Synthesis, anti-hypertensive effect of a novel angiotensin II AT 1 receptor antagonist and its anti-tumor activity in prostate cancer

Article abstract of DOI:10.1055/s-0032-1329952

Since the first non-peptide Ang II receptor antagonist was originally reported, it has become the most common target in the development of new treatments for hypertension. In recent years, all components of the classical RAS have been reported in the pros

Full text of DOI:10.1055/s-0032-1329952

Original Article 637
Synthesis, Anti-hypertensive Effect of a Novel
Angiotensin II AT1 Receptor Antagonist and its Anti-
tumor Activity in Prostate Cancer
Authors
Y.-J. Da, W.-D. Yuan, L.-F. Zhu, Z.-L. Chen
Affiliation
Department of Pharmaceutical Science and Technology, College of Chemistry and Biology, Donghua University,
Shanghai, China
Key words
Abstract
▼
anti-tumor activity. This new compound showed
high affinity to AT1 receptor and anti-hyperten-
sive activity in spontaneously hypertensive rats
and renal hypertensive rats. Moreover, in human
prostate cancer cells and in athymic nude mice
bearing human prostate cancer cells, we observed
this new compound had an efficient antiprolifer-
ative activity in vitro and anti-tumor activity in
vivo. The preliminary pharmacological charac-
teristics with oral acute toxicity test suggested
that this new compound can be considered as a
candidate for both anti-hypertensive and anti-
tumor drug.
▶
angiotensin II
●
▶
AT1 receptor
synthesis
hypertension
prostate
●
Since the first non-peptide Ang II receptor antag-
onist was originally reported, it has become the
most common target in the development of new
treatments for hypertension. In recent years, all
components of the classical RAS have been
reported in the prostate, these results suggest the
possibility that ARB is a novel therapeutic class of
agents for prostate cancer. In this study, a new
compound 2-(4-((2-propyl-5-nitro-1H-benzo[d]
imidazol-1-yl) methyl)-1H-indol-1-yl) benzoic
acid was synthesized and evaluated as a novel
angiotensin II AT1 receptor antagonist by radioli-
gand binding assays, anti-hypertensive assays in
vivo and oral acute toxicity test. MTT assays and
tests in nude mice were used to demonstrate its
▶
●
▶
●
▶
●
▶
●
cancer
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
amf
received 16.07.2012
accepted 15.10.2012
Introduction
of various cancers. For instance, AT1 receptor
antagonist suppress the growth of human pan-
creatic cancer cells [5] and, like ACE inhibitors,
inhibit tumor angiogenesis growth and lung
metastasis resulting from injection of lung can-
cer cell lines [6]. Rivera et al. also revealed that
AT1 receptor antagonist reduced tumor volume,
vascular density, mitotic index and cell prolifera-
tion by injection of glicoma cells [7]. It is also
reported that the AT1 receptor antagonist had the
potential to inhibit the growth of prostate cancer
cells and tumors through the AT1 receptor [8,9].
Most of ARBs came from the transformation of
Losartan’s structure. In the structure of Losartan,
the acidic group tetrazole have some disadvan-
tages in chemical synthesis. For example, it could
be dangerous due to the use of toxic such as
sodium azide. So we tried to find an acidic group
in place of tetrazole. Carboxyl group was then
found to be used as a suitable substitute. Because
carboxyl group is friendly to environment, more-
over synthesis of carboxyl group has a higher
yield. Poss developed a series of N-phenyl groups
▼
Bibliography
The renin-angiotensin system (RAS) is known to
play a pivotal role in our body balance. In this
system, angiotensin II (Ang II) receptor is the final
site of action. It plays an important pharmaco-
logical part in the treatment of cardiovascular
disorders [1]. Such as hypertension, cardiac
hypertrophy, diabetic nephropathy, arrhythmia
and heart failure. However, angiotensin II type 1
(AT1) receptor antagonists, namely the angi-
otensin II type 1 receptor blockers (ARBs), are
selective for AT1 receptors, and act independ-
ently on the Ang II synthetic pathway [2]. This
provides a more specific attempt to inhibit the
activity of the RAS and has become the main
pharmacological approach [3].
DOI http://dx.doi.org/
10.1055/s-0032-1329952
Published online:
November 30, 2012
Arzneimittelforschung 2012;
62: 637–643
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Correspondence
Z.-L. Chen
Department of Pharmaceutical
Science & Technology
College of Chemistry and
Biology
Donghua University
2999 North Renmin Road
201620 Shanghai
Since a low prevalence of cancer in hypertensive
patients receiving angiotensin converting enzyme
inhibitors was reported, a biological effect of Ang II
on the development or progression of cancer has
been drawn attention [4]. There is emerging evi-
dence that Ang II is involved in the development
China
Tel.: +86/21/6779 2743
Fax: +86/21/6779 2743
zlchen1967@yahoo.com
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

638 Original Article
0.487mmol) in acetone (10mL) was added K2CO3 ( 87mg,
0.630mmol), and stirred for 30min at 50°C, then 4 (153mg,
0.487mmol) was added. The resulting mixture was refluxed for
6h. The mixture was diluted with water and extracted with
ethyl acetate. The organic layer was washed with water, dried
over MgSO4. After filtration, the solvent was removed under
reduced pressure. The residue was purified by flash column
chromatography with petroleum ether/ethyl acetate (7: 2) as
eluant. A yellow solid 76 mg was obtained. Yield: 35.7%. mp:
97.5–99.7°C.
Fig. 1 The chemical
structure of com-
pound 1.
O2N
N
N
N
COOH
1-((1H-indol-4-yl)methyl)-2-propyl-5-nitro-1H-benzo[d]imida-
zole (9) To a solution of 8 (318mg, 0.73mmol) in methanol
(12mL) was added 12mL of 2M sodium hydroxide, and refluxed
for 3h. The solvent was removed in vacuo. The mixture was
diluted with water and extracted with dichloromethane. The
organic layer was washed with brine, dried over MgSO4. After
filtration, the solvent was removed under reduced pressure. The
residue was purified by flash column chromatography with
dichloromethane/methanol (250: 1 ) as eluant. A yellow solid
207mg was obtained. Yield: 85.7%. 1H-NMR (400MHz, CDCl3) δ:
8.72 (1H, d, J=1.9 Hz, 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=2.8Hz, 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, Ph-CH2), 2.93 (2H, t, J=7.5 Hz, -CH2CH2CH3), 1.92 (2H, m,
J=7.4Hz, -CH2CH2CH3), 1.03 (3H, t, J=7.3Hz, -CH2CH2CH3). MS
(ESI) m/z: 335.6[M+H]+.
instead of the structure indole groups. And indole groups can
extend the duration of the drug in human body. We can under-
stand them through the patents EP0488532, US5208235,
US5212177 and US5374615 [10–13]. Through benzimidazole
structure in place of imidazole structure of losartan, cande-
sartan and pomisartan [14,15] were developed. At last we used
5-nitro-1H-benzo[d]imidazole to replace the imidazole. Based
on the above design solutions, a novel angiotensin II AT1 receptor
antagonist compound 1 ( Fig. 1), 2-(4-((2- propyl -5-nitro-1H-
benzo[d]imidazol-1-yl) methyl)-1H-indol-1-yl) benzoic acid
was designed and synthesized.
In radioligand binding assays, this compound showed high
a ffinity to AT1 receptor. Then its antihypertensive activity
was investigated in spontaneously hypertensive rats and renal
hypertensive rats. In these assays, compound 1 showed efficient,
stable and durable antihypertensive effects. In addition, antipro-
liferative activity in prostate cancer cells and anti-tumor activity
in nude mice were observed then. All these preliminary phar-
macological characteristics with oral acute toxicity test sug-
gested that compound 1 can be considered as a candidate for
anti-hypertensive and anti-tumor drug.
▶
●
2-(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl) methyl)-1H-
indol-1-yl) benzonitrile (10) To a solution of 9 (158mg,
0.47mmol) K2CO3 (135mg, 0.98mmol) in DMF (10mL) was
added 2-Fluorobenzonitrile (0.08mL, 0.74mmol) under nitro-
gen, and the mixture was refluxed for 5h. The resulting mixture
was washed with water and brine, and then the organic layer
was dried over MgSO4. After filtration, the solvent was removed
under reduced pressure. The residue was purified by flash col-
umn chromatograpy with petroleum ether/ethyl acetate (2: 1)
1
Materials and Methods
as eluant. A yellow solid 163mg was obtained. Yield: 79.3%. H
-NMR (400MHz, CDCl3) δ: 8.67 (1H, d), 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=7.7
Hz, 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=2.1 Hz, Ph-H),
7.24 (1H, s, Ph-H), 7.09 (1H, t, J=7.6 Hz, indo-H), 6.98 (1H, d,
J1 =2.1Hz, J2 =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/z: 436.5[M+H]+.
▼
Chemical
All chemicals used were of reagent grade. Yields refer to purified
products are not optimized. 1H NMR spectra were measured on
a Bruker 400MHz spectrometer using Me4Si as internal stand-
ard. ESI-MS spectra were recorded on a Micromass triple quad-
rupole mass spectrometer. Column chromatography was
performed using silic gel H (300–400).
(4-methyl-1H-indol-1-yl) (phenyl)methanone (3) 3 was pre-
pared according to the procedure described by Daljit [16].
2-(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl) methyl)-1H-
indol-1-yl) benzoic acid (1)
A solution of 10 (107mg,
(4-bromomethyl)-1H-indol-1-yl)(phenyl)methanone (4) 4 was
prepared according to the procedure described by Daljit [16].
0.25mmol) and 10mL of 5M NaOH in methanol (10mL) were
refluxed for 34h. The solvent was removed in vacuo. The pH
value of the mixture was adjusted to 6 by careful addition of 6M
aqueous hydrochloride. The mixture was extracted with dichlo-
romethane, washed with water. The organic phase was dried
over MgSO4. After filtration, the solvent was removed under
reduced pressure. The residue was purified by flash column
chromatography with dichloromethane/methanol (40: 1) as
eluant. A yellow solid 57mg was obtained. Yield: 51.2%.
1H-NMR(DMSO, 400MHz)δ: 12.9(1H, s, -COOH), 8.52(1H, d,
J=2.15Hz, Ph-H), 8.13(1H, dd, J1 =2.2Hz, J2 =8.9Hz, Ph-H),
N-(2-amino-5-nitrophenyl) butyramide (6) 6 was prepared by
the general procedure described by Baker [17].
2-propyl-5-nitro-1H-benzo[d]imidazole (7) 7 was prepared by
the general procedure described by Baker [17].
(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)methyl)-1H-in-
dol-1-yl)(phenyl)methanone (8) To a solution of 7 (100mg,
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

Original Article 639
8.09(1H, d, Ph-H), 7.70–7.51 (4H, t, Ph-H),7.36(1H, indo-H),
7.28–7.14(3H, t, Ph-H), 6.30(1H, J=4.3 Hz, 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, 125MHz) 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. ESI-MS (m/z): 455.2[M+H]+,
477.1[M+Na]+; HR-MS: 477.15106.
high-dose groups). Each compound was suspended in a 0.5%
solution of sodium carboxymethyl cellulose and administered
orally at the dose of 5mg/kg and 10mg/kg separately. Losartan
(10mg/kg) (SanXin Zhujiang Chemical Engineering Company)
was taken as positive control. The negative control group was
administered the same volume of sodium carboxymethyl cellu-
lose solution. To measure the blood pressure, the animals were
anesthetized with sodium pentobarbital (40mg/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 sig-
nal analysis system (MPA-2000, Alcott Biotech, and China).
Blood pressure and heart rate were monitored at 0–10h and 24h
after the administration in spontaneous hypertensive rats and
renal hypertensive rats. MAP was calculated using the formula:
MAP=DBP+(SBP−DBP)/3. Results of the study were expressed
as mean±SD. A probability level of less than 0.05 was considered
significant. The completed animal research here adhered to the
“Principles of Laboratory Animal Care” and was approved by
IACUC.
Radioligand binding assays in VSMC
Cell lines and cell culture
Primary vascular smooth muscle cells (VSMCs) were obtained
from thoracic aorta of SD rats and cultured by the tissue explants
methods [18]. 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 37°C. The homogenate was
centrifuged at 10000×g for 5min. They were then incubated
with 1ml of DMEM supplemented with 15% fetal bovine serum
(FBS) at 37°C in 95% air 5% CO2. Cells at passage 3–7 were used
for the experiments.
Antiproliferative activity in prostate cancer cells
The MTT assays were performed as previously described [21].
Briefly, LNCap cells (Institute of Biochemistry and Cell Biology,
China) were seeded in 24-well culture plates at a density of 104
to 105 cells/well. Cells were cultured in phenol red-free
RPMI+0.1% BSA for 18–24h at 37°C, 5% CO2 atmosphere before
experiments and then treated with Ang II (0.1μM), Ang II (1μM),
losartan (10μM), compound 1 (10μM), AngII (1μM)+losartan
(10μM), AngII (1μM)+ compound 1 (10μM), respectively. The
control group received drug-free medium with 0.05% v/v DMSO.
Subsequently, MTT (5mg/mL) was added to each well on day 2.
After incubation at 37°C for 4h, the medium was discarded and
150μL DMSO was added into each well. The optical density was
measured by a micro-plate reader (Bio-Rad, California, USA) at a
test wavelength of 570nm.
Saturation study
50μL test compounds (1nM) (Department of Medicinal Chemis-
try, DongHua University) and 50μL
(Northern Biotechnology Company) were incubated in 24-well
[125]
Ang II (0.03–10.00nM)
plates with VSMCs for 60min at 37°C. Nonspecific binding was
[125]
defined as
Ang II bounding in the presence of unlabeled
10μM [Sar1, Ile8] Ang II (GL Biochem, Shanghai). After the reaction,
removed the liquid immediately, washed 5 times with PBS, and
digested cells with 0.1M NaOH for 10min. The radioactivity was
counted with a γ-counter (Wallac 1470 Wizard, PerkinElmer,
Finland). The dissociation constant (Kd value) and the receptor
density (Bmax value) were estimated by Scatchard analysis [19].
Competition study
The inhibitory efficacy of the antagonists was determined under
Antitumor activity in nude mice
the same conditions as in the saturation study. The VSMCs con-
The antitumor activity of compound 1 was determined in ath-
ymic nude mice bearing LNCap tumors. LNCap cells (107) were
injected into the flank region of athymic nude mice (4–6 weeks
old), and treatment was started on day 8 when the tumor meas-
ured 5mm in diameter [22]. Each mouse received one of 2
different doses of compound 1 (5.0 or 10.0mg/kg/day) and losa-
rtan (5.0 or 10.0mg/kg/day) was taken as positive group. The
control group received water containing sodium hypochlorite
(10ppm). Each group consisted of 10 animals. Tumors were
measured with a caliper every 7 days. The volume of the
tumor was calculated using the formula: tumor volume
(mm3)=length×(width) 2 ×0.5.
[125]
taining 0.1nM
Ang II were incubated with various concen-
trations of competitors for 60min at 37°C. The final
concentrations were 1×10–6~1×10–12M. The inhibitory concen-
tration of test compound that caused 50% inhibition if the spe-
cific binding of Ang II (IC50) was determined by regression
analysis of displacement curves. The inhibition constant (Ki
value) was calculated from the formula Ki =IC50/(1+[L]/kd) [20],
where [L] was the concentration of radioligand present in tubes.
Antihypertensive effects in rats
The compound 1 were subjected to biological evaluation for
their effects on systolic blood pressure (SBP) and diastolic blood
pressure (DBP) in spontaneous hypertensive rats (SHRs) (250–
300g, Second military medical university, China) and renal
hypertensive rats. For preparing renal hypertensive rats, the left
renal arteries of Sprague-Dawley rats (300–350g) were com-
pletely ligated under sodium pentobarbital (40mg/kg) anesthe-
sia. Thereafter, the rats with SBP higher than 160mmHg were
selected and used as renal hypertensive rats. Than both sponta-
neous and renal hypertensive rats were randomly divided into
different experimental groups of 10 animals (negative control
group, positive control groups, compound low-dose groups and
Acute toxicity studies
The toxicity of compound 1 was determined in normal ICR rats
(6–8 weeks, Academia Sinica, China), which were weighed indi-
vidually. LD50 was delivered via intragastric administration (ig)
at doses of 5000.0, 3823.6.0, 2924.0, 2236.1, 1710.0, 1000.0mg/
kg. Survival was assessed daily for 2 weeks. The lethal dose
(LD50) and 95% confidence limits were determined from logistic
regression analysis (GLM) curve fitting of the 14 days mortality
data. They were observed continuously and recorded systemati-
cally for the physical signs of toxicity including skin changes,
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

640 Original Article
Fig. 2 Reagent and conditions: a benzoyl
chloride, Et3N, DMAP, CH2Cl2; b NBS, AIBN, CCl4,
reflux; c PrCOCI,Et3N, THF; d HCl, C2H5OH, reflux;
e K2CO3, CH3COCH3, reflux; f NaOH, H2O, CH3OH,
reflux; g 2-fluorobenzonitrile, K2CO3, DMF, reflux;
h NaOH, H2O, CH3OH, reflux; i NaOH, H2O,
CH3OH, reflux.
Br
b
a
N
N
O
N
H
O
e
2
3
4
H
O2N
O2N
NH2
O2N
N
N
c
d
NH 2
O
NH2
NH
5
6
7
O2N
N
N
O2N
N
O2N
N
N
N
h
f
g
N
N
N
H
CN
O
8
N
9
10
O2N
O2N
N
N
N
i
N
N
CONH
COOH
2
11
1
mobility, aggressiveness and respiratory movements. Finally, the
survivals were dissected and examined pathological changes of
organs.
Then 8 was hydrolyzed with aqueous NaOH to provide com-
pound 9. Compound 9 with 2-fluorobenzonitrile using K2CO3 in
DMF yielded the compound 10, which was amidated to give the
compound 11. The final compound was obtained through the
hydrolysis of compound 11.
Statistics
Results are expressed as means±standard error of the means.
Data were analyzed by one-way analysis of variance. When over-
all statistical significance was achieved (P<0.05). Student’s t-test
was used to compare each of the doses to the vehicle control.
Probability values less than 0.05 were considered to be signifi-
cant. Binding isotherms from competition studies were obtained
using the nonlinear regression program Graphpad Prism 4 soft-
ware.
Biological evaluation
Radioligand binding assays in VSMC
Compound 1 displaced the specific binding of [125I] [Sar1, Ile8]
angiotensin II to a single population of binding site (angiotensin
AT1 receptor) in rat vascular smooth muscle cell (VSMC). The kd
and Bmax values were 0.34±0.023nM and 5.242±0.75fmol/mg
protein. In competition study, compound 1 and Losartan com-
peted dose-dependently with 125I-Ang II ( Fig. 3). Compound 1
▶
●
and Losartan displayed high specific affinity for the AT1 recep-
tor (Ki =0.473±0.28nM, 1.71±0.12nM, IC50 =0.277±0.136nM,
2.36±0.091nM, respectively). It showed that compound 1 exhib-
ited more affinity to AT1 receptors than Losartan because its Ki
and IC50 values were significantly much lower compared with
Losartan’s.
Results
▼
Chemistry
The target compound was prepared according to the route
described in Fig. 2.
▶
●
Treatment of 4-methyl-1H-indole 2 with benzoyl chloride in
methylene chloride in the presence of triethylamine and
4-(dimethylamino) pyridine (DMAP) gave (4-methyl-1H-in-
dol-1-yl) (phenyl)methanone 3, which was brominated to pro-
duce (4-bromomethyl)-1H-indol-1-yl)(phenyl)methanone 4.
4-nitrobenzene-1,2-diamine 5 in THF with butyryl chloride
using triethylamine as base gave the secondary amines 6, which
was cyclized to produced benzoimidazoles 7. Subsequently
compound 8 was obtained using4 and 7 in the presence of K2CO3.
Antihypertensive effects in rats
The effects of compound 1 (5, 10mg/kg) and Losartan (10mg/kg)
on the reduction of mean arterial pressure (MAP) in spontane-
ously hypertensive rats (SHR) and renal hypertensive rats (RHR)
▶
are shown in Fig. 4, 5 respectively (n=10). The results sug-
●
gested that compounds 1 caused significant, durable and stable
reduction in blood pressure in vivo.
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

Original Article 641
10
0
125
100
75
50
25
0
losartan
compound 1
–10
–20
–30
–40
–50
–60
*
*
*
**
**
**
**
**
*
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
0
1
2
3
4
5
6
7
8
9
10
24
–12
–10
–8
–6
Time(h)
Concentration of compound,Log(M)
control
losartan 10mg/kg
compound 1 5mg/kg
compound 1 10mg/kg
Fig. 3 Inhibitory effects of compound 1 and losartan (10–6~10–12 M) on
the specific binding of 125I-Ang II to AT1 receptors in VSMCs.
Fig. 5 Reduction of mean arterial pressure in renal hypertensive rats
(n=10). Values are means±standard error of means for experiments.
*, ** Significant difference from the control, p<0.05 and p<0.01, respec-
tively.
10
0
39mmHg for 5mg/kg and 49mmHg for 10mg/kg, respectively.
At 24h, the reduction of compound 1 is 22mmHg for 5mg/kg
and 33mmHg for 10mg/kg, respectively. It is a significant differ-
ence from the control. Losartan (10mg/kg) decreased MAP at 4h
after taken to reach the maximum (26mmHg). At 24h, the
reduction of Losartan returned to 10mmHg. The result indicated
that in RHR, compound 1 also had a better effect than Losartan.
All the compounds did not influence heart rate of the rats (data
not shown).
–10
*
*
–20
–30
–40
–50
–60
**
**
**
**
**
**
**
**
*
*
**
**
**
**
**
**
**
**
**
*
**
**
**
**
**
**
**
**
**
0
1
2
3
4
5
6
7
8
9 10
24
Time(h)
Antiproliferative activity in prostate cancer cells
control
losartan 10mg/kg
compound 1 5mg/kg
compound 1 10mg/kg
The effect of the compound 1 on cell viability was assessed by
MTT assay. Treatment of human prostate cancer cell line LNCaP
cells with Ang II at the concentrations of 0.1μM and 1μM
resulted in an increase in the viability of cells. The proliferative
effect of Ang II is dose-manner. A significant reduction in the
viability as compared to DMSO-treated controls was observed
after 24h of treatment with Losartan and compound 1 at the
concentration of 10μM treat with Ang II (1μM). However, Losar-
tan and compound 1 could not influence the growth of LNCaP
Fig. 4 Reduction of mean arterial pressure in spontaneously hyperten-
sive rats (n=10). Values are means±standard error of means for experi-
ments. *, ** Significant difference from the control, p<0.05 and p<0.01,
respectively.
In SHR, compared to the control group, compound 1 (5mg/kg,
▶
10mg/kg) decreased the MAP in a dose-dependent manner
cells without treated with Ang II (● Fig. 6). The result suggested
▶
(
Fig. 4). The maximal reduction was observed at 4h after
that compound 1 has antiproliferative activity in prostate cancer
●
intragastric administration, namely, 34mmHg for 5mg/kg and
51mmHg for 10mg/kg, respectively. This reduction can remain
for more than 10h. Then at 24h compound 1 still had significant
difference from the control. The reduction of compound 1 at 24h
was 23mmHg for 5mg/kg and 30mmHg for 10mg/kg, respec-
tively. Losartan (10mg/kg) decreased MAP at 4h after taken to
reach the maximum (26mmHg) and than blood pressure
returned slowly, at 24h it only decreased MAP 13mmHg. Com-
pared to Losartan, compound 1 has both stronger and longer
anti-hypertensive effect, and this effect maintained for more
than 24h. The results indicated that compound 1 had a more
favorable, stable and durable blood pressure-lowering effect
than Losartan. All the compounds did not influence heart rate of
the rats (data not shown).
cells.
Antitumor activity in nude mice
To determine whether the in vitro antiproliferative activity of
compound 1 could be translated to antitumor activity in vivo,
LNCap cells were established as xenografts in nude mice (n=10).
When the tumors reached about 5 mm in diameter, the animals
were given compound 1 at 5.0 or 10mg/kg/day. Losartan at 5.0
or 10mg/kg/day were used as positive control group. The nega-
tive control group received water containing sodium hypochlo-
▶
rite (10ppm). As shown in ● Fig. 7, at 4 weeks, control group
had developed large tumor of 29.899±2.011 relative volume
compared with those at 0 week. Mice treated with compound 1
at 5.0 or 10mg/kg/day show more inhibition of tumor relative
volume at 4 weeks by 14.712±2.129, 9.449±3.871, respectively
In RHR, compound 1 (5mg/kg, 10mg/kg) also decreased the
▶
MAP in a dose-dependent manner ( Fig. 5). The maximal
compared with tumor relative volume at 4 weeks by
●
reduction was observed at 4h after administration, namely,
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

642 Original Article
Fig. 6 Cell viability effect of AngII, losartan
and compound 1 in prostate carcinoma cell
lines (LNCaP) measured by MTT. Values are
means±standard error of means for experiments.
* P<0.05, compared with control.
160
140
120
100
80
*
*
60
40
20
0
Discussion
35
▼
control
In this study, a new angiotensin II AT1 receptor antagonist, com-
pound 1, 2-(4-((2-propyl-5-nitro-1H-benzo[d]imidazol-1-yl)
methyl)-1H-indol-1-yl) benzoic acid was designed and synthe-
sized.
1:5.0mg
30
25
20
15
10
5
1:10mg
losartan:5.0mg
losartan:10mg
*
The binding assay in the receptor affinity presented that com-
pound 1 exhibited more affinity to AT1 receptors than losartan,
which suggested that compound 1 could specifically and com-
petitively antagonize Ang II to AT1 receptors. Its tight receptor
binding ability might be expected to produce potent and long-
lasting antihypertensive effects in preclinical and clinical set-
tings. The anti-hypertension tests in spontaneously and renal
hypertensive rats showed that compound 1 had an obvious and
continuous reduction in blood pressure. This reduction can last
more than 24h. And this anti-hypertension effect is dose-
dependent. Acute toxicity test revealed that the highly active
compound 1 showed higher LD50 values compared to losartan,
which suggested that this new compound was safer.
Prostate cancer is highly prevalent in society, and its early stages
can be treated by androgen ablation therapy, but there is no
effective treatment for the cancer eventually regresses. There-
fore, clinical development of agents that are relatively safe but
can delay onset and progression of human prostate cancer is
highly desirable. There is an increasing number of evidence that
Ang II may have an important role in both normal prostatic
growth and the development of a range of prostatic diseases in
addition to its well known actions. In our study, the MTT assay in
prostate cancer cells (LNCap) showed that compound 1 possessed
the significant effects against carcinoma of prostate in vitrio. In
vivo assay further displayed that compound 1 has anti-tumor
effect in nude mice with LNCaP cells. The results of the present
work further demonstrated that drugs target AT1 receptor might
be effective in the treatment of carcinoma of prostate in clinic.
Some evidence suggests that AT1 receptor blockade can reduce
tumor volume, vascular density, mitotic index and cell prolifera-
tion through the suppression of the mitogen-activated protein
kinase (MAPK) or the Janus tyrosine kinase-signal transducer
and activator of transcription (STAT) phosphorylation. Samir et
al. considered Ang II might reduce the aberrant methylation and
accordingly reduced the risk of cancer development [23], the
**
**
*
**
**
**
0
0
1W
2W
Date
3W
4W
Fig. 7 Anti-tumor activity of compound 1 and losartan (5mg/kg/
day,10mg/kg/day) in nude mice, tumor growth of LNCap xenografts was
measured at indicated times. Values are means±standard error of means
for experiments. *, ** Significant difference from the control, p<0.05 and
p<0.01, respectively.
22.789±5.782, 17.674±1.344, resectively treated with losartan
at 5.0 or 10mg/kg/day.
Acute toxicity via intragastric administration
Acute toxicity testing suggested that the LD50 value of compound
1 was 2573.2mg/kg and the 95% confidence interval was
2167.1~3061.5 ( Table 1). The LD of losartan was 2248mg/
kg. At all dose levels except for 1000.0mg/kg, rats were observed
to be hypokinetic beginning at approximately 8h after intragas-
tric administration, and some of them died. Rats treated at all
doses survived through the 2 weeks observation period could
maintain weight without obvious untoward effects. The results
showed that compound 1 was a potent AT1-receptor antagonist
with low acute toxicity.
▶
●
50
Da Y-J et al. A novel angiotensin II receptor… Arzneimittelforschung 2012; 62: 637–643

Original Article 643
Table 1 Acute Toxicity Test of Compound 1 (ig) Determined by lethal dose (LD50).
Compound
Dose (mg/kg)
Mortality (%)
LD50 (mg/kg)
95% confidence interval (mg/kg)
1000.0
1710.0
2236.1
2924.0
3823.6
5000.0
0
30
50
60
80
1
2573.2
2167.1~3061.5
100
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Acknowledgment
▼
Chinese National Natural Science Foundation (No.30973611;811
02407;81101298), Foundation of Shanghai government (No.
10DZ0502300;10ZZ45), Foundation of Donghua University (No.
11D10501;11D10518;12D10515;105-10-0044030;LK0902;
BC201128), Foundation of Yiwu government (2011-G1-15).
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Conflict of Interest Statement
▼
We declare that we have no conflict of interest.
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