, the
animals were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The right carotid artery
and jugular vein were cannulated for arterial pressure measurement and drug administration,
respectively. The arterial catheter was connected to a pressure transducer and displayed on a
computer and analyzed with a biological signal analysis system (MPA-2000, Alcott Biotech,
21

China). The parameters were measured continuously for 10 h, and again at 24 h 12.
Acknowledgements:
This work was supported by Chinese National Natural Science Foundation (No. 21372042,
81301878, 81101298), Foundation of Shanghai Government (No. 13431900700, 13430722300,
13ZR1441000, 13ZR1440900, 201370), Foundation of Donghua University (No. 11D10501;
12D10515) and Foundation of Yiwu Government (2011-G1-15；2013623).
Abbreviations used
RAS, rennin-angiotensin system; AII, angiotensin II; ARBs, angiotensin II receptor blockers;
FDA, US Food and Drug Administration; DMAP, 4-(dimethylamino)pyridine; NMR, nuclear
magnetic resonance; HRMS, high-resolution mass spectrometry; ESI, electrospray ionization;
MS, mass spectrometry; DMSO, dimethylsulfoxide; DMF, N,N-dimethylformamide; THF,
tetrahydrofuran; AIBN, azodiisobutyronitrile; NBS, bromosuccinimide; NOESY, nuclear
overhauser effect spectroscopy; VSMC, vascular Smooth Muscle Cell; IC50, half maximal
inhibitory concentration; SHR, spontaneously hypertensive rats; MBP, mean blood pressure;
RHR, renal hypertensive rats; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine
serum.
References:
1. Hong, K.; Kim, C.; Lee, S.; Yoo, S. E. Fundam. Clin. Pharmacol. 1998, 12, 64.
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Y.; Chatani, F.; Iwao, H. J. Pharmacol. Exp. Ther. 1995, 273, 509.
3. Chiu, A. T.; McCall, D. E.; Price, W. A.; Wong, P. C.; Carini, D. J.; Duncia, J. V.; Wexler, R.;
Yoo, S.; Johnson, A.; Timmermans, P. B. J. Pharmacol. Exp. Ther. 1990, 252, 711.
4. Siragy, H. Am. J. Cardiol. 1999, 84, 3.
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Sharma, A.; Singh, M. Bioorg. Med. Chem. Lett. 2005, 15, 3962.
22

6. Katritzky, A. R.; Witek, R. M.; Rodriguez-Garcia, V.; Mohapatra, P. P.; Rogers, J. W.;
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23

a.
c.
b.
L3
L3
L3
O
H
O
H
H
O
O
N
O
N
O
N
L1
L1
L1
N
N
N
N
N
N
L2
L2
L2
N
N
O
O
O
O
O
O
H
H
H
Fig.1 (a) Binding profile of 7 where L1–L2 are lipophilic pockets, L3 is a lipophilic or H-bond donor
pocket, and H is H-bond donor site. (b) Binding profile of 3 where L1–L2 are lipophilic pockets, L3 is a
lipophilic or H-bond donor pocket, and H is H-bond donor site. (c) Binding profile of 6 where L1–L2 are
lipophilic pockets, L3 is a lipophilic or H-bond donor pocket, and H is H-bond donor site.
24

Fig.2 Energy-minimized conformation of 7, 3, 6 and their overlay conformation
25

H
N
H
N
O2N
O2N
R
R
N
H
N
Fig.3 Tautomeric forms of 10a-d
26

4
4
7
5
O2N
N
N
5
N
N
2
2
6
O2N
6
7
N
H
N
H
16
14b
Fig.4 compounds 14b and 16
27

Fig.5 The NOESY of 14b (left) and 16(right).
28

4
125
100
75
50
25
0
losartan
3
-13 -12 -11 -10 -9
-8
-7
-6
-5
compound, log(M)
Fig.6 Inhibitory effects of 3, 4 and Losartan (10-6-10-12M) on the specific binding of 125I-AII to AT1
receptors in VSMCs
29