Synthesis and evaluation of novel angiotensin II receptor 1 antagonists as anti-hypertension drugs

Article abstract of DOI:10.1016/j.bmc.2016.03.028

Three new angiotensin II receptor 1 antagonists, 1, 2 and 3 were designed, synthesized and evaluated. The AT₁ receptor-binding assays in vitro showed that all the synthesized compounds had nanomolar affinity for the AT₁ receptor. Fro

Full text of DOI:10.1016/j.bmc.2016.03.028

Accepted Manuscript
Synthesis and Evaluation of Novel Angiotensin II Receptor 1 Antagonists as
Anti-hypertension Drugs
Xiaolu Bao, Weibo Zhu, Ruijing Zhang, Caihong Wen, Li Wang, Yijia Yan,
Hesheng Tang, Zhilong Chen
PII:
S0968-0896(16)30177-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.028
BMC 12877
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 February 2016
16 March 2016
16 March 2016
Please cite this article as: Bao, X., Zhu, W., Zhang, R., Wen, C., Wang, L., Yan, Y., Tang, H., Chen, Z., Synthesis
and Evaluation of Novel Angiotensin II Receptor 1 Antagonists as Anti-hypertension Drugs, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.03.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Evaluation of Novel Angiotensin II Receptor 1 Antagonists as
Anti-hypertension Drugs
1
Xiaolu Bao , Weibo Zhu , Ruijing Zhang , Caihong Wen , Li Wang , Yijia Yan , Hesheng Tang , Zhilong
2
2
2
2
3
4
1
,2*
Chen
1
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials
Science and Engineering,Donghua University, Shanghai 201620, P.R.China
2
Department of Pharmaceutical Science and Technology, College of Chemistry and Biology, Donghua
University, Shanghai 201620, P.R.China
3
Ningbo Dongmi Pharmaceutical Co., Ltd. Ningbo 315899, Zhejiang, P.R.China
4
Shanghai Xianhui Pharmaceutical Co., Ltd. Shanghai 200433, P.R.China
Corresponding author: Zhilong Chen, Donghua University, 2999 North Renmin Road, Shanghai 201620,
P.R.China
Email: zlchen1967@qq.com
Abstract:
Three new angiotensin II receptor 1 antagonists, 1, 2 and 3 were designed, synthesized and
evaluated. The AT
1
receptor-binding assays in vitro showed that all the synthesized
receptor. From which compound 3 was found
compounds had nanomolar affinity for the AT
1
to be the most potent ligands with an IC50 value of 2.67 ± 0.23 nM. Biological evaluation in
vivo revealed that all the compounds could cause significant decrease on MBP in a dose
dependent manner in spontaneously hypertensive rats, and compound 3 especially showed an
efficient and long-lasting effect in reducing blood pressure, whose maximal response lowered
41 mmHg of MBP at 10 mg/kg and 62 mmHg at 15 mg/kg after oral administration, the
significant anti-hypertensive effect lasted beyond 12 h, which is better than the reference
compound losartan. The pharmacokinetic experiments showed that compound 3 could be
absorbed efficiently and metabolized smoothly both in blood and in tissues in Wistar rats. The
acute toxicity assay suggested that it has low toxicity with the LD50 value of 2974.35 mg/kg.
1
These results demonstrate that compound 3 is a potent angiotensin AT receptor antagonist
which could be considered as a novel anti-hypertension candidate and deserved for further
investigation.
Key words
1
Hypertension; Anti-hypertension; Angiotensin II; AT receptor antagonist
1. Introduction
Hypertension is a complex multifactorial, polygenic disease that worsens with age, and leads
to target-organ complications which span a spectrum comprising cardiac hypertrophy, heart
failure, exacerbation of coronary heart disease, stroke and hypertensive chronic kidney
disease[1]. Although, there are a number of pharmaceuticals such as diuretics, beta blockers,
angiotensin converting enzyme inhibitors (ACE inhibitors), calcium channel antagonists,
angiotensin receptor blockers (ARBs) have been invented for treating this disease,
approximately one-third of the hypertensive population is still not adequately treated, and it
remains one of the largest unmet medical needs in the 21st century[2, 3].

The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in blood pressure
regulation and electrolyte homeostasis. Angiotensin II (Ang II), a vasoconstrictive peptide
hormone, is the effector molecule of the renin-angiotensin system. Classically, Ang II
mediates its action via two major receptors, namely type 1 (AT
angiotensin AT receptor is closely associated with the regulation of blood pressure and
electrolyte balance. The search for Angiotensin II receptor 1 antagonists as potential
antihypertensive agents started 20 years ago[6], nowadays, several Ang II receptor 1 (AT
1 2
) and type 2 (AT )[4, 5]. The
1
1
)
antagonists have been discovered and widely accepted as novel antihypertensive drug
clinically because of its less side effects and good therapeutic profiles than ACE inhibitors[7].
Losartan (Figure 1) is the first Ang II receptor 1 antagonist launched which is widely used
in reducing blood pressure. Based on the structure-activity relationship of losartan, we
designed and synthesized
3
new compounds 1, 2,
3
(Figure 1) in which
5
-oxo-1, 2, 4-oxadiazole in the structure formed the electron transport system and C- terminal
carboxylic acid which is the charge area, n-butyl is the drain water area, diphenyl methyl is
the intermediate connecting part, the fifth position of imidazole ring is alcohol aldehyde, acid,
respectively. And then, the pharmacological profile of these compounds was investigated by
receptor binding studies, antihypertension effect in spontaneously hypertensive rats (SHRs) in
vivo. What’s more, the pharmacokinetic characteristics and acute toxicity of selected
compound were further investigated.
Figure 1. The chemical structure of losartan, compound 1, 2 and 3
2. Materials and methods
2.1 Chemistry
All chemical reagents were of highest commercially available quality and applied without
further purification. Yields referring to purified products were not optimized. Melting points
(
1
m.p.) were measured on an electro thermal melting point apparatus and were uncorrected.
4
H-NMR spectra were measured on a Bruker 400 MHz Spectrometer using TMS (Me Si) 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 meshes).
4
'-((2-butyl-4-chloro-5-formyl-1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-carbonitrile
6)
3
A solution of compound 4 (87.5 mg, 0.47 mmol) and K (135 mg, 0.98 mmol) in DMF
(
2
CO
(
10 mL) was treated with compound 5 (200.5 mg, 0.74 mmol), and the mixture was stirred at
o
10 C under nitrogen for 12 h. The resulting mixture was diluted with water and extracted
-
with ethyl acetate (30 mL × 4). The organic layer was washed with saturated salt water (40

4
mL × 4) and dried over MgSO . After filtration, the solvent was removed under reduced
pressure. The resulting residue was purified by column chromatography to give 6 (163 mg) as
1
a yellow solid. Yield: 73.2%. H NMR (400MHz, CDCl
3
, ppm) ꢀ: 9.78 (s, 1H), 7.76-7.43 (m,
H), 7.18 (d, 2H), 5.62 (s, 2H), 2.68 (t, 2H), 1.71 (m, 2H), 1.36 (m, 2H), 0.89 (t, 3H). MS
6
+
(
ESI): [M + H] calcd 378.1; found 378.2.
'-((2-butyl-4-chloro-5-(hydroxymethyl)-1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-car
bonitrile (7)
A solution of compound 6 (500 mg, 1.33 mmol) in MeOH was treated with NaBH
4
4
(13 mg,
.34 mmol) at 0 C and stirred for 0.5 h. After the reaction was completed, the resulting
mixture was diluted with water and extracted with ethyl acetate (30 mL × 4). The organic
layer was washed with saturated salt water (40 mL × 4) and dried over MgSO . After filtration,
o
0
4
the solvent was removed under reduced pressure. The residue was purified by column
1
chromatography to give 422 mg compound 7 as a yellow solid. Yield: 83.7%. H NMR
(
3
400MHz, CDCl ) ꢀ: 7.77-7.44 (m, 6H), 7.12 (d, 2H), 5.29 (s, 2H), 4.52 (s, 2H), 2.59 (t, 2H),
1
3
2
1
7
4
.03 (s, 1H), 1.68 (m, 2H), 1.33 (m, 2H), 0.88 (t, 3H).
3
C NMR (100MHz, CDCl ) ꢀ: 148.6,
44.6, 137.8, 136.8, 133.8, 132.9, 129.9, 129.4, 127.8, 127.3, 126.3, 124.9, 118.5, 111.2, 77.3,
+
7.1, 76.7, 53.0, 47.2, 29.7, 26.7, 22.4, 13.7. MS (ESI): [M + H] calcd 380.2; found 380.3.
'-((2-butyl-4-chloro-5-((methoxymethoxy)methyl)-1H-imidazol-1-yl)methyl)-[1,1'-biphe
nyl]-2-carbonitrile (8)
A solution of compound 7 (500 mg, 1.32 mmol) in DCM was treated with chloromethyl
methyl ether (126 mg, 1.58 mmol) and DIPEA (340 mg, 2.64 mmol) at r.t. and stirred for 3.5
h. The resulting mixture was diluted with water and extracted with DCM (30 mL × 4). The
4
organic layer was washed with saturated salt water (40 mL × 4) and dried over MgSO . After
filtration, the solvent was removed under reduced pressure. The residue was purified by
1
column chromatography to give 403 mg compound 8 as a yellow solid. Yield: 72.2%. H
NMR (400 MHz, CDCl
3
, ppm) ꢀ: 7.72-7.42(m, 6H), 7.09(d, 2H), 5.22(s, 2H), 4.57(s, 2H),
.45(s, 2H), 3.33(s, 3H), 2.57(t, 2H), 1.65(m, 2H), 1.33(m, 2H); 0.85(t, 3H). MS (ESI): [M +
4
+
H] calcd 424.2; found 424.2 [M + 1].
'-((2-butyl-4-chloro-5-((methoxymethoxy)methyl)-1H-imidazol-1-yl)methyl)-N'-hydrox
4
y-[1,1'-biphenyl]-2-carboximidamide (9)
A solution of compound 8 (500 mg, 1.18 mmol) in EtOH (50 mL) was treated with
o
(651 mg, 4.72 mmol) at 90 C
hydroxylamine hydrochloride (244 mg, 3.54 mmol) and K
2
CO
3
and stirred for 24 h. The resulting mixture was diluted with water (100 mL) and extracted
with ethyl acetate (30 mL × 4). The organic layer was washed with saturated salt water (40
4
mL × 4) and dried over MgSO . After filtration, the solvent was removed under reduced
pressure. The resulting residue was purified by column chromatography to give 400 mg
+
compound 9 as a yellow solid. Yield: 74.4%. MS (ESI): [M + H] calcd 457.2; found 457.2
[
M + 1].
'-((2-butyl-4-chloro-5-((methoxymethoxy)methyl)-1H-imidazol-1-yl)methyl)-N'-((isobut
4
oxycarbonyl)oxy)-[1,1'-biphenyl]-2-carboximidamide(10)
A solution of compound 9 (500 mg, 1.10 mmol) in DMF was treated with isobutyl
o
chloroformate (180 mg, 1.32 mmol) and pyridine (174 mg, 2.20 mol) at 0 C and stirred for 6
h under nitrogen. After the reaction was completed, the resulting mixture was diluted with
water and extracted with ethyl acetate (30 mL × 4). The organic layer was washed with

saturated salt water (40 mL × 4) and dried over MgSO
4
. After filtration, the solvent was
removed under reduced pressure. The residue was purified by column chromatography to give
1
4
7
2
0
3
3
81 mg compound 10 as a yellow solid. Yield: 78.7%. H NMR (400 MHz, CDCl , ppm) ꢀ:
.63-7.30 (m, 6H), 7.01 (d, 2H), 5.18 (s, 2H), 4.68 (s, 2H), 4.57 (s, 2H), 4.42 (s, 2H), 4.00 (d,
H), 3.34 (s, 3H), 2.55 (t, 2H), 2.00 (m, 1H), 1.64 (m, 2H), 1.36-1.27 (m, 2H), 0.95 (d, 6H),
+
.84 (t, 3H). MS (ESI): [M + H] calcd 557.2; found 557.4 [M + 1] .
+
-(4'-((2-butyl-4-chloro-5-((methoxymethoxy)methyl)-1H-imidazol-1-yl)methyl)-[1,1'-bip
henyl]-2-yl)-1,2,4-oxadiazol-5(4H)-one (11)
A solution of compound 10 (500 mg, 0.90 mmol) in 50 mL xylene was refluxed for 6 h under
nitrogen. The solvent was removed under reduced pressure and the residue was purified by
1
column chromatography to give 420 mg compound 11 as a yellow solid. Yield: 96.8%. H
NMR (400 MHz, CDCl
3
, ppm) ꢀ: 7.72-7.25 (m, 6H), 6.88 (d, 2H), 5.18 (s, 2H), 4.48 (s, 2H),
.27 (s, 2H), 3.26 (s, 3H), 2.33 (t, 2H), 1.52 (m, 2H), 1.28 (m, 2H); 0.84 (t, 3H). MS (ESI):
4
+
[
M + H] calcd 483.2; found 483.3.
-butyl-4-chloro-1-((2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-yl)met
2
hyl)- 5-hydroxymethyl-1H-imidazole(1)
A solution of compound 11 (500 mg, 1.04 mmol) in 50 mL DCM was treated with TFA (10
mL) and stirred at r.t. for 28 h. The resulting mixture was diluted with water and extracted
with ethyl acetate (30 mL × 4). The organic layer was washed with saturated salt water (40
4
mL × 4) and dried over MgSO . After filtration, the solvent was removed under reduced
pressure. The residue was purified by column chromatography to give 228 mg compound 1 as
1
a yellow solid. Yield: 50.1%. H NMR (400MHz, dimethyl sulfoxide(DMSO), ppm) ꢀ:
7
.73-7.38 (m, 6H), 7.16 (d, 2H), 5.88 (s, 2H, -NCH
2
-), 2.67 (t, 2H), 2.61 (s, 2H), 1.62 (m, 2H),
+
.36 (m, 2H), 0.92 (t, 3H). MS (ESI): [M + H] calcd 439.2; found 439.3. HRMS (ESI): m/z
1
+
-1
4 3
calculated for C23H24ClN O [M + H] : 439.1537; Found: 438.1459. IR (KBr, cm ): 3462.37,
2959.32, 2870.49, 1758.72, 1464.03, 1258.02, 1016.75, 756.46.
4
'-((2-butyl-4-chloro-5-(1,3-dioxolan-2-yl)-1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-ca
rbonitrile(12)
A solution of compound 6 (500 mg, 1.33 mmol) in toluene was treated with ethylene glycol
(
247 mg, 3.99 mmol) and PPTS (33 mg, 0.13 mmol), the solution was refluxed for 7 h. The
resulting mixture was diluted with water and extracted with ethyl acetate (30 mL × 4). The
organic layer was washed with saturated salt water (40 mL × 4) and dried over MgSO . After
4
filtration, the solvent was removed under reduced pressure. The residue was purified by
1
column chromatography to give 542 mg compound 12 as a yellow solid. Yield: 96.8%. H
NMR (400MHz, CDCl
.98-3.93 (m, 4H), 2.47 (t, 2H), 1.59 (m, 2H), 1.28 (m, 2H), 0.82 (t, 3H). MS (ESI): [M + H]
calcd 422.2; found 422.2.
'-((2-butyl-4-chloro-5-(1,3-dioxolan-2-yl)-1H-imidazol-1-yl)methyl)-N'-hydroxy-[1,1'-bi
3
, ppm) ꢀ: 7.75-7.42 (m, 6H), 7.20 (d, 2H), 5.92 (s, 1H), 5.26 (s, 2H),
+
3
4
phenyl]-2-carboximidamide(13)
A solution of compound 12 (500 mg, 1.19 mmol) in ethanol (50 mL) was treated with
o
(657 mg, 4.76 mmol) at 90 C
hydroxylamine hydrochloride (246 mg, 3.57 mmol) and K
2
CO
3
and stirred for 24 h. The resulting mixture was diluted with water (100 mL) and extracted
with ethyl acetate (30 mL × 4). The organic layer was washed with saturated salt water (40
4
mL × 4) and dried over MgSO . After filtration, the solvent was removed under reduced

pressure. The resulting residue was purified by column chromatography to give 390 mg
1
compound 13 as a yellow solid. Yield: 72.3%. H NMR (400MHz, CDCl , ppm) ꢀ: 7.65-7.33
3
(
m, 6H), 7.13 (d, 2H), 5.92 (s, 1H), 5.22 (s, 2H), 4.41 (s, 2H), 3.98-3.92 (m, 4H), 2.47 (t, 2H),
+
.57 (m, 2H), 1.26 (m, 2H), 0.87 (t, 3H). MS (ESI): [M + H] calcd 455.2; found 455.3.
1
4
'-((2-butyl-4-chloro-5-(1,3-dioxolan-2-yl)-1H-imidazol-1-yl)methyl)-N'-((isobutoxycarbo
nyl)oxy)-[1,1'-biphenyl]-2-carboximidamide(14)
A solution of compound 13 (500 mg, 1.10 mmol) in DMF (50 mL) was treated with isobutyl
o
chloroformate (180 mg, 1.32 mmol) and pyridine (174 mg, 2.20 mol) at 0 C and stirred for 6
h under nitrogen. After the reaction was completed, the resulting mixture was diluted with
water and extracted with ethyl acetate (30 mL × 4). The organic layer was washed with
saturated salt water (40 mL × 4) and dried over MgSO
4
. After filtration, the solvent was
removed under reduced pressure. The residue was purified by column chromatography to give
1
2
7
2
3
52 mg compound 14 as a yellow solid. Yield: 41.3%. H NMR (400MHz, CDCl , ppm) ꢀ:
.67-7.34 (m, 6H), 7.14 (d, 2H), 5.92 (s, 1H), 5.24 (s, 2H), 4.03 (d, 2H), 4.04-3.95 (m, 4H),
.54 (t, 2H), 2.04-2.00 (m, 1H), 1.59 (m, 2H), 1.28 (m, 2H), 0.97 (d, 6H), 0.83 (t, 3H). MS
+
ESI): [M + H] calcd 455.2; found 555.3.
(
3
-(4'-((2-butyl-4-chloro-5-(1,3-dioxolan-2-yl)-1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2
-
yl)-1,2,4-oxadiazol-5(4H)-one(15)
A solution of compound 14 (500 mg, 0.90 mmol) in xylene (50 mL) was refluxed for 6 h
under nitrogen. After the reaction was completed, the solvent was removed under reduced
pressure. The residue was purified by column chromatography to give 400 mg compound 15
1
as a yellow solid. Yield: 92.8%. H NMR (400 MHz, CDCl
3
,) ꢀ: 7.82-7.00 (m, 8H), 5.79 (s,
H), 5.22 (s, 2H), 3.89 (s, 4H), 2.38 (t, 2H), 1.52 (m, 2H), 1.25 (m, 2H), 0.82 (t, 3H). MS
1
+
(
ESI): [M + H] calcd 481.2; found 481.3.
-butyl-4-chloro-1-((2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-yl)met
2
hyl)-1H-imidazole-5-carbaldehyde(2)
A solution of compound 15 (500 mg, 1.04 mmol) in DCM (50 mL) was treated with PPTS
(
261 mg, 1.04 mmol) and stirred at r.t. for 3 h. After the reaction was completed, the resulting
mixture was diluted with water and extracted with ethyl acetate (30 mL × 4). The organic
layer was washed with saturated salt water (40 mL × 4) and dried over MgSO . After filtration,
4
the solvent was removed under reduced pressure. The residue was purified by column
1
chromatography to give 303 mg 2 as a yellow solid. Yield: 66.8%. H NMR (400 MHz,
DMSO, ppm) ꢀ: 9.65 (s, 1H), 8.95 (s, H), 7.78-7.08 (m, 8H), 5.54 (s, 2H), 2.66 (t, 2H), 1.69
+
m, 2H), 1.36 (m, 2H), 0.90 (t, 3H). MS(ESI): [M + H] calcd 437.2; found 437.3. IR (KBr,
(
-1
cm ): 3431.17, 2959.71, 2860.32, 2745.12, 2667.68, 1773.81, 1670.76, 1598.95, 1522.89,
1492.38, 1462.05, 1378.28, 1278.41, 1221.56, 941.26, 868.10, 763.50, 723.22.
2
-butyl-4-chloro-1-((2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-yl)met
hyl)-1H-imidazole-5-carboxylic acid(3)
A solution of compound 2 (500 mg, 1.14 mmol) in butanol (50 mL) was treated with NaClO
2
(
2 4
1.03 g, 11.4 mmol) and NaH PO (1.37 g, 11.4 mmol) in 20 mL water, and the solution was
stirred at r.t. for 5 h. After the reaction was completed, the resulting mixture was diluted with
water and extracted with ethyl acetate (30 mL × 4). The organic layer was washed with
saturated salt water (40 mL × 4) and dried over MgSO
4
. After filtration, the solvent was
removed under reduced pressure. The residue was purified by column chromatography to give

1
4
35 mg 3 as a yellow solid. Yield: 84.6%. H NMR (400 MHz, DMSO, ppm) ꢀ: 12.42 (s, 1H);
7
.72-7.14 (m, 8H), 5.62 (s, 2H), 2.58 (t, 2H), 1.46 (m, 2H), 1.24 (m, 2H), 0.80 (t, 3H). MS
+
ESI): [M + H] calcd 453.1; found 453.3. HRMS (ESI): m/z calculated for C23
(
H
22
N
4
O
4
Cl [M
+
-1
H] : 453.1330; found 452.1244. IR (KBr, cm ): 3461.39, 2958.23, 2933.47, 2872.61,
+
1756.6, 1499.70, 1464.10, 1259.31, 1020.62, 1007.46, 757.19.
2
2
.2 Biological evaluation
.2.1 Binding affinities to Ang II (AT
1
) receptor in vitro
The affinity toward AT
1
receptor of the new compounds were tested by their ability to
1
25
displace [ I]-Ang II from its specific binding sites in vascular smooth muscle cells(VSMCs,
Abcore-inc, co., Ltd, Shanghai, China) line of rats. 3 - 6 generations of VSMCs were used for
experiments. Losartan (Shanghai Zhongkang Weiye Biological Technology Co., Ltd) and new
-
10
compounds 1, 2, 3 were dissolved in DMSO and diluted to different concentrations (10
-
-
0 M) with PBS before experiments. I-Ang II (Zhongshan Hospital, Fudan University,
6
Shanghai, China) was dissolved with PBS and diluted to 0.1 nM. VSMCs (10 cells / well,
4
125
1
o
5
2
00 µL) were seeded into 24 - well plates and cultured in 37 C, 5% CO . After the cells
125
adhered to the wall, they were washed and incubated in PBS containing 0.1 nM I-Ang II
and compounds of diffident concentrations and then cultivated 4 ℃ for 150 min. The final
-12
-6
concentrations of the compounds were 10 - 10 M. And then nonspecific binding
represented 5 - 10% of total binding which was measured in presence of 1 ꢁM Ang II. The
resulting VSMCs were washed 3 times with PBS and digested for 10 min with 0.1 M NaOH[8,
1
25
9
]. These cells bound by I-Ang II were counted by ꢂ - counter (SN - 682, Ri Huan
Company, Shanghai, China). IC50 value and the half inhibition constant of the combination of
1
compounds 1, 2, 3 with AT receptor were estimated by the nonlinear portion of the
competition curves.
2.2.2 Anti - hypertension effect in spontaneously hypertensive rats
Compounds 1, 2, 3 were subjected to biological evaluation for their effects on blood pressure
of spontaneous hypertensive rats (SHRs) (250 ± 20g, Charles River Experimental Animal
Technology Co. Ltd., Beijing, China). The systolic blood pressure (SBP) and diastolic blood
pressure (DBP) of SHRs were measured by noninvasive tail artery manometry under
conscious state. 48 male SHRs were divided into eight experimental groups randomly:
negative control group, positive control group (losartan 10 mg/kg), compound 1 low-dose and
high-dose groups, compound 2 low-dose and high-dose groups, compound 3 low-dose and
high-dose groups. Compounds 1, 2, 3 and losartan were suspended in DMSO and oleic acid
(
V
1
: V
Rats in compound 1, 2, 3 groups were administered orally with compound at the dose of
0 mg/kg and 15 mg/kg respectively. Rats in positive control group were administrated with
2
= 1 : 4).
1
losartan (10 mg/kg). And rats in negative control group were administered with the same
volume of the solvent. The blood pressure and heart rates were monitored before and after the
administration by a biological signal analysis system (MPA - 2000, Alcott Biotech, Shanghai,
China). Six determinations were made in every session of blood pressure measurements and
the means of the six values were taken as the SBP level and DBP level, respectively. The
mean blood pressure (MBP) was calculated by the formula: MBP = (SBP - DBP) / 3 + DBP

[
10], and results were expressed as mean ± SEM. A probability level of less than 0.05 was
considered significant.
2.2.3 Pharmacokinetic assays
High performance liquid chromatography (HPLC) method was used to analyze the drug
concentrations in plasma. The method was developed, validated and operates on two separate
but similar Waters-brand systems. The primary system comprised an Alliance 2489
separations module, equipped with quaternary pump, on-line degasser and auto sampler, and a
600 photodiode array ultraviolet detector. Instrument control, data acquisition and data
processing were achieved with Waters Empower software. The flow rate was 0.8 mL/min and
the injection volume was 20 ꢁL. Separations were performed at ambient temperature on a
reverse phase WondasiliconC18-WR column (150 mm × 4.6 mm, 5 ꢁm). Mobile phases A
was 0.1% formic acid contained 2 mM ammonium acetate (20%) and Mobile phases B was
methanol (80%).
2.2.3.1 Drug concentration in plasma
8
wistar rats were administrated with compound 3 at dose of 10 mg/kg. 0.5 mL venous
blood was taken before administration and 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72h after
administration. Plasma was extracted by centrifuging at 4000 × g, 4℃ for 10 minutes. Then
200 ꢁL plasma samples for analysis were placed into a 1.8 mL polypropylene microfuge tube
followed by 100 ꢁL of internal standard. Acetonitrile (200 ꢁL) was added to precipitate
proteins and the tube vortex mixed for 30s. Precipitated proteins were separated by
centrifugation for 5 min at 8,000 × g. The supernatant was filted by needle filter of 0.45 ꢁL
and then 20 ꢁL filtrate was taken to analyze in HPLC. Linearity for compound 3 was tested
by extracting plasma standards spiked at nominal concentrations of 1, 5, 10, 50, 100, 500
ꢁg/mL (1, 5, 10, 50, 100, 500 ꢁg/mL for compound 3). The calibration line was generated by
least squares linear regression of the peak height ratio (PHR) of analyte/internal standard
-
against nominal concentration with a weighting of concentration [11].
2
2.2.3.2 Drug distribution in tissues
66 Wistar rats were administrated with compound c at the dose of 10 mg/kg. Rats were
killed by cutting the abdominal aorta, and the heart, spleen, kidney, brain and stomach were
quickly excised at each designated time point (0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72h after
administration). The tissues were cut into small pieces and then grinding at 4°C with normal
saline using a Polytron homogeniser (PRO, USA) to prepare a 25% (w/v) homogenate
dilution. Compound 3 and its metabolites in solution (final concentration of 0.01, 0.05, 0.1,
0.5, 1 and 5 ꢁg/mL) were added to the tissue homogenate and vortexed at 4 °C for 5 min.
Then the tissue homogenate dilution were extracted by centrifuging at 3000 × g, 4 ℃ for 10
min, supernatant fluid was collected and analyzed by HPLC[12, 13].
2.2.4 Acute toxicity experiments
The toxicity of compound 3 was determined in sixty normal ICR mice with half males and
half females (20 ± 2 g, Academia Sinica, Shanghai, China). The lethal dose (LD50) was
delivered via intragastric administration at doses of 1200, 1600, 2133.3, 2844.40, 5056.79,

6742.38 mg/kg respectively. Survival rate was assessed daily for two weeks. The LD50 value
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
systematically for the physical signs of toxicity including skin changes, mobility,
aggressiveness, respiratory movements and so on. The survivals were dissected and their
organs and tissues were examined for pathological changes on the fifteenth day.
2.5 Statistics
Results were expressed as means ± standard error of the means. Data were analyzed by
one-way analysis of variance. When overall 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 significant. Binding isotherms from competition
studies were obtained using the nonlinear regression program GraphPad Prism 5 software
(
(
Network of Science Software of China) and LD50 were obtained using SPSS Statistics 20
International Business Machines Corporation, New York, USA). The completed animal
research here adhered to the “Principles of Laboratory Animal Care” and was approved by
IACUC.
3
Results
3.1 Chemistry
The preparation of imidazole derivatives 1-3 were performed by means of a multistep
procedure described in Scheme 1-2.
The synthesis of compound 1 was accomplished starting from the suitable commercially
available 2-butyl-4-chlorine-5-formyl imidazole (4), which reacted with 2- 4’- bromomethyl
2 3
phenyl benzonitrile (5) in DMF using K CO as base to give imidazole 6. Compound 6 was
hydrogenated and the resulting hydroxy group was protected by dimethoxymethane to give
compound 8. After reaction with hydroxylamine and then isobutyl chloroformate, the cyano
group of 8 was transferred into 5-oxo-1, 2, 4-oxadiazol group of 11. The protecting group
methoxymethoxy of 11 was removed with TFA to generate target compound 1.

o
Scheme 1. Reagents and reaction conditions: (a) K
2
CO
, ((CH
, EtOH, 90 C, 24h, 74.4%; (e) ClCOOCH
N, DMF, 0 C, 6h, 78.7% ; (f) xylene, reflux, 6h, 96.8%; (g)CF COOH, CH
3
, DMF, -10 C, 12h, 73.2%; (b)
CH) NCH CH , CH Cl , R.T.,
CH(CH
Cl , R.T.,
o
NaBH
4
, CH
3
OH, 0 C, 0.5h, 83.7%; (c) ClCH
2
OCH
3
3
)
2
2
2
3
2
2
o
3.5h, 72.2%; (d) NH
2
OH·HCl, K CO
2
3
2
3 2
) ,
o
C
6
H
5
3
2
2
28h, 50.1%.
The synthesis of compounds 2 and 3 was started from the protection reaction of aldehyde
group of compound 6 with ethylene glycol. The resulting compound 12 was reacted with
hydroxylamine and then isobutyl chloroformate to give 5-oxo-1,2,4-oxadiazol 15. Compound
15 was deprotected by PPTS in acetone to generate 2. Compound 2 was then oxidized to give
the target compound 3.

Scheme 2. Reagents and reaction conditions: (a) HOCH
2
CH
, EtOH, 90 C, 24 h, 72.3%; (c) ClCOOCH
pydridine, DMF, 0 C, 6 h, 41.3%; (d) xylene, reflux, 6 h, 92.8%; (e) PPTS, CH Cl
2
OH, PPTS, toluene, reflux, 7 h,
CH(CH
, reflux,
o
9
6.8%; (b) NH
2
OH·HCl, K
2
CO
3
2
3 2
) ,
o
2
2
3
h, 66.8%; (f) NaClO
2
, NaH
2
PO
4 2
, n-BuOH, H O, 84.6%
3
.2 Biological evaluation
.2.1 Binding affinities to Ang II (AT
3
1
) receptor in vitro
Radioligand binding assay showed that the compounds 1, 2, 3 had nanomolar affinity to
angiotensin type 1 receptor (Table 1, Figure 2). In competition experiments, compounds 1, 2,
1
25
3
and losartan could compete dose - dependently with I - Ang II. Compound 3 displayed
the highest specific affinity to the AT receptor with the IC50 value of 2.67 ± 0.23 nM and the
Ki value of 2.0 ± 0.17 nM which was more affinity than losartan (IC50 = 19.15 ± 0.15 nM, Ki
14.36 ± 0.11 nM).
1
=
Table 1. Radioligand binding assay (binding IC50) of new compounds 1, 2, 3 and losartan.
Compound
IC50 ± SEM (nM)
5.94 ± 0.22
Ki (nM)
4.30 ± 0.16
3.13 ± 0.11
2.29 ± 0.01
13.86 ± 0.15
1
2
3
4.32 ± 0.15
3.16 ± 0.19
Losartan
19.15 ± 0.21

-6
-12
Figure 2. Inhibitory effects of new compounds 1, 2, 3 and losartan (10 - 10 M) on the
125
specific binding of I-Ang II to AT
1
receptors in VSMCs.
3.2.2 Antihypertensive effects in vivo
The effects of compound 1, 2, 3 (10, 15 mg/kg), losartan(10 mg/kg) on the mean blood
pressure (MBP) in vivo after oral administration in spontaneously hypertensive rats (SHRs)
were shown in Figure 3. The results indicated that all the compounds could decrease the blood
pressure significantly compared with the negative control group, among which compound 3
played more effective than compound 1 and 2. The maximal response of compound 3 (10, 15
mg/kg) was observed at 2h after dosing with reduction of 41 mmHg and 62 mmHg of MBP
respectively which were superior than that of losartan at 10 mg/kg. The significant (p < 0.05)
anti-hypertensive effect of compound 3 lasted for at least 12h. Besides, compound 3 (15
mg/kg) was more effective than losartan (10 mg/kg), and it did not influence heart rates of the
rats. Thus, compound 3 may have the potent to be a novel anti-hypertension candidate and
deserved for further investigation.

Figure 3. Effects of compound 1, 2, 3 (10 mg/kg, 15 mg/kg) and losartan (10 mg/kg) on mean
* **
blood pressure (MBP) in spontaneously hypertensive rats (n = 6). , Significant difference
from the control, p﹤0.05 and p﹤0.01, respectively.
3.2.3 Pharmacokinetic study
3.2.3.1 Drug concentration in plasma
The analytical procedures described were used to quantify compound 3 in rat plasma samples
obtained from 6 male Wistar rats which were orally administered with compound 3 solution.
Microsoft excel program, GraphPad Prism software and DAS 2.0 were used to calculate the
pharmacokinetic parameters. The mean concentration - time curve of compound 3 was shown
in Figure 4. The area under the concentration - time curve from 0 - 72 h (AUC0~72) was
estimated by linear trapezoidal rule. Maximum concentration (Cmax) and time to reach Cmax
(
T
max) were obtained directly from the observed concentration - time curves. Terminal half -
life (t1/2) was calculated as t1/2 = 0.693/k and k was determined by linear regression of the
e
e
logarithmical plasma concentration vs time for the last four data points in the
concentration-time curve[14]. The pharmacokinetic parameters of compound 3 were shown in
table 2. The results showed that compound 3 absorbed quickly and metabolized slowly in
animals and worthy of further researching.
Figure 4. The mean concentration - time curve in rat plasma after oral administration of
compound 3.

Table 2. Pharmacokinetic parameters of compound 3 on plasma after oral administration.
Parameter
Unit
h
Compound 3
2.00 ± 0.20
T
max
max
C
ng/mL
10.30 ± 0.05
0.04 ± 0.005
18.17 ± 0.36
295.80 ± 2.35
-1
h
k
e
t
1/2
h
AUC0~72
ng/mL·h
3.2.3.2 Drug distribution in tissues
The time courses of the concentration of compound 3 and its metabolites in the heart, liver,
spleen, lung, kidney, brain and stomach following oral administration to rats were shown in
Figure 5. The main parameters including AUC values in each tissue were shown in Table 3.
The AUC value of compound 3 in the liver was larger than those in other tissues, and the
AUC values in brain were the minimum (table 3). The distribution of compound 3 to the heart,
liver，kidney and stomach tissues was rapid, and these concentrations peaked within 0.5 h
(
Figure 5). From the results shown in table 4, the compound 3 was highly distributed in spleen
and liver.
Table 3. Pharmacokinetic parameters of compound 3 on tissue after oral administration to rats
Parameter
Unit
Heart
Liver
Spleen
Lung
Kidney
Brain
Stomach
0.5±0.16
T
max
h
0.5±0.32
0.5±0.21
4±0.36
1±0.29
0.5±0.15
2±0.59
C
max
ng/g
8.87±0.53
0.076±0.01
9.13±0.89
200.41±0.69
9.56±0.84
0.07±0.02
10.69±0.03
260.05±1.99
8.84±0.12
0.079±0.01
8.74±0.03
275.21±0.64
9.16±0.59
10.46±0.49
0.04±0.005
15.61±0.05
202.97±7.34
5.80±0.54
0.041±0.01
16.9±0.01
123.07±2.61
8.91±0.21
-
1
ke
h
0.059±0.03
12.36±2.09
213.96±7.67
0.05±0.004
15.06±0.005
221.03±107.6
t
1/2
h
AUC0~72
ng/g·h
9
(
A)
(B)

(
(
(
C)
E)
G)
(D)
(F)
Figure 5. The mean concentration-time curves in the heart tissue (A), liver tissue (B), spleen
tissue (C), lung tissue (D), kidney tissue (E), brain tissue (F), stomach tissue (G), of rats after
administration. Each point represents the mean ± SEM (n = 5).

3.2.4 Acute toxicity experiments
The acute toxicity assay of compound 3 proved its low acute toxicity. The LD50 value of
compound 3 was 2974.35 mg/kg and the 95% confidence interval was 2479.79 - 3694.45
mg/kg (Table 4). There was no physiological abnormalities appeared in mice administered at
dose of 1200 mg/kg. However, hyperthyroidism at 5 h after administration appeared in others
groups, and some mice died. There was no significant change in weight of survival mice after
two weeks observation and no obvious untoward reaction. Besides, no abnormality was
observed with the naked eye of the survival mice after dissection.
Table 4. The lethal dose (LD50) of compound 3 determined by acute toxicity test
Dose(mg/kg)
Log(dose)
Mortality (%)
LD50
and
95%
confidence interval
(
(
mg/kg)
1
200.00
3.08
0
1600.00
2133.30
2844.40
5056.79
6742.38
3.20
3.33
3.45
3.70
3.83
10
20
2
974.35
2479.79 - 3694.45)
40
90
100
4
. Discussion and Conclusion
In this study, three new AT receptor antagonists（1, 2, 3）were designed, synthesized and
evaluated. All the synthesized compounds showed nanomolar affinity for the AT receptor in
1
1
radioligand binding assay in vitro and could cause decrease on MBP in a dose dependent
manner in spontaneously hypertensive rats in vivo. Among them compound 3 showed highly
1
competitive and specific affinity antagonist of AT receptor, and also it caused significant and
continuous reduction in blood pressure which could last more than 12 h in spontaneously
hypertensive rats at 10 mg/kg. Pharmacokinetic experiments in Wistar rats showed that
compound 3 could be absorbed rapidly and metabolized gradually. Besides, compound 3 was
safe with an LD50 value of 2974.35 mg/kg in acute toxicity assay. From the present tests,
compound 3 could be considered as a candidate of antihypertensive drug with effective,
long-lasting and low toxic characteristics, and worthy of further investigation.
5. Acknowledgement
This work was supported by funds of Science and Technology Commission of Shanghai
Municipality (Grants No. 13431900700, 14431906200), found of Science and Technology
Bureau of Ningbo (Grant No. 2015A610274), and fundamental research fund of the Central
Universities (Grant No. 16D310611).
6. Conferences
1.
Herrera, V.L.M. and N. Ruiz-Opazo, Rat as a model system for hypertension drug discovery. Drug
Discovery Today: Disease Models, 2008. 5(3): p. 179-184.
2.
Casimiro-Garcia, A., et al., Discovery of a series of imidazo[4,5-b]pyridines with dual activity at

angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-gamma. J Med Chem,
011. 54(12): p. 4219-33.
2
3.
Natesan Murugesan, J.E.T., Zhengxiang Gu, Bridgette L. Kunst, Leena Fadnis, Lyndon A. Cornelius, Rose
Ann F. Baska, Yifan Yang, Sophie M. Beyer, Hossain Monshizadegan, Kenneth E. Dickinson, Balkrushna
Panchal, Maria T. Valentine, Saeho Chong, Richard A. Morrison, Kenneth E. Carlson, and S.M. James R.
Powell, Joel C. Barrish, Mark C. Kowala, and John E. Macor*, Discovery of N-Isoxazolyl
Biphenylsulfonamides as Potent Dual Angiotensin II and Endothelin A Receptor Antagonists. J. Med.
Chem, 2002. 45: p. 3829-3835.
4.
Marie-Odile Guimond, C.W., Mathias Alterman, Anders Hallberg, Nicole Gallo-Payet Comparative
functional properties of two structurally similar selective nonpeptide drug-like ligands for the
angiotensin II type-2 (AT2) receptor. Effects on neurite outgrowth in NG108-15 cells. European Journal
of Pharmacology, 2013. 699 p. 12.
5.
Renal pharmacology of GR138950, a novel non-peptide angiotensin AT1 receptor antagonist. European
Journal of Pharmacology, 1995. 280: p. 9.
6
.
Design and Synthesis of Nonpeptide Angiotensin II Receptor Antagonists Featuring Acyclic
Imidazole-mimicking Structural Units. Bioorganic & Medicinal Chemistry 1998. 6: p. 15.
Alka Bali, Y.B., M. Sugumaran, Jatinder Singh Saggu, P. Balakumar, Gurpreet Kaur, Gulshan Bansal, Ajay
Sharma and Manjeet Singh, Design, synthesis, and evaluation of novelly substituted benzimidazole
compounds as angiotensin II receptor antagonists. Bioorganic & Medicinal Chemistry Letters 2005. 15:
p. 4.
7.
8.
Nie, Y.Y., et al., Synthesis and biological evaluation of novel potent angiotensin II receptor antagonists
with anti-hypertension effect. Bioorg Med Chem, 2012. 20(8): p. 2747-61.
9.
Zheng, H.l., et al., Synthesis and Biological Evaluation of a New Angiotensin II Receptor Antagonist.
Drug Res (Stuttg), 2014. 64(12): p. 656-662.
10.
Da, Y.J., et al., Synthesis and biological evaluation of new fluorine substituted derivatives as angiotensin
II receptor antagonists with anti-hypertension and anti-tumor effects. Bioorg Med Chem, 2012. 20(24):
p. 7101-11.
1
1.
2.
McWhinney, B.C., et al., Analysis of 12 beta-lactam antibiotics in human plasma by HPLC with
ultraviolet detection. J Chromatogr B Analyt Technol Biomed Life Sci, 2010. 878(22): p. 2039-43.
Togami, K., Y. Kanehira, and H. Tada, Pharmacokinetic evaluation of tissue distribution of pirfenidone
and its metabolites for idiopathic pulmonary fibrosis therapy. Biopharm Drug Dispos, 2015. 36(4): p.
1
205-15.
1
3.
4.
Fretellier, N., et al., Distribution profile of gadolinium in gadolinium chelate-treated renally-impaired
rats: Role of pharmaceutical formulation. Eur J Pharm Sci, 2015. 72: p. 46-56.
1
Yuan, Y., et al., Influence of compound danshen tablet on the pharmacokinetics of losartan and its
metabolite EXP3174 by liquid chromatography coupled with mass spectrometry. Biomed Chromatogr,
2013. 27(9): p. 1219-24.

Tables
Table 1. Radioligand binding assay (binding IC50) of new compounds 1, 2, 3 and losartan.
Table 2. Pharmacokinetic parameters of compound 3 on plasma after oral administration.
Table 3. Pharmacokinetic parameters of compound 3 on tissue after oral administration to rats
Table 4. The lethal dose (LD50) of compound 3 determined by acute toxicity test
Table 1
Compound
IC50 ± SEM (nM)
5.94 ± 0.22
Ki (nM)
4.30 ± 0.16
3.13 ± 0.11
2.29 ± 0.01
13.86 ± 0.15
1
2
3
4.32 ± 0.15
3.16 ± 0.19
Losartan
19.15 ± 0.21
Table 2
Parameter
Unit
h
Compound 3
2.00 ± 0.20
T
max
max
C
ng/mL
10.30 ± 0.05
0.04 ± 0.005
18.17 ± 0.36
295.80 ± 2.35
-1
h
k
e
t
1/2
h
AUC0~72
ng/mL·h
Table 3
Parameter
Unit
h
Heart
Liver
0.5±0.21
9.56±0.84
0.07±0.02
Spleen
Lung
Kidney
Brain
2±0.59
Stomach
0.5±0.16
T
max
0.5±0.32
8.87±0.53
0.076±0.01
9.13±0.89
200.41±0.69
4±0.36
1±0.29
0.5±0.15
C
max
ng/g
8.84±0.12
0.079±0.01
8.74±0.03
275.21±0.64
9.16±0.59
10.46±0.49
0.04±0.005
15.61±0.05
202.97±7.34
5.80±0.54
0.041±0.01
8.91±0.21
0.05±0.004
-1
h
ke
0.059±0.03
12.36±2.09
213.96±7.67
t
1/2
h
10.69±0.03
16.9±0.01
15.06±0.005
221.03±107.
AUC0~72
ng/g·h
260.05±1.99
123.07±2.61
6
9
Table 4.
Dose(mg/kg)
Log(dose)
Mortality (%)
LD50
and
95%
confidence interval

(
(
mg/kg)
1
200.00
3.08
0
1600.00
2133.30
2844.40
5056.79
6742.38
3.20
3.33
3.45
3.70
3.83
10
20
2
974.35
2479.79 - 3694.45)
40
90
100

Graphical abstract
ꢇꢌ
ꢋ
ꢄ
ꢋ
Anti-hypertensive
effect in vivo
AT
1
receptor-binding
assays in vitro
ꢋ
1ꢃ ꢄꢅꢆꢇꢈ ꢊꢈ
ꢉ
ꢊ
ꢊ
2ꢃ ꢄꢅꢆꢇꢈꢊ
ꢈꢋ
3
ꢃ ꢄꢅꢆꢇꢊꢊꢈ
Acute toxicity of
compound 3
Pharmacokinetic
characteristics of
compound 3
Compound with significant
anti-hypertensive effect was
selected (compound 3)