Synthesis and biological evaluation of novel potent angiotensin II receptor antagonists with anti-hypertension effect

Article abstract of DOI:10.1016/j.bmc.2012.02.003

A series of novel angiotensin II type 1 receptor antagonists were prepared. Radioligand binding assay suggested that compounds 1b and 1c could be recognized by the AT₁ receptor with an IC₅₀ value of 1.6 ± 0.09 nM and 2.64 ± 0.7 nM, respectively. In vivo anti-hypertension experiments showed that compounds (1a, 1b, 1c, 1e) elicited a significant decrease in SBP and DBP of spontaneous hypertensive rats (SHRs). The antihypertensive effects maintained for 10 h, which indicated that these compounds had a favorable blood pressure-lowering effect. Acute toxicity testing suggested that the LD₅₀ value of compound 1b was 2316.8 mg/kg which was lower than valsartan (LD₅₀ = 307.50 mg/kg) but higher than losartan (LD₅₀ = 2248 mg/kg). So they could be considered as novel anti-hypertension candidates and deserved for further investigation.

Full text of DOI:10.1016/j.bmc.2012.02.003

Bioorganic & Medicinal Chemistry 20 (2012) 2747–2761
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel potent angiotensin II receptor
antagonists with anti-hypertension effect
Yong-yan Nie, Ya-jing Da, Hao Zheng, Xiao-xia Yang, Lin Jia, Cai-hong Wen, Li-sha Liang, Juan Tian,
⇑
Zhi-long Chen
Department of Pharmaceutical Science and Technology, College of Chemistry and Biology, Donghua University, Shanghai 201600, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel angiotensin II type 1 receptor antagonists were prepared. Radioligand binding assay sug-
gested that compounds 1b and 1c could be recognized by the AT1 receptor with an IC50 value of
1.6 0.09 nM and 2.64 0.7 nM, respectively. In vivo anti-hypertension experiments showed that com-
pounds (1a, 1b, 1c, 1e) elicited a significant decrease in SBP and DBP of spontaneous hypertensive rats
(SHRs). The antihypertensive effects maintained for 10 h, which indicated that these compounds had a
favorable blood pressure-lowering effect. Acute toxicity testing suggested that the LD50 value of com-
pound 1b was 2316.8 mg/kg which was lower than valsartan (LD50 = 307.50 mg/kg) but higher than
losartan (LD50 = 2248 mg/kg). So they could be considered as novel anti-hypertension candidates and
deserved for further investigation.
Received 21 October 2011
Revised 31 January 2012
Accepted 1 February 2012
Available online 11 February 2012
Keywords:
Angiotensin II receptor antagonists
Hypertension
Synthesis
Acute toxicity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
candesartan, irbesartan, telmisartan, and olmesartan are also being
used in the clinics and lots of other congeners are being developed.
The rennin angiotensin system (RAS) plays a central role in the
regulation of blood pressure and electrolyte/fluid homeostasis. It
consists of a cascade of enzymatic reactions producing angiotensin
II (Ang II), which is an octapeptide that presents a wide range of
physiological actions on cardiovascular, renal, endocrine and cen-
tral nervous systems.1,2
Most of ARBs have acidic group tetrazole ring, such as valsartan,
losartan, candesartan, ibersartan. The tetrazole group had some dis-
advantages in chemical synthesis and biometabolism. For example,
the synthesis of tetrazole derivatives could be dangerous due to the
use of toxic and explosive azide compounds such as sodium azide or
trialkytin azide. So it is important to find an acidic group in place of
tetrazole. 5-Oxo-1,2,4-oxadiazol was found to be a suitable substi-
tute because it is easy to be prepared with high yield, and friendly
to environment. So another new ARB azilsartan medoxomil (Edar-
bi-Takeda) was developed and approved in clinics by FDA in 2011
for oral treatment of hypertension, either alone or combined with
other drugs. azilsartan medoxomil at its maximal dose had superior
efficacy to both valsartan and olmesartan at their maximal approved
doses without increasing adverse events.6 Bakris also reported that
azilsartan medoxomil was well tolerated and more efficacious at its
maximal dose than the highest dose of olmesartan.7
In the present work 5-oxo-1,2,4-oxadiazol was used to displace
tetrazole group of valsartan which is appeared in some ARBs in
clinics.8 So a series of new compounds 1a–g with N-acylated ami-
noacid moiety and biphenyl moiety were synthesized (Table 1).
Fluorine atoms was also introduced in para-position of 5-oxo-
1,2,4-oxadiazol to raise the acidity of the molecular 2. In addition,
the preliminary pharmacological characteristics including the ef-
fect on hypertension, and acute toxicity of compound 1b was also
presented to the development of novel anti-hypertension.
Ang II is produced in vivo from angiotensin I by the angiotensin
converting enzyme (ACE). ACE inhibitors, such as Enalapril and
Captopril, are widely used in clinic to control hypertension. But
ACE has its limitation in clinical use for some of the major side ef-
fects, such as dry cough, angioedema and rashes, resulted from lack
of specificity to ACE for Ang I.3 However, angiotensin II type 1 (AT1)
receptor antagonists, namely the angiotensin II type 1 receptor
blockers (ARBs), are selective for AT1 receptors, and act indepen-
dently on the Ang II synthetic pathway.4 AT1 receptor antagonists
are the most specific means presently available to block the re-
nin-angiotensin enzymatic cascade. In addition, studies in clinic
and experiments have demonstrated the protective effect of AT1
blockers on cardiovascular morbidity and mortality.5 The discovery
of potent and orally active nonpeptide Ang II antagonists with few
side effects such as valsartan, and losartan has encouraged the
design of a large number of similar compounds. Among them,
⇑
Corresponding author. Address: 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.
E-mail address: zlchen1967@yahoo.com (Z. Chen).
The dominant conformations of compound 1a and valsartan are
shown as Figure 1 using software Spartan 8. We found that the
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.003

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Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
Table 1
Oxadiazole compounds 1a–f
with aqueous LiOH to provide the final biphenylyloxadiazol com-
pounds 1a–g.11
For the 50-fluoro-biphenylyloxadiazol compound 2, we chose a
different synthetic route because the key intermediate 40-(bromo-
methyl)-5-fluoro-biphenyl-2-carbonitrile 12 was commercially
unavailable. As outlined in Scheme 2, compound 11 was prepared
from 2-bromo-4-fluorobenzonitrile which was subjected to palla-
dium coupling with p-toylboronic acid in the presence of a base
under the conditions described by Suzuki in 81.1% yield. After rad-
ical bromination of 11 with NBS in chloroform 40-(bromomethyl)-
5-fluoro-biphenyl-2-carbonitrile 12 was obtained in 78% yield after
recrystallization.12 The following synthetic steps was similar to
that of 1a–g.
O
R1
R2
N
COOH
R3
N
O
O
NH
Compound R1
R2
R3 Mp (°C)
Formula
½
a 2D4
ꢀ
(c3, EtOH)
2.2. Biological evaluation
1a
1b
1c
1d
1e
1f
iPr
iPr
iPr
iBu
iBu
nBu
nPr
Et
H
H
H
H
H
H
H
F
112–113
86–88
79–81
123–124
115–117
ꢁ55
ꢁ48
ꢁ45
ꢁ67
ꢁ86
C25H29N3O5
C24H27N3O5
C23H25N3O5
C26H31N3O5
C25H29N3O5
C29H29N3O5
C28H27N3O5
C24H26FN3O5
2.2.1. Effects of the new compounds on specific Ang II binding
to AT1 receptors in VSMCs
nBu
nPr
In vascular smooth muscle cell (VSMC) cultures, the specific
binding of 125I-Ang II was inhibited in a concentration-dependent
manner by compounds 1a, 1b, 1c, 1d, 1e, 1f, 1g, compounds 2
and valsartan, irbesartan (Fig. 2). The IC50 values of compounds
1b, 1c and valsartan were 0.49 0.1 nM, 1.6 0.09 nM and
2.64 0.7 nM, respectively (Table. 2). It showed that compounds
1b, 1c exhibited more affinity to AT1 receptors than valsartan be-
cause their IC50 values were significantly lower compared with val-
sartan’s (P <0.05). So compounds 1b and 1c showed much stronger
inhibitory ability than compounds1a, 1d, 1e, 1f, 1g and 2 in recep-
tor binding assay which indicated that compounds 1b and 1c
might have better antihypertensive activity in vivo antihyperten-
sive experiments.
PhCH2 nBu
PhCH2 nPr
166–167 ꢁ101
1g
2
153–154
157–159
ꢁ92
ꢁ47
iPr
npr
minimal energy of 1a is lower than that of varsartan. The energy-
minimized stereo-conformation of 1a fits perfectly with valsartan.
2. Result and discussion
2.1. Chemistry
Compounds with biphenylyloxadiazol group (1a–g) were pre-
pared according to the route described in Scheme 1. Natural amino
acid, Val, Leu and Phe, were reacted with methanol and acetyl chlo-
ride to give aminoacid esters (4a–c). Alkylation of 4a–c in DMF
with 40-(bromomethy)-biphenyl-2-carbonitrile using triethyl-
amine as base gave the secondary amines 5a–c.9 Subsequently
compounds 6a–g were obtained by the acylation of 5a–c with dif-
ferent acyl chlorides in the presence of 4-N,N-dimethylamino pyri-
din and triethylamine.
In order to obtain the oxadiazole compounds, the cyano com-
pounds 6a–g were firstly converted into the amidoximes 7a–g in
DMSO by reacting with hydroxylamine hydrochloride in presence
of triethylamine.10 Then the amidoximes 7a–g were reacted with
isobutyl chloroformate using pyridine as a base in DMF to give
the o-acyl amidoxime compounds 8a–g. The cyclized oxadiazole
compounds 9a–g were easily obtained from 8a to 8g in xylene
under refluxing. These ester derivatives 9a–g were hydrolyzed
2.2.2. Effects of the compounds on blood pressure of SHR
Compounds 1a, 1b, 1c, 1e and 2 caused significant reduction of
SBP (Fig. 3A) and DBP (Fig. 3B) in spontaneously hypertensive rats,
but compounds 1d, 1f and 1g did not decreased blood pressure sig-
nificantly, there were no differences between the experimental
groups and the negative control group (Fig. 3C). Compounds 1a,
1b, 1c and 1e at the dose of 15 mg/kg had the antihypertensive
activities equal to valsartan and better than irbesartan. But com-
pound 2 at 15 mg/kg had little effect on SBP and DBP. Biological
study of compounds 1a, 1b, 1c, 1e and 2 at 30 mg/kg on hyperten-
sive rats also revealed the higher antihypertensive activity of these
compounds than irbesartan. The maximal effects of the com-
pounds on the reduction of SBP and DBP were presented in 3–4 h
after administration. The antihypertensive effects maintained for
10 h, which indicated that the compounds had a favorable blood
Figure 1. Energy-minimized conformation of valsartan (135.0750 kJ/mol), compound 1a (112.3563 kJ/mol) and their overlay conformation.

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2749
R
HN
COOCH
3
R
R
c
a
b
i
3a:R= Pr
H N
2
COOH
i
H N
2
COOCH
3
3b: R= Bu
3c: R=PhCH
NC
2
3 a-c
4a-c
5a-c
O
R
1
O
R
1
R
N
COOCH
N
R
N
COOCH
2
3
2
3
i
n
6a: R = Pr, R2= Bu
1
e
d
i
6b: R = Pr, R2=Pr
1
i
6c: R = Pr, R2=Et
1
i
n
6d: R = Bu, R2= Bu
1
HO
NC
6a-g
i
6e: R = Bu, R2=Pr
1
NH
2
n
6f: R =PhCH , R2= Bu
6g: R =PhCH , R2=Pr
1
2
7a-g
1
2
O
R
1
O
R
1
O
R
1
R
2
N
COOCH
R
2
N
COOH
3
R
N
O
COOCH
N
2
3
g
f
N
N
O
O
O
O
NH
NH
NH
2
O
O
1a-g
8a-g
9a-g
Scheme 1. Reagents and conditions: (a) CH3OH, SOCl2, 0 °C; (b) 20-cyano-4-bromomethylbiphenyl, DMF, Et3N, reflux; (c) R2COCl (R2
(d) NH2OHꢂHCl, DMSO, Et3N, reflux; (e) isobutyl chloroformate, pyridine, DMF; (f) xylene, reflux; (g) LiOH, CH3OH, H2O.
=
nBu, Pr, Et), DMAP, Et3N, CH2Cl2, reflux;
pressure-lowering effect. All the compounds did not influence
heart rate of the rats.
settings. The anti-hypertension test showed that compounds 1a,
1b, 1c, 1e had obvious reduction in the blood pressure in spontane-
ously hypertensive rats. Acute toxicity test revealed that the highly
active compound 1b showed higher LD50 values compared to losar-
tan, which suggested that the new compound was safer. So the new
compounds could be considered as candidate with high perfor-
mance and fewer adverse effects for novel anti-hypertension drugs.
From both the in vitro and in vivo data it can be seen that the
nature of the amino acid side chain is crucial for activity. The com-
pounds with Val as side chain was better than the compounds with
Leu and Phe. These results were in agreement with the molecular
modeling studies.
2.2.3. Toxicity of compound 1b via intragastric administration
Acute toxicity testing suggested that the LD50 value of com-
pound 1b was 2316.8 mg/kg and the 95% confidence interval was
1878.8–2856.8 mg/kg (Table 3). It was well known that the LD50
of losartan was 2248 mg/kg and the LD50 of valsartan was
307.50 mg/kg. At all dose levels except for 1000.0 mg/kg, rats were
observed to be hypokinetic beginning at approximately 5 h after
intragastric administration, and some of them died. Rats treated
at all doses survived through the two weeks observation period
could maintain weight without obvious untoward effects. The re-
sults showed that compound 1c was a potent AT1-receptor antag-
onist with low acute toxicity.
4. Experimental section
All chemicals used were of reagent grade. Yields refer to puri-
fied products and are not optimized. All melting points were mea-
sured using an Electrothermal 9200 apparatus and are uncorrected.
1H NMR spectra were measured on a Bruker 400 MHz spectrome-
ter using Me4Si as internal standard. ESI-MS spectra were recorded
on a Micromass triple quadrupole mass spectrometer. Optical rota-
tions were measured at 25 °C on a Jasco-P2000 polarimeter. Col-
umn chromatography was performed using silica gel H (300–400).
3. Conclusions
In this work a series of new compounds with 5-oxo-1,2,4-oxa-
diazole group were designed and synthesized avoiding the use of
hazardous azide and tin compounds existing in the preparing of
losartan, valsartan and some other ARBs.
The binding assay in the receptor affinity presented that com-
pounds 1b, 1c exhibited more affinity to AT1 receptors than valsar-
tan, which suggested that compounds 1b and 1c could specifically
and competitively 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
4.1. L-Valine methyl ester hydrochloride (4a)
To 5 mL of ice-bath cooled methanol was added dropwise 1 mL
of SOCl2, the resulting mixture was stirred for 0.5 h and 1.01 g

2750
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
Br
HN
COOCH3
Br
j
CN
i
h
CN
CN
F
F
F
F
NC
13
10
11
12
O
O
N
COOCH3
N
COOCH3
k
m
l
F
F
N
NC
HO
15
NH2
14
O
O
O
O
N
COOH
N
COOCH3
N
COOCH3
o
n
F
F
F
H
N
H
N
O
O
O
N
O
O
NH
NH
NH2
O
17
16
2
Scheme 2. Reagents and conditions: (h) p-tolylboronic acid, Pd(OAc)2, K2CO3, PPh3, 1,4-dioxane, H2O, reflux; (i) NBS, AIBN, CHCl3, reflux; (j) DIPEA, CH2Cl2, reflux; (k) butyryl
chloride, DIPEA, CH2Cl2, Et3N; (l) NH2OHꢂHCl, DIPEA, CH3CH2OH, reflux; (m) isobutyl chloroformate, DIPEA, CH2Cl2; (n) xylene, reflux; (o) LiOH, H2O, CH3OH, reflux.
Table 2
1f
valsartan
irbesartan
1a
1b
1c
1d
Radioligand binding assay (binding IC50) of compounds 1a–f and 2.
1g
1e
2
125
100
75
50
25
0
Binding IC50
Compounds
IC50 SEM (nM)
Ki (nM)
1a
1b
1c
1d
1e
1f
1g
2
9.82 4.20
0.49 0.10
1.60 0.09
10.18 0.02
35.97 0.21
17.99 0.13
26.52 0.09
20.81 0.12
2.64 0.70
0.58 0.22
6.65 2.50
0.33 0.12
1.03 0.59
6.88 0.11
24.30 0.25
12.16 0.47
17.92 0.17
14.06 0.33
2.22 0.50
0.41 0.12
Valsartan
Irbesartan
-13 -12 -11 -10
-9
-8
-7
-6
-5
compound, log(M)
Figure 2. Inhibitory effects of compounds1a, 1b, 1c, 1d, 1e, 1f, 1g, 2, valsartan and
irbesartan (10ꢁ6 to 10ꢁ12 M) on the specific binding of 125I-Ang II to AT1 receptors in
VSMCs.
4.3. L-Phenylalanine methyl ester hydrochloride (4c)
Compound 4c was prepared according to the procedure de-
scribed for the preparation of 4a. Yield 96.4%. Mp: 173.2–
175.3 °C. The spectral data were consistent with that reported in
the literature.8
(8.55 mmol) of L-Valine was added. The stirring was continued
overnight, and the solvent was removed under reduced pressure.
The residue was purified by flash column chromatograph (eluent:
methanol/ether = 1/5, v/v) to afford a colorless pinch-like crystal.
Mp: 128–132 °C. Yield 98.8%. The spectral data were consistent
with that reported in the literature.8
4.4. N-((20-Cyanobiphenyl-4-yl) methyl)-
L-Valine methyl ester
(5a)
4.2. L-Isoleucine methyl ester hydrochloride (4b)
4.6 mL Triethylamine (36.00 mmol) was added dropwise to an
ice-bath cooled solution of 4a (2.01 g, 12.0 mmol) in 15 mL of
DMF under nitrogen followed by 20-cyano-4-bromomethylbiphe-
nyl (2.99 g, 11.00 mmol). The mixture was heated to 70 °C and
was monitored by TLC. After completion of the reaction, the
Compound 4b was prepared according to the procedure for the
preparation of 4a. Yield 97.5%. Mp: 158–160 °C. The spectral data
were consistent with that reported in the literature.8

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2751
valsartan
valsartan
irbesartan
1b
5
irbesartan
5
0
1a
control
0
control
-5
-10
-15
-20
-25
-30
-35
-40
-5
-10
-15
-20
-25
-30
-35
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1a 15mg/kg
Compound 1b 15mg/kg
valsartan
irbesartan
1b
valsartan
irbesartan
1a
0
-10
-20
-30
-40
0
-10
-20
-30
-40
control
control
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1a 30mg/kg
Compound 1b 30mg/kg
Figure 3A. Systolic Blood Pressure (SPB) and diastolic blood pressure (DBP) development during the experiment. Compounds 1a, 1b, 1c, 1e (15 mg/kg and 30 mg/kg) and 2
(30 mg/kg) presented favorable antihypertensive activities in SPB of spontaneous hypertensive rats (SHRs). The equal doses of valsartan and irbesartan were taken as positive
controls.
solution was washed with water and the organic layer was evapo-
rated. The residue was purified by column chromatograph to afford
a colorless oil. Yield 81.8%.1H NMR (CDCl3, 500 MHz) d: 7.78–7.25
(8H, m, Ph), 3.90, 3.65 (2H, two d, J = 13.5 Hz, PhCH2), 3.74 (s,
OCH3), 3.05 (1H, d, J = 6.3 Hz, NCH), 1.94 (1H, m, Me2CH), 0.96,
0.97 (3H, two d, 6H, J = 6.6 Hz, 2 ꢃ CH3); 13C NMR (CDCl3,
125 MHz) d: 175.5, 145.2, 140.7, 136.7, 133.6, 132.6, 129.9, 128.5,
128.3, 127.3, 118.6, 111.0, 66.6, 52.0, 51.3, 31.6, 19.2, 18.5; ESI-
MS (m/z): 323.2[M+1]+, 345.2[M+Na]+.
55.4, 38.6, 29.6, 19.4, 16.2 ESI-MS (m/z): 337.2[M+1]+,
359.2[M+Na]+.
4.6. N-((20-Cyanobiphenyl-4-yl) methyl)-
ester (5c)
L-Phenylalanine methyl
Compound 5c was prepared according to the procedure de-
scribed for the preparation of 5a. Yield 67.3%, 1H NMR (CDCl3,
500 MHz) d: 7.67 (1H, d, J = 8.56 Hz, Ph), 7.54 (1H, t, J = 7.68 Hz,
Ph), 7.45–7.41 (3H, m, Ph), 7.35–7.31 (3H, m, Ph), 7.28–7.25 (2H,
m, Ph), 7.21–7.16 (3H, m, Ph), 3.85 (1H, d, J = 13.6 Hz, NCH2),
3.67 (d, J = 13.6 Hz, NCH2), 3.63 (3H, s, COOCH3), 3.56 (2H, t,
J = 6.8 Hz, NCH), 2.97 (2H, m, PhCH2), 1.96 (1H, s, NH); 13C NMR
(CDCl3, 125 MHz) d: 174.5, 144.8, 140.1, 137.0, 136.5, 133.4,
132.5, 129.6, ESI-MS (m/z): 371.2[M+1]+, 393.2[M+Na]+.
4.5. N-((20-Cyanobiphenyl-4-yl)methyl)-
L-Isoleucine methyl
ester (5b)
Compound 5b was prepared according to the procedure de-
scribed for the preparation of 5a. Yield 62.1%, 1H NMR (CDCl3,
500 MHz) d: 7.78–7.23 (8H, m, Ph-H), 3.89, 3.62 (2H, two d,
J = 13.5 Hz, J = 13.7 Hz, –NH–CH2–), 3.72 (3H, s, –OCH3), 3.03 (1H,
d, J = 6.3 Hz, –CO–CH–N–), 1.95 (1H, m, –CH–CH3), 1.26 (2H, m, –
CH–CH2CH3), 0.96, 0.97 (6H, two d, J = 6.6 Hz, –CHCH3); 13C NMR
(CDCl3, 125 MHz) d: 177.0, 145.1, 141.1, 139.9, 138.3, 137.5,
133.6, 133.6, 133.1, 133.1, 132.2, 132.2, 121.5, 116.4, 70.3, 57.5,
4.7. N-Butyryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Valine
methyl ester (6b)
To 31 mL of dichloromethane was added methyl ester 5b
(3.97 g, 11.8 mmol), TEA (5 mL, 39.10 mmol) and DMAP (0.09 g,
0.7 mmol). To the mixture was added dropwise carbonyl chloride

2752
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
valsartan
irbesartan
1c
control
valsartan
irbesartan
1e
5
0
5
0
control
-5
-5
-10
-15
-20
-25
-30
-35
-40
-10
-15
-20
-25
-30
-35
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1c 15mg/kg
Compound 1e 15mg/kg
valsartan
irbesartan
1c
valsartan
irbesartan
1e
0
-10
-20
-30
-40
control
0
-10
-20
-30
-40
control
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1c 30mg/kg
Compound 1e 30mg/kg
Fig. 3A (continued)
(2.1 mL, 35.60 mmol) under nitrogen. After stirring for 1 h, the
temperature was raised to 45 °C, and the mixture was monitored
by TLC till completion. To the mixture was added 10 mL of 1 M di-
lute aqueous hydrochloride. The organic phase was separated,
washed with saturated NaHCO3 solution, and dried over MgSO4.
After filtration, the solvent was removed under reduced pressure,
and the residue was purified by flash column chromatograph to af-
ford a colorless oil. Yield 74.9%. 1H NMR (CDCl3, 300 MHz) d: 7.72–
7.26 (8H, m, Ph-H), 5.08 (d, J = 15.6 Hz), 5.00 (1H, d, J = 10.5 Hz, –
CO–CH–N–), 4.68 (d, J = 17.9 Hz), 4.26 (d, J = 15.6 Hz), 4.04 (2H, d,
J = 10.8 Hz, –N–CH2–), 3.43 (s), 3.35 (3H, s, –OCH3), 2.43–2.30
(3H, m, –CO–CH2–, –CHCH3), 1.28–0.84 (9H, m,); 13C NMR (CDCl3,
125 MHz) d: 178.2, 175.0, 145.1, 141.1, 139.9, 138.3, 137.5, 130.6,
130.6, 130.1, 130.1, 129.2, 129.2, 121.5, 116.4, 66.8, 55.8, 55.4,
39.3, 30.2, 21.1, 20.9, 20.9, 18.6. ESI-MS (m/z): 379.2[M+1]+,
401.2[M+Na]+; HR-MS: 378.1944, C23H26N2O3.
7.54–7.37 (4H, m, Ph-H), 7.34–7.28 (2H, m, Ph-H), 5.08 (d,
J = 15.4 Hz), 4.97 (1H, d, J = 10.3 Hz, –CO–CH–N–), 4.72 (d, J =
17.9 Hz), 4.26 (d, J = 15.4 Hz), 4.11 (2H, d, J = 11.1 Hz, –N–CH2–),
3.40 (s), 3.35 (3H, s, –OCH3), 2.58–2.18 (3H, m, –CO–CH2–, –
CHCH3), 1.85–1.13 (4H, m, –CH2CH2CH3), 0.83–0.99 (9H, m,); 13C
NMR (CDCl3, 125 MHz) d: 180.2, 177.0, 145.1, 141.1, 139.9, 138.3,
137.5, 133.6, 133.6, 136.1, 136.1, 135.2, 135.2, 121.5, 116.4, 68.8,
55.8, 55.4, 35.8, 33.5, 30.2, 27.1, 21.9, 21.9, 18.3. ESI-MS (m/z):
407.3[M+1]+, 429.3[M+Na]+; HR-MS: 406.2261, C25H30N2O3.
4.9. N-Propionyl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Valine
methyl ester (6c)
Compound 6c was prepared according to the procedure de-
scribed for the preparation of 6b. Yield 74.9%. 1H NMR (CDCl3,
500 MHz) d: 7.72–7.26 (8H, m, Ph-H), 5.08 (d, J = 15.6 Hz), 5.00
(d, 1H, J = 10.5 Hz, –CO–CH–N–), 4.68 (d, J = 17.9 Hz), 4.26 (d,
J = 15.6 Hz), 4.04 (2H, d, J = 10.8 Hz, –N–CH2–), 3.43 (s), 3.35 (3H,
s, –OCH3), 2.43–2.30 (3H, m, –CO–CH2–, –CHCH3), 1.28–0.84 (9H,
m,); 13C NMR (CDCl3, 125 MHz) d: 178.9, 177.0, 145.1, 141.4,
139.9, 138.3, 137.5, 133.6, 133.6, 133.1, 133.1, 132.2, 132.2,
121.5, 116.4, 68.8, 55.8, 55.4, 30.2 29.9, 21.9, 21.9, 15.0. ESI-MS
(m/z): 379.2[M+1]+, 401.2[M+Na]+; HRMS: 378.1944, C23H26N2O3
4.8. N-Valeryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Valine
methyl ester (6a)
Compound 6a was prepared according to the procedure de-
scribed for the preparation of 6b. Yield 75.5%. 1H NMR (CDCl3,
500 MHz) d: 7.75–7.67 (1H, m, Ph-H), 7.63–7.58 (1H, m, Ph-H),

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2753
55.4, 39.8, 36.8, 33.5, 27.1, 18.3. ESI-MS (m/z): 455.3[M+1]+,
valsartan
irbesartan
2
477.2[M+Na]+; HR-MS: 454.2256, C29H30N2O3.
5
0
control
4.13. N-Butyryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Phenylal
-5
anine methyl ester (6g)
-10
-15
-20
-25
-30
-35
-40
Compound 6g was prepared according to the procedure de-
scribed for the preparation of 6b, Yield 75.5%. 1H NMR (CDCl3,
500 MHz) d: 7.77 (1H, d, Ph-H), 7.50–7.43 (4H, m, Ph-H), 7.31–7.08
(8H, m, Ph-H), 4.49 (d, 1H, J = 17.7 Hz, –CO–CH–N–), 4.37 (m), 3.87
(2H, d, J = 16.5 Hz, –N–CH2–), 3.67 (3H, s, –OCH3), 3.40–3.36 (m),
3.29–3.24 (2H, m, –N–CHCH2), 2.35–2.25 (2H, m, –CO–CH2–), 1.69
(2H, m, –CH2CH2CH3), 0.89 (3H, t, J = 7.4 Hz); 13C NMR (CDCl3,
125 MHz) d: 173.7, 170.8, 144.8, 138.0, 137.4, 137.1, 133.7, 132.8,
130.0, 129.9, 129.2, 129.1, 128.9, 128.7, 128.5, 128.3, 127.6, 127.4,
127.3, 126.6, 118.5, 111.2, 77.3, 77.0, 76.7, 61.1, 52.0, 51.8, 35.4,
35.2, 18.5, 13.8; ESI-MS (m/z): 441.3[M+1]+, 463.3[M+Na]+.
0
2
4
6
8
10
12
time(hour)
Compound 2 15mg/kg
4.14. N-Propionyl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
methyl]-L-Valine methyl ester (7c)
valsartan
irbesartan
2
0
-10
-20
-30
-40
To a solution of nitrile 6c (3.75 g, 9.90 mmol) in 60 mL of DMSO
was added hydroxylamine hydrochloride (2.76 g, 39.70 mmol) and
TEA (10.1 mL, 61.20 mmol). The mixture was stirred for 16 h at
90 °C. After cooling to rt, the mixture was diluted with water,
extracted with ethyl acetate, dried, the solvent was removed under
reduced pressure. The residue was purified by flash column chro-
matograph to afford a white solid. Yield 34.4%. 1H NMR (CDCl3,
300 MHz) d: 7.71–7.15 (8H, m, Ph-H), 4.99 (d, J = 15.3 Hz), 4.92
(1H, d, J = 10.3 Hz, –CO–CH–N–), 4.76 (m), 4.26 (d, J = 15.3 Hz),
4.04 (2H, d, J = 10.8 Hz –N–CH2–), 4.43 (2H, s, –NH2), 3.39 (s),
3.36 (3H, s, –OCH3), 2.62–2.25 (3H, m, –CO–CH2–, –CHCH3),
1.27–0.86 (9H m,); 13C NMR (CDCl3, 125 MHz) d: 178.9, 175.0,
169, 141.1, 139.9, 139.0, 136.8, 135.4, 133.6, 133.6, 132.5, 132.5,
132.2, 132.2, 131.4, 68.8, 55.8, 55.4, 30.2, 29.9, 21.9, 21.9, 15.0.
LRMS (m/z): 410[Mꢁ1]ꢁ; ESI-MS: 411.2152, C23H29N3O4.
control
0
2
4
6
8
10
12
time(hour)
Compound 2 30mg/kg
4.15. N-Valeryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
Fig. 3A (continued)
methyl]-L-Valine methyl ester (7a)
4.10. N-Valeryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L
-Isoleucine
Compound 7a was prepared according to the procedure
described for the preparation of 7c. Yield 75.5%. 1H NMR (CDCl3,
500 MHz) d: 7.58 (m, 1H, Ph-H), 7.57–7.37 (5H, m, Ph-H), 7.21
(2H, m, Ph-H), 5.02 (d, J = 15.3 Hz), 4.92 (d, 1H, J = 10.3 Hz, –CO–
CH–N–), 4.67 (m), 4.30 (d, J = 15.3 Hz), 4.07 (2H, d, J = 10.8 Hz –
N–CH2–), 4.43 (2H, s, –NH2), 3.46 (s), 3.40 (3H, s, –OCH3),
2.55–2.24 (3H, m, –CO–CH2–, –CHCH3), 1.82–1.58 (2H, m, –
CH2CH2CH3), 1.48–1.25 (2H, m, –CH2CH2CH3), 1.03–0.82 (9H, m);
13C NMR (CDCl3, 125 MHz) d: 178.2, 177.0, 169, 141.1, 139.9,
139, 136.8, 135.4, 133.6, 133.6, 132.5, 132.5, 132.2, 132.2, 131.4,
68.8, 55.8, 55.4, 36.8, 31.5, 30.2, 27.1, 21.1, 21.9, 21.9, 18.3. LRMS
(m/z): 440.3[M+1]+; HR-MS: 439.2446, C25H33N3O4.
methyl ester (6d)
Compound 6d was prepared according to the procedure
described for the preparation of 6b. Yield 74.4%. The spectral data
were consistent with that reported in the literature.8
4.11. N-Butyryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Isoleucine
methyl ester (6e)
Compound 6e was prepared according to the procedure de-
scribed for the preparation of 6b. Yield 75.2%. The spectral data
were consistent with that reported in the literature.8
4.12. N-Valeryl-N-((20-cyanobiphenyl-4-yl)methyl)-
L-Phenylal
anine methyl ester (6f)
4.16. N-Butyryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
methyl]-L-Valine methyl ester (7b)
Compound 6f was prepared according to the procedure described
for the preparation of 6b. Yield 75.5%, 1H NMR (CDCl3, 500 MHz) d:
7.77 (1H, m, Ph-H), 7.50–7.43 (4H, m, Ph-H), 7.31–7.08 (8H, m,
Ph-H) 4.50 (1H, d, J = 17.1 Hz, –CO–CH–N–), 4.37 (m), 3.90 (2H, d,
J = 17.2 Hz, –N–CH2–), 3.67 (3H, s, –OCH3), 3.40–3.36 (m), 3.29–
3.24 (2H, m, –N–CHCH2), 2.35–2.25 (2H, m, –CO–CH2–), 1.63 (2H,
m, –CH2CH2CH2–), 1.35–1.24 (m, 2H, –CH2CH2CH3), 0.85 (3H, t,
J = 7.4 Hz); 13C NMR (CDCl3, 125 MHz) d: 178.2, 177.0, 145.2,
145.1, 141.1, 139.9, 138.3, 137.5, 133.6, 133.6, 133.4, 133.4, 133.1,
133.1, 132.9, 132.9, 132.2, 132.2, 130.7, 121.5, 116.4, 64.1, 55.5,
Compound 7b was prepared according to the procedure
described for the preparation of 7c. Yield 75.5%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
7.68–7.15 (8H, m, Ph-H), 5.03 (d, J = 15.3 Hz), 4.97 (1H, d,
J = 10.8 Hz, –N–CH–), 4.45 (2H, s, –NH2), 4.75–4.57 (m), 4.28 (d,
J = 15.3 Hz), 4.06 (d, J = 10.8 Hz, –CH2–N–), 3.45 (s), 3.39 (3H, s, –
OCH3), 2.65–2.21 (3H, m, –CO–CH2–, –CH–CH3), 1.70 (2H, m),
1.07–0.91 (9H, m); 13C NMR (CDCl3, 125 MHz) d: 173.6, 170.8,
153.0, 139.8, 139.4, 137.9, 135.4, 131.4, 130.1, 129.9, 129.6, 129.1,

2754
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
valsartan
irbesartan
1b
valsartan
irbesartan
1a
0
control
0
-10
-20
-30
-40
control
-10
-20
-30
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1a 15mg/Kg
Compound 1b 15mg/Kg
valsartan
irbesartan
1b
5
0
valsartan
irbesartan
1a
5
0
control
control
-5
-5
-10
-15
-20
-25
-30
-35
-40
-10
-15
-20
-25
-30
-35
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1a 30mg/Kg
Compound 1b 30mg/Kg
Figure 3B. Systolic Blood Pressure (SPB) and diastolic blood pressure (DBP) development during the experiment. Compounds 1a, 1b, 1c, 1e (15 mg/kg and 30 mg/kg) and 2
(30 mg/kg) also caused obvious reduction in DBP of SHRs.
128.9, 128.6, 128.3, 127.4, 127.1, 126.5, 60.7, 51.9, 51.8, 35.3, 35.1,
NMR spectra are complicated due to amide rotomers. 1HNMR
(CDCl3, 500 MHz) d: 11.08 (1H, s), 7.29–7.89 (8H, m), 6.90 (2H, s),
4.46 (2H, s), 4.41 (1H, d), 3.68 (3H, s), 2.53 (1H, m), 2.05 (2H, t),
1.55–1.68 (4H, m), 1.11 (3H, d), 0.90 (6H, t). 13C NMR (CDCl3,
125 MHz) d: 178.2, 177.0, 169, 131.1, 139.9, 139.0, 136.8, 135.4,
133.6, 133.6, 132.5, 132.5, 132.2, 132.2, 131.2, 66.4, 55.8, 55.4,
37.3, 35.9, 29.6, 24.1, 19.4, 18.0, 16.2. ESI-MS (m/z):
440.25[M+1]+, C25H34N3H4.
29.5, 18.4, 14.0, 13.7; ESI-MS (m/z): 426.2[M+1]+, 448.3[M+Na]+.
4.17. N-Valeryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
methyl]-L-Isoleucine methyl ester (7d)
Compound 7d was prepared according to the procedure described
for the preparation of 7c. Yield 46.5%. Mp 98.2–100.6 °C. NMR spectra
are complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
11.08 (1H, s), 7.29–7.89 (8H, m), 6.90 (2H, s), 4.46 (2H, s), 4.41 (1H, d),
3.68 (3H, s), 2.53 (1H, m), 2.05 (2H, t), 1.31–1.55 (6H, m), 1.11 (3H, d),
0.90 (6H, t). 13C NMR (CDCl3, 125 MHz) d: 178.2, 177.0, 169, 141.1,
139.9, 139.0, 136.8, 135.4, 133.6, 133.6, 132.5, 132.5, 132.2, 132.2,
131.4, 66.3, 55.8, 55.4, 36.8, 35.9, 33.5, 29.5, 27.1, 19.4, 18.3, 16.2.
ESI-MS (m/z): 454.26[M+1]+.
4.19. N-Valeryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
methyl]-L-Phenylalanine methyl ester (7f)
Compound 7f was prepared according to the procedure described
for the preparation of 7c. Yield 46.5%. NMR spectra are complicated
due to amide rotomers.1H NMR (CDCl3, 300.75 MHz) d: 7.57–7.13
(13H, m, Ph-H), 4.47–4.40 (4H, m, –CO–CH–N–, –NH2, –N–CH2–),
3.90 (1H, d, J = 16.9 Hz, –N–CH2–), 3.65 (3H, s, –OCH3), 3.42–3.24
(2H, m, –N–CHCH2), 2.31–2.28 (2H, m, –CO–CH2–), 1.64–1.60 (2H,
m, –CH2CH2CH3), 1.34–1.30 (2H, m, –CH2CH2CH3), 1.05–0.87 (3H,
m); 13C NMR (CDCl3, 125 MHz) d: 178.2, 177.0, 169, 145.2, 141.1,
4.18. N-Butyryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-yl)
methyl]-L-Isoleucine methyl ester (7e)
Compound 7e was prepared according to the procedure de-
scribed for the preparation of 7c. Yield 43.2%. Mp 97.5–99.7 °C.

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2755
valsartan
irbesartan
1c
valsartan
irbesartan
1e
0
-10
-20
-30
-40
control
0
-10
-20
-30
-40
control
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1e 15mg/Kg
Compound 1c 15mg/Kg
valsartan
irbesartan
1c
valsartan
irbesartan
1e
5
0
5
0
control
control
-5
-5
-10
-15
-20
-25
-30
-35
-40
-10
-15
-20
-25
-30
-35
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 1e 30mg/Kg
Compound 1c 30mg/Kg
Fig. 3B (continued)
139.9, 139.0, 136.8, 136.0, 135.4, 133.6, 133.6, 133.4, 133.4, 132.9,
132.9, 132.5, 132.5, 132.2, 132.2, 131.2, 131.4, 64.1, 55.5, 55.4,
36.8, 33.5, 27.1, 18.3. ESI-MS (m/z): 488.3[M+1]+, 510.3[M+Na]+;
HR-MS: 487.2468, C29H33N3O4.
4.21. N-propionyl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamim
idoyl)biphenyl-4-yl)methyl]- -Valine methyl ester (8c)
L
To an ice-bath cooled solution of hydroxide 7c (1.81 g,
4.4 mmol) and pyridine (0.43 mL, 5.30 mmol) dissolved in 20 mL
of DMF was added dropwise isobutyl carbonochloridate (0.53 mL,
4.40 mmol). After stirring for 2 h, the mixture was diluted with
water and extracted with ethyl acetate. The organic phase was
dried over MgSO4. After filtration, the solvent was removed under
reduced pressure. The residue was purified by flash column chro-
matography to afford a white solid. Yield 87.4%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3,
300.75 MHz) d: 7.64–7.15 (8H, m, Ph-H), 5.01–4.96 (1H, m, –CO–
CH–N–), 4.69–4.64 (m), 4.28 (2H, d, J = 15.6 Hz, –N–CH2–), 4.11–
4.00 (m, 2H, –OCH2–), 3.44 (s), 3.37 (3H, s, –OCH3), 2.38–2.25
(2H, m, –CHCH3 ꢃ 2), 2.03–1.99 (2H, m, –CO–CH2–), 1.27–0.83
(m, 15H); 13C NMR (CDCl3, 125 MHz) d: 178.9, 177.0, 169, 156.5,
141.1, 139.9, 139.0, 136.8, 135.4, 133.6, 133.6, 132.5, 132.5,
132.2, 132.2, 131.4, 84.6, 68.8, 55.8, 55.4, 32.8, 30.2, 29.9, 23.5,
23.5, 21.9, 21.9, 15.0. ESI-MS (m/z): 512[M+1]+.
4.20. N-Butyryl-N-[(20-(hydroxycarbamimidoyl)biphenyl-4-
yl)methyl]-L-Phenylalanine methyl ester (7g)
Compound 7g was prepared according to the procedure
described for the preparation of 7c. Yield 46.5%. NMR spectra are
complicated due to amide rotomers.1H NMR (CDCl3, 300.75 MHz)
d: 7.58–7.19 (13H, m, Ph-H), 4.98 (d, J = 16.0 Hz), 4.97 (1H, d,
J = 10.7 Hz, –CO–CH–N–), 4.66 (m), 4.31 (d), 4.05 (2H, d,
J = 17.9 Hz, –N–CH2–), 3.63 (3H, s, –OCH3), 3.40–3.20 (2H, m, –N–
CHCH2), 2.61–2.18 (m, 2H, –CO–CH2–), 1.69–1.61 (2H, m, –
CH2CH2CH3), 1.04–0.86 (3H m,); 13C–NMR (CDCl3, 125 MHz) d:
171.2, 153.4, 140.0, 139.4, 138.7, 136.6, 131.6, 130.3, 129.9,
129.8, 128.8, 128.4, 127.6, 127.4, 126.1, 77.3, 77.0, 76.7, 65.8,
61.6, 51.8, 51.6, 48.1, 46.2, 45.4, 35.7, 35.5, 30.9, 27.7, 27.5, 19.9,
18.8, 18.7, 13.9, 11.5; ESI-MS (m/z): 474.2[M+1]+, 496.3[M+Na]+.

2756
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
valsartan
irbesartan
2
valsartan
irbesartan
1d
0
-10
-20
-30
-40
0
-10
-20
-30
-40
control
1g
1f
control
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 2 15mg/Kg
Compound 1d 1f 1g(15mg/Kg)
valsartan
irbesartan
2
valsartan
irbesartan
1d
5
0
5
0
control
1g
1f
control
-5
-5
-10
-15
-20
-25
-30
-35
-40
-10
-15
-20
-25
-30
-35
-40
0
2
4
6
8
10
12
0
2
4
6
8
10
12
time(hour)
time(hour)
Compound 2 30mg/Kg
Compound 1d 1f 1g(15mg/Kg)
Fig. 3B (continued)
Figure 3C. Systolic Blood Pressure (SPB) and diastolic blood pressure (DBP)
development during the experiment. No significant alteration between the negative
control group and the groups treated with 15 mg/kg (ig) of compounds 1d, 1f, 1g
(⁄P <0.05; +P <0.01; versus control; &: P <0.05 vs irbesartan).
4.22. N-Valeryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamim
idoyl)biphenyl-4-yl)methyl]- -Valine methyl ester (8a)
L
55.8, 55.4, 36.8, 33.5, 32.8, 30.2, 27.1, 23.5, 23.5, 21.9, 21.9, 18.3.
ESI-MS (m/z): 540.4[M+1]+, 562.4[M+Na]+.
Compound 8a was prepared according to the procedure
described for the preparation of 8c. Yield 87.6%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
7.64–7.20 (8H, m, Ph-H), 4.97–4.94 (1H, m, –CO–CH–N–),
4.65–4.61 (m), 4.07–4.04 (2H, m, –N–CH2–), 4.02 (2H, d,
J = 6.8 Hz, –OCH2–), 3.45 (s), 3.39 (3H, s, –OCH3), 2.48–2.02 (4H,
m, –CO–CH2–, –CHCH3 ꢃ 2), 1.78–1.60 (2H, m, –CH2CH2CH3), 1.32
(2H, m, –CH2CH2CH3), 1.03–0.84 (15H, m); 13C NMR (CDCl3,
125 MHz) d: 178.2, 177.0, 169, 156.5, 141.1, 139.9, 139.0, 136.8,
135.4, 133.6, 133.6, 132.5, 132.5, 132.2, 132.2, 131.4, 84.6, 68.8,
4.23. N-Butyryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamimidoyl)
biphenyl-4-yl)methyl]-L-Valine methyl ester (8b)
Compound 8b was prepared according to the procedure de-
scribed for the preparation of 8c. Yield 87.6%. NMR spectra are com-
plicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d: 7.65–
7.19 (8H, m, Ph-H), 4.98–4.96 (1H, m, –CO–CH–N–), 4.66–4.62 (m),
4.31 (d, J = 15.4 Hz, –N–CH2–), 4.07–4.03 (2H, m, –OCH2), 3.46 (s),
3.40 (3H, s, –OCH3), 2.63–2.03 (4H, m, –CO–CH2–, –CHCH3 ꢃ 2),
Table 3
Acute toxicity test of compound 1c (ig) determined by median lethal dose (LD50
)
Compound
Dose (mg/kg)
Mortality (%)
LD50 (mg/kg)
2573.2
95% confidence interval (mg/kg)
2167.1–3061.5
1b
1000.0
1710.0
2236.1
2924.0
3823.6
5000.0
0
30
50
60
80
100

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2757
1.84–1.65 (2H, m, –CH2CH3), 1.05–0.85 (15H, m); 13C NMR (CDCl3,
4.28. N-Propionyl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]- -Valine methyl ester (9c)
125 MHz) d: 174.4, 171.2, 157.2, 140.0, 138.8, 136.9, 130.6, 130.3,
129.9, 128.8, 128.4, 127.6, 127.4, 126.2, 77.3, 77.0, 76.7, 74.4, 61.7,
51.8, 51.5, 48.2, 35.7, 35.5, 27.8, 27.7, 27.5, 19.9, 18.9, 18.7, 13.9,
13.8; LRMS (m/z): 526.5[M+1]+, 548.4[M+Na]+.
L
To 10 mL of xylene dried over anhydrous calcium dichloride
was added 8c (1.46 g, 2.85 mmol). The solution was stirred over
reflux for 5 h. The solvent was removed under reduced pressure
to afford a yellow oil. The residue was recrystallized to afford the
4.24. N-Valeryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamimidoyl)
biphenyl-4-yl)methyl]-L-Isoleucine methyl ester (8d)
product as
a
white crystal. Yield 78.7%. 1H NMR (CDCl3,
300.75 MHz, doubling due to amide rotamers) d: 9.26 (1H, s, –
NH–), 7.76–7.18 (8H, m, Ph-H), 4.88–4.83 (1H, m, –CO–CH–N–),
4.63 (s), 4.27 (d, J = 15.6 Hz), 4.02 (2H, d, J = 10.5 Hz, –N–CH2–),
3.43 (s), 3.41 (3H, s, –OCH3), 2.57–2.03 (3H, m, –CO–CH2–, –
Compound 8d was prepared according to the procedure
described for the preparation of 8c. Yield 85.6%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
7.29–7.89 (8H, m), 6.90 (2H, s), 4.46 (2H, s), 4.41 (1H, d), 3.94
(2H, d), 3.68 (3H, d), 2.53 (1H, m), 1.97 (1H, m), 1.31–1.55 (6H,
m), 1.11 (3H, d), 0.91 (6H, d), 0.90 (6H, t). 13C NMR (CDCl3,
125 MHz) d: 178.2, 177.0, 169, 156.5, 141.1, 139.0, 136.8, 135.4,
133.6, 133.6, 132.5, 132.5, 132.2, 131.4, 84.6, 66.9, 55.8, 55.4,
36.8, 35.9, 32.8, 29.6, 27.1, 23.5, 23.5, 19.4, 18.3, 16.2. ESI-MS (m/
z): 554.32[M+1]+.
CHCH3), 1.15–0.82 (9H, m);ESI-MS (m/z): 438[M+1]+, 460[M+Na]+
.
4.29. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Valine methyl ester (9a)
Compound 9a was prepared according to the procedure
described for the preparation of 9c. Yield 78.3%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
9.60 (s, 1H, –NH–), 7.70–7.16 (m, 8H, Ph-H), 4.85–4.80 (m, 1H, –
CO–CH–N–), 4.63–4.62 (m), 4.27 (d, J = 15.4 Hz), 4.12 (d,
J = 10.1 Hz, –N–CH2–), 3.49 (s), 3.43 (3H, s, –OCH3), 2.40–2.34
(3H, m, –CO–CH2–, –CHCH3), 1.67–1.58 (2H, m, –CH2CH2CH3),
1.34–1.25 (2H, m, –CH2CH2CH3), 1.03–0.82 (9H, m); 13C NMR
(CDCl3, 125 MHz) d: 174.8, 174.6, 170.9, 170.1, 159.6, 159.5,
157.9, 157.7, 141.2, 141.0, 137.9, 137.8, 137.1, 131.8, 130.9,
129.7, 129.0, 128.6, 127.8, 127.6, 127.5, 126.1, 121.9, 65.9, 61.8,
51.9, 51.6, 48.1, 45.4, 33.2, 27.6, 27.5, 27.4, 27.1, 22.3, 22.2, 19.7,
18.5, 13.7; ESI-MS (m/z): 466.5[M+1]+, 488.4[M+Na]+.
4.25. N-Butyryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamimidoyl)
biphenyl-4-yl)methyl]-L-Isoleucine methyl ester (8e)
Compound 8e was prepared according to the procedure
described for the preparation of 6c. Yield 84.9%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500.12 MHz)
d: 7.29–7.89 (8H, m), 6.90 (2H, s), 4.46 (2H, s), 4.41 (1H, d), 3.94
(2H, d), 3.68 (3H, d), 2.53 (1H, m), 2.05 (2H, t), 1.97 (1H, m), 1.55–
1.68 (4H, m), 1.11 (3H, d), 0.91 (6H, d), 0.90 (6H, t). 13C NMR (CDCl3,
125 MHz) d: 178.2, 177.0, 169, 156.5, 141.1, 139.9, 136.8, 135.4,
133.6, 133.6, 132.5, 132.5, 132.2, 132.2, 131.4, 84.6, 66.3, 55.8,
55.4, 39.3, 35.9, 29.6, 32.8, 29.6, 24.1, 23.5, 23.5, 19.4, 18.0, 16.2.
ESI-MS (m/z): 540.30[M+1]+
4.30. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Valine methyl ester (9b)
Compound 9b was prepared according to the procedure de-
scribed for the preparation of 9c. Yield 78.3%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
9.43 (1H, s, –NH–), 7.76–7.19 (8H, m, Ph-H), 4.87–4.86 (1H, m, –
CO–CH–N–), 4.72–4.05 (2H, m, –NCH2), 3.48–3.42 (3H, m, –
OCH3), 2.58–2.17 (3H, m, –CO–CH2–, –CHCH3), 1.84–1.65 (2H, m,
–CH2CH3), 1.04–0.92 (m, 9H); 13C NMR (CDCl3, 125 MHz) d:
174.5, 174.4, 171.1, 170.3, 159.4, 159.3, 157.7, 157.6, 141.1,
140.9, 138.5, 137.7, 137.6, 137.0, 132.0, 131.9, 130.9, 129.8,
129.1, 128.7, 128.1, 127.9, 127.7, 126.4, 121.9, 66.0, 61.8, 52.0,
51.7, 48.1, 45.6, 35.6, 35.4, 27.7, 19.8, 19.7, 18.8, 18.7, 18.6, 13.9,
13.8; ESI-MS (m/z): 452.2[M+1]+, 474.2[M+Na]+.
4.26. N-Valeryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamimidoyl)
biphenyl-4-yl)methyl]-L-Phenylalanine methyl ester (8f)
Compound 8f was prepared according to the procedure
described for the preparation of 8c. Yield 85.6%. 1H NMR (CDCl3,
500 MHz) d: 7.76–7.14 (13H, m, Ph-H), 4.49 (1H, d, J = 17.1 Hz, –
CO–CH–N–), 4.39 (m), 3.88 (2H, d, J = 17.0 Hz, –N–CH2–), 4.02 (d,
2H, J = 6.8 Hz, –O–CH2–), 3.65 (3H, s, –OCH3), 3.41–3.33 (m),
3.26–3.19 (2H, m, –N–CH–CH2), 2.33–2.27 (2H, m, –CO–CH2–),
2.02 (1H, m, –CHCH3), 1.62 (2H, m, –CH2CH2CH3), 1.36–1.24 (2H,
m, –CH2CH2CH3), 0.95–0.87 (9H, m); ESI-MS (m/z): 588.4[M+1]+,
610.4[M+Na]+.
4.31. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Isoleucine methyl ester (9d)
4.27. N-Butyryl-N-[(20-(N0-(isobutoxycarbonyloxy)carbamimidoyl)
biphenyl-4-yl)methyl]-L-Phenylalanine methyl ester (8g)
Compound 9d was prepared according to the procedure
described for the preparation of 9c. Yield 71.8%. 1H NMR (CDCl3,
500 MHz) 7.29–7.89 (8H, m), 4.46 (2H, s), 4.41 (1H, d), 3.68 (3H,
s), 2.53 (1H, m), 2.05 (2H, t), 2.0 (1H, s), 1.31–1.55 (6H, m), 1.11
(3H, d), 0.90 (6H, t). ESI-MS (m/z): 479. 6[M+1]+.
Compound 8g was prepared according to the procedure de-
scribed for the preparation of 8c. Yield 85.6%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500 MHz) d:
7.76–7.14 (13H, m, Ph-H), 4.50 (d, J = 17.0 Hz, –CO–CH–N–),
4.40–4.37 (m), 3.90 (2H, d, J = 16.9 Hz, –N–CH2–), 4.02 (2H, d,
J = 6.7 Hz, –OCH2–), 3.66 (3H, s, –OCH3), 3.42–3.35 (m), 3.26–3.21
(2H, m, –N–CH–CH2), 2.30–2.26 (2H, m, –CO–CH2–), 2.04–2.00
(1H, m, –CHCH3), 1.70–1.64 (m, 2H, –CH2CH2CH3), 0.98–0.86 (9H,
m); 13C NMR (CDCl3, 125 MHz) d: 173.7, 170.9, 157.2, 153.9,
145.8, 140.0, 139.0, 138.0, 136.0, 130.8, 130.6, 130.3, 129.8,
129.1, 128.8, 128.5, 128.0, 127.6, 127.3, 119.8, 65.2, 61.0, 52.0,
51.9, 42.0, 35.5, 35.3, 31.9, 29.7, 27.8, 23.8, 22.7, 18.9, 18.5, 15.4,
13.8, 10.9; ESI-MS (m/z): 574.5[M+1]+, 596.5[M+Na]+.
4.32. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Isoleucine methyl ester (9e)
Compound 9e was prepared according to the procedure de-
scribed for the preparation of 9c. Yield71.8%. 1H NMR (CDCl3,
500 MHz)7.29–7.89 (8H, m), 4.46 (2H, s), 4.41 (1H, d), 3.68 (3H,
s), 2.53 (1H, m), 2.05 (2H, t), 2.0 (1H, s), 1.55–1.68 (6H, m), 1.11
(3H, d), 0.90 (6H, t). ESI-MS (m/z): 465.54[M+1]+.

2758
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
4.33. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]- -Phenylalanine methyl ester (9f)
4.37. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]- -Valine (1b)
L
L
Compound 9f was prepared according to the procedure de-
scribed for the preparation of 9c. Yield 78.3%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 500.12 MHz)
d: 7.78–7.15 (m, 13H, Ph-H), 4.64–4.44 (m, 1H, –CO–CH–N–), 4.27
(m), 3.75 (2H, d, J = 17.1 Hz, –N–CH2–), 3.64 (3H, s, –OCH3),
3.35–3.24 (2H, m, –N–CH–CH2), 2.31 –2.21 (2H, m, –CO–CH2–),
1.65–1.59 (2H, m, –CH2CH2CH3), 1.35–1.30 (2H, m, –CH2CH2CH3),
0.92–0.85 (3H m,); 13C NMR (CDCl3, 125 MHz) d: 175.9, 174.9,
172.5, 171.8, 160.7, 157.9, 157.7, 140.9, 140.7, 138.5, 137.9, 136.5,
136.3, 132.1, 131.9, 130.7, 130.5, 129.7, 129.4, 129.2, 128.6, 128.3,
127.9, 127.8, 127.0, 121.9, 121.4, 68.0, 65.2, 60.3, 38.5, 33.6, 33.2,
29.5, 28.7, 27.2, 26.8, 22.8, 27.2, 27.1, 26.8, 22.8, 22.3, 19.6, 19.5,
18.7, 18.4, 14.0, 13.7, 13.6; ESI-MS (m/z): 514.3[M+1]+, 536.3
[M+Na]+
Compound 1b was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 85.6%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 9.96 (d, J = 7.8 Hz),
9.17 (d, 1H, J = 7.6 Hz), 7.66–7.56 (m, 2H, CM, Cm, 7.46–7.41 (m,
2H, CM, Cm), 7.25–7.23 (m, 1H, CM, Cm), 7.21–7.18 (m, 2H, CM,
Cm), 7.14–7.12 (m, 1H, CM, Cm), 5.24–5.20 (d, J = 15.2HZ, 1H,
CM, Cm), 4.70–3.96 (2H, m, CM, Cm), 2.61–2.30 (m, 3H, CM, Cm),
1.67–1.52 (m, 2H), 1.52–0.0.81 (m, 9H); 13C NMR (CDCl3,
100 MHz) d: 176.0, 175.2, 172.2, 160.9, 160.3, 158.2, 141.0, 138.2,
136.8, 136.4, 132.4, 132.2, 130.8, 129.5, 129.0, 128.2, 127.3,
122.1, 121.6, 66.5, 65.5, 65.4, 51.6, 45.2, 35.9, 35.7, 27.4, 20.0,
18.8, 18.7, 14.0; ESI-MS (m/z): 438.2[M+1]+, 460.2[M+Na]+. HR-
MS: 460.1835.
4.34. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
4.38. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Phenylalanine methyl ester (9g)
yl)biphenyl-4-yl)methyl]-L-Isoleucine (1d)
Compound 9g was prepared according to the procedure de-
scribed for the preparation of 9c. Yield 78.7%. 1H NMR (CDCl3,
500.12 MHz) d: 9.26 (1H, s, –NH–), 7.76–7.18 (8H, m, Ph-H),
4.88–4.83 (1H, m, –CO–CH–N–), 4.63 (s), 4.27 (d, J = 15.6 Hz),
4.02 (2H, d, J = 10.5 Hz, –N–CH2–), 3.43 (s), 3.41 (s, 3H, –OCH3),
2.57–2.03 (m, 3H, –CO–CH2–, –CHCH3), 1.15–0.82 (m, 9H); ESI-
MS (m/z): 438[M+1]+, 460[M+Na]+
Compound 1d was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 87.2%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 10.08–9.44 (m, 2H,),
7.64–7.55 (m, 2H, CM, Cm), 7.44–7.39 (m, 2H, CM, Cm), 7.24–
7.21 (m, 4H, CM, Cm), 5.25–4.68 (d, J = 15.4 Hz, 1H, –CM, Cm,
4.53–4.03 (m, 2H, CM, Cm), 2.58–2.33 (m, 2H, CM, Cm), 2.10–
1.93 (m, 1H, CM, Cm), 1.57–1.45 (m, 2H, CM, Cm), 1.36–1.27 (m,
2H, CM, Cm) 1.25–1.20 (m, 2H, CM, Cm) 0.89–0.78 (m, 9H, (m,
2H CM, Cm); 13C NMR (CDCl3, 100 MHz) d: 176.5, 175.5, 173.3,
172.2, 160.7, 160.2, 158.1, 158.0, 141.1, 140.9, 138.5, 138.1,
136.8, 136.4, 129.9, 128.8, 126.9, 121.9, 121.5, 64.6, 51.0, 33.8,
33.4, 33.3, 27.3, 25.1, 24.8, 22.4, 22.3, 15.9, 15.7, 14.9, 13.9, 13.8,
11.2, 11.0, 10.9; ESI-MS (m/z): 466.4[M+1]+, 488.4[M+Na]+. HR-
MS: 488.2155.
4.35. N-Propionyl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Valine (1c)
To a solution of 9c (0.96 g, 2.20 mmol) dissolved in DMF was
added 10 mL of 2 M aqueous sodium hydroxide and slight amount
of methanol. The mixture was stirred for 3 h at 70 °C. The pH value
of the mixture was adjusted to 3 by careful addition of 2 M aque-
ous hydrochloride. The mixture was extracted with ethyl acetate,
and the organic phase was dried over MgSO4. After filtration, the
residue was purified by flash column chromatograph to afford a
white solid. Yield 16.3%. Mp: 79.1–82.4 °C. 1H NMR (CDCl3,
300.75 MHz, doubling due to amide rotamers) (CM: major rot-
amer; Cm: minor rotamer) d: 7.98–7.20 (m, 8H, CM, Cm), 4.70–
4.58 (d, J = 15.0 Hz, 1H, CM, Cm), 4.22–4.10 (m), 3.98–3.71 (2H,
m, CM, Cm), 2.81–2.42 (m, 3H, CM, Cm), 1.23–0.96 (m, 9H, CM,
4.39. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Isoleucine (1e)
Compound 1e was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 83.6%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 10.15–9.44 (br s,
2H), 7.67–7.54 (m, 2H, CM, Cm), 7.38–7.37 (m, 2H, CM, Cm),
7.20–7.08 (m, 4H, CM, Cm), 4.94–4.91 (d, J = 16.2 Hz, 1H, CM,
Cm), 4.50–4.01 (m, 2H, CM, Cm) 2.26 (m, 2H, CM, Cm), 2.08 (m,
2H, CM, Cm) 1.58–1.48 (m, 2H, CM, Cm), 1.33 (m, 2H, CM, Cm),
0.87–0.77 (m, 9H, CM, Cm); 13C NMR (CDCl3, 100 MHz) d: 176.23,
176.0, 175.4, 173.4, 173.2, 160.7, 160.2, 158.1, 158.0, 141.2,
141.0, 138.4, 138.0, 136.8, 136.4, 136.3, 132.2, 132.1, 130.8,
129.9, 129.3, 128.8, 128.3, 127.9, 127.0, 126.9, 122.0, 121.6, 64.1,
53.6, 51.0, 45.5, 35.8, 35.7, 35.6, 33.6, 33.3, 31.0, 26.5, 25.1, 24.8,
18.7, 15.9, 15.8, 14.9, 14.6, 13.9, 13.7, 11.2, 11.0; ESI-MS (m/z):
452.3[M+1]+, 474.3[M+Na]+. HR-MS: 474.2009.
Cm) ESI-MS (m/z): 424[M+1]+, 446[M+Na]+
.
4.36. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Valine (1a)
Compound 1a was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 78.7%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 8.14 (br s, 1H),
7.56–7.52 (m, 2H, CM, Cm), 7.41–7.39 (m, 1H, CM, Cm), 7.35–
7.33 (m, 1H, CM, Cm), 7.25–7.23 (m, 1H, CM, Cm), 4.76–4.60 (d,
J = 17.2 Hz, 1H, CM, Cm), 4.02–3.73 (m, 2H, CM, Cm), 2.75–2.55
(m, 2H, CM, Cm) 1.77 (m, 2H, CM, Cm), 1.50–1.43 (M, 2H, CM,
Cm), 1.27 (M, 1H, CM, Cm), 1.01–0.96 (9H, m, CM, Cm); IR (KBr,
cmꢁ1): 3432.21, 2962.79, 2873.10, 1775.81, 1620.17, 1466.11,
1207.30, 945.89, 762.93, 660.77, 577.82. 13C NMR (CDCl3,
125 MHz) d: 175.9, 174.9, 172.5, 171.8, 160.7, 160.1, 157.9, 141.0,
138.6, 137.9, 136.5, 132.1, 131.9, 130.8, 130.6, 129.8, 129.4,
129.2, 128.6, 128.3, 127.9, 127.8, 126.9, 121.9, 121.4, 68.0, 65.2,
60.3, 38.6, 33.6, 33.2, 29.5, 28.8, 27.2, 27.1, 26.8, 22.8, 22.3, 22.2,
19.6, 19.5, 18.8, 18.4, 14.0, 13.7, 13.6 ESI-MS (m/z): 452.3[M+1]+,
474.3[M+Na]+; HR-MS: 474.2007.
4.40. N-Valeryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Phenylalanine (1f)
Compound 1f was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 89.5%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 7.74–7.72 (m, 1H,
CM, Cm), 7.64–7.60 (m, 1H, CM, Cm), 7.51–7.43 (m, 2H, CM, Cm),
7.32–7.25 (m, 5H, CM, Cm), 7.18–7.13 (m, 4H, CM, Cm), 4.44–
4.40 (d, J = 16.8HZ, 1H, CM, Cm), 4.17 (m, 1H, CM, Cm), 3.57–3.52

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2759
(m, 1H, CM, Cm), 3.27–3.26 (m, 2H, CM, Cm), 2.33–2.28 (m, 2H, CM,
Cm), 1.54–1.50 (m, 2H, CM, Cm)1.31–1.25 (m, 2H, CM, Cm), 0.89–
0.85 (m, 3H, CM, Cm); 13C NMR (CDCl3, 125 MHz,) d: 175.8, 174.9,
174.8, 174.7, 173.5, 160.1, 157.9, 157.8, 140.9, 137.9, 137.5, 135.6,
131.8, 130.6, 129.7, 129.0, 128.9, 128.5, 128.4, 127.7, 127.6, 126.6,
121.7, 61.3, 52.6, 34.5, 33.1, 31.7, 31.2, 29.9, 29.4, 29.2, 29.1, 28.7,
27.0, 26.9, 22.4, 22.1, 20.5, 13.9, 13.5; IR (KBr, cmꢁ1): 3431.01,
3029.60, 2957.90, 2925.82, 2858.36, 1772.16, 1620.14, 1492.83,
1464.04, 1215.21, 947.01, 759.88, 703.20. ESI-MS (m/z):
500.3[M+1]+, 522.4[M+Na]+; HR-MS: 522.2011.
was washed with water for 5 times (50 mL ꢃ 5) and brine for 1
time (50 mL), then organic layer dried with MgSO4, and concen-
trated in vacuo. The product was purified by chromatography with
petroleum ether/ethyl acetate (40:1) as eluant. A yellow solid
3.65 g was obtained. Yield: 50.7%. 1HNMR (400 MHz, CDCl3) d:
7.77 (1H, dd, J1 = 8.8 Hz, J2 = 5.6 Hz, Ph0-H), 7.53–7.48 (4H, m, Ph-
H), 7.22 (1H, dd, J1 = 9.2 Hz, J2 = 2.4 Hz, Ph0-H), 7.14 (1H, dt,
J1 = 2.6 Hz, J2 = 8.0 Hz, Ph0-H), 3.92 and 3.66 (2H, two d,
J = 13.6 Hz, –NH–CH2–), 3.75 (3H, s, –OCH3), 3.06 (1H, d,
J = 6.0 Hz, –CO–CH–N–), 2.00–1.89 (2H, m, –CH(CH3)2, –NH–),
0.99–0.96 (6H, m, CH(CH3)2); ESI-MS (m/z): 341.1[M+H]+.
4.41. N-Butyryl-N-[(20-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-
yl)biphenyl-4-yl)methyl]-L-Phenylalanine (1g)
4.45. (S)-Methyl-2-(N-((20-cyano-50-fluorobiphenyl-4-yl)
methyl)butyramido)-3-methylbutanoate (14)
Compound 1g was prepared according to the procedure de-
scribed for the preparation of 1c. Yield 91.2%. NMR spectra are
complicated due to amide rotomers. 1H NMR (CDCl3, 400 MHz)
(CM: major rotamer; Cm: minor rotamer) d: 7.93–7.77 (m, 1H,
CM, Cm), 7.67–7.60 (m, 1H, CM, Cm), 7.51–7.49 (m, 1H, CM, Cm),
7.44–7.40 (m, 1H, CM, Cm), 7.32–7.30 (m, 2H, CM, Cm), 7.27–
7.24 (M, 3H, CM, Cm), 7.20–7.16 (m, 4H, CM, Cm), 4.44–4.40 (d,
J = 16.8HZ, 1H, CM, Cm), 4.26–4.11 (m, 1H, 4.44–4.40 (d,
J = 16.8HZ, 1H, CM, Cm)), 3.58–3.54 (d, 1H, 4.44–4.40 (d,
J = 16.8HZ, 1H, CM, Cm), 3.66–3.54 (m, 1H, CM, Cm), 3.26 (m, 2H,
CM, Cm), 2.43–2.05 (m, 2H, CM, Cm), 1.59–1.26 (m, 2H, CM, Cm),
0.92–0.86 (m, 3H, CM, Cm); 13C NMR (CDCl3, 100 MHz) d: 174.6,
172.2, 161.5, 157.2, 140.8, 139.6, 136.6, 136.4, 132.5, 132.3,
130.6, 130.5, 129.8, 129.6, 129.5, 128.9, 128.5, 128.3, 128.2, 88.3,
36.3, 35.6, 29.7, 26.6, 26.2, 19.8, 19.4, 18.8, 18.7, 18.4, 14.0, 13.9,
36.3, 35.6, 29.7, 26.6, 26.2, 19.8, 19.5, 18.8, 18.7, 18.4, 14.0, 13.9;
ESI-MS (m/z): 486.1[M+1]+, 508.2[M+Na]+. HR-MS: 508.1849.
To a solution of 12 (3.65 g, 10.72 mmol), DIPEA (1.95 mL,
11.76 mmol) in CH2Cl2 (60 mL) was added n-butyryl chloride
(1.66 mL, 16.04 mmol) at 0 °C, and the mixture was stirred for
6 h at room temperature. Then the reaction solution was poured
into the ice water, the organic layer was washed with diluted
hydrochloric acid, KHCO3 aqueous and brine, dried by MgSO4,
and concentrated in vacuo. The product was purified by chroma-
tography with petroleum ether/ethyl acetate (15:1) as eluant. A
yellow oil 2.80 g (yield 63.6%) was obtained. 1HNMR (400 MHz,
CDCl3) d: 7.78–7.16 (m, 7H, Ph-H), 5.08 and 4.70 (1H, two d,
J1 = 18.4 Hz, J2 = 16.0 Hz, –NCH2–), 4.98 and 4.07 (1H, two d,
J = 10.8 Hz, –N–CH–), 4.65 and 4.28 (1H, two d, J1 = 18.4 Hz,
J2 = 15.6 Hz, –NCH2–), 3.46 and 3.38 (3H, two s, –OCH3), 2.34–
2.25 (3H, m, –CH2CH2CH3 and –CH(CH3)2), 1.71–1.68 (2H, m, –
CH2CH2CH3), 1.05–0.87 (9H, m); ESI-MS (m/z): 433.2[M+H]+.
4.46. (S)-Methyl 2-(N-((50-fluoro-20-(N0-hydroxycarbamimidoyl)
biphenyl-4-yl)methyl) butyramido)-3-methylbutanoate (15)
4.42. 5-Fluoro-40-methylbiphenyl-2-carbonitrile (11)
To
a
solution of 2-bromo-4-fluorobenzonitrile (6.82 g,
A solution of 13 (2.80 g, 6.82 mmol), hydroxylamine hydrochlo-
ride (1.42 g, 20.46 mmol) and DIPEA (3.57 mL, 20.46 mmol) in
EtOH (50 mL) was refluxed for 23 h under nitrogen. After removal
of solvent in vacuo, the residue was dissolved in EtOAc, then
washed with water and brine, dried by MgSO4, and concentrated
in vacuo. The product was purified by chromatography with petro-
leum ether/ethyl acetate (1:1) as eluant. A yellow oil 1.02 g was
obtained. Yield 33.7%. 1HNMR (400 MHz, CDCl3) d: 7.54–7.01 (7H,
m, Ph-H), 5.01 and 4.65 (1H, two d, J1 = 18.4 Hz, J2 = 15.6 Hz, –
NCH2–), 4.95 and 4.07 (1H, two d, J = 10.4 Hz, –N–CH–), 4.63 and
4.28 (1H, two d, J1 = 18.4 Hz, J2 = 15.6 Hz, –NCH2–), 4.46 (2H, d,
J = 8.4 Hz, –NH2–), 3.42 and 3.37 (3H, two s, –OCH3), 2.34–2.25
34.10 mmol), p-tolylboronic acid (5.75 g, 42.29 mmol), PPh3
(1.76 g, 6.33 mmol) and Pd(OAc)2 (28 mg, 0.13 mmol) in 1,4-diox-
ane (96 mL) was added K2CO3 (11.67 g, 84.60 mmol) in 24 mL H2O.
The mixture was refluxed for 4 h under nitrogen. Then the mixture
was diluted with EtOAc, washed with 5% NaOH and brine, dried
with MgSO4, and concentrated in vacuo. Chromatographied with
petroleum ether/ethyl acetate (150:1) to yield the desired product
as white solid (5.84 g, 81.1%). 1HNMR (400 MHz, CDCl3) d: 7.79
(1H, dd, J1 = 8.4 Hz, J2 = 5.2 Hz, Ph0-H), 7.40 (2H, d, J = 8.4 Hz, Ph-
H), 7.29 (2H, d, J = 8.4 Hz, Ph-H), 7.22 (1H, dd, J1 = 9.6 Hz,
J2 = 2.8 Hz, Ph0-H), 7.14–7.10 (1H, m, Ph0-H), 2.44 (3H, s, Ph-CH3).
ESI-MS (m/z): 211.23[M+1]+.
(3H, m, –CH2CH2CH3 and –CH(CH3)2), 1.71–1.68 (m, 2H,
–
CH2CH2CH3), 1.05–0.87 (m, 9H); ESI-MS (m/z): 444.2[M+H]+.
4.43. 40-(Bromomethyl)-5-fluorobiphenyl-2-carbonitrile (12)
4.47. (S)-Methyl-2-(N-((50-fluoro-20-(N0-(isobutoxycarbonyloxy)
carbamimidoyl) biphenyl-4-yl) methyl)butyramido)-3-methyl
butanoate (16)
A solution of 11 (5.83 g, 27.60 mmol), NBS (5.40 g, 30.33 mmol),
AIBN (0.68 g, 4.14 mmol) in CCl4 (100 mL) was refluxed for 3 h,
then the mixture was diluted with CH2Cl2, washed with brine,
dried with MgSO4, and concentrated in vacuo. The product was
purified by chromatography with petroleum ether/ethyl acetate
(150:1) as eluant. A yellow viscous oil 6.25 g was obtained. Yield:
78%. 1HNMR (400 MHz, CDCl3) d: 7.79 (1H, dd, J1 = 8.4 Hz,
J2 = 5.6 Hz, Ph0-H), 7.55 (4H, m, Ph-H), 7.28–7.15 (1H, m, Ph0-H),
4.56 (2H, s, Ph-CH2-Br). ESI-MS (m/z): 290.13[M+1]+.
To
a solution of 15 (1.02 g, 2.30 mmol), DIPEA (0.45 mL,
2.53 mmol) in 50 mL CH2Cl2 was added isobutyl carbonochloridate
at 0 °C, and the mixture was stirred for 4 h at room temperature.
The reaction was washed with water and brine, dried by MgSO4,
and concentrated in vacuo. The product was purified by chroma-
tography with petroleum ether/ethyl acetate (2.5:1) as eluant. A
yellow viscous oil 1.02 g was obtained. Yield 81.6%. 1HNMR
(400 MHz, CDCl3) d: 7.58–7.00 (m, 7H, Ph-H), 4.96 and 4.63 (1H,
two d, J1 = 18.4 Hz, J2 = 15.6 Hz, –NCH2–), 4.89 and 4.06 (1H, two
d, J = 10.4 Hz, –N–CH–), 4.62 and 4.25 (1H, two d, J1 = 18.4 Hz,
J2 = 15.6 Hz, –NCH2–), 3.98 (2H, d, J = 6.8 Hz, –NH2–), 3.41 and
3.36 (two s, 3H, –OCH3), 3.82 (d, 2H, J = 4.0 Hz, –OCH2–CH–),
2.27–1.98 (4H, m, –CH2CH2CH3 and 2 ꢃ –CH(CH3)2), 1.77–1.62
4.44. (S)-Methyl 2-((20-cyano-50-fluorobiphenyl-4-yl)
methylamino)-3-methylbutanoate (13)
A solution of 12 (6.14 g, 21.16 mmol), Valine methyl ester
(7.80 g, 46.52 mmol), DIPEA (10.48 mL, 63.44 mmol) in CH2Cl2
(100 mL) was stirred at 50 °C for 11 h under nitrogen. The mixture

2760
Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
(2H, m, –CH2CH2CH3), 1.00–0.82 (15H, m); ESI-MS (m/z):
0.1 nM. Quiescent and confluent cells were cultured in 24-well
plates. After the cells adhibited to the walls, they were washed
and incubated for 1 h in PBS containing 0.1 nM 125I-Ang II and
new compounds of different concentrations. The final concentra-
tions were 1 ꢃ 10ꢁ6 to 1 ꢃ 10ꢁ12 M. Binding of 125I-Ang II to VSMCs
544.3[M+H]+.
4.48. (S)-Methyl 2-(N-((50-fluoro-20-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)biphenyl-4-yl)methyl) butyramido)-3-methyl
butanoate (17)
was performed at 4 °C for 2.5 h.15 Total reaction volume was 500
lL
consisting of VSMCs, 125I-Ang II and various concentrations of com-
pounds. Nonspecific binding was measured in the presence of
1 ꢃ 10ꢁ6 M and represented 5–10% of total binding.16 The reaction
was terminated by removing the PBS and washing the cells thrice.
The attached cells were dissolved in 0.5 ml of 0.1 M NaOH. The
cells bound by Ang II were quantified by radioactivity counting
in a gamma spectrometer (SN-682, Ri Huan Company, Shanghai).
The IC50 value (concentration for 50% displacement of the specifi-
cally bound 125I-Ang II) was estimated by the linear portion of
the competition curves.
The solution of 16 (1.02 g, 1.88 mmol) in 50 mL xylene was re-
fluxed for 5 h under nitrogen. After the reaction was completed,
the solvent was removed in vacuo, the residue was dissolved in
EtOAc, then washed with water and brine, dried by MgSO4, and
concentrated in vacuo. The product was purified by chromatogra-
phy with petroleum ether/ethyl acetate (1:1) as eluant. A yellow
oil748 mg was obtained. Yield 88.9%. ESI-MS m/z: 468.2[MꢁH]ꢁ.
8.0 (1H, s), 7.20–7.87 (8H, m), 4.46 (2H, s), 4.41 (1H, d), 3.06 (1H,
m), 2.05 (2H, t), 1.68 (2H, m), 0.91 (6H, d), 0.90 (3H, t) ESI-MS
(m/z): 469.51[M+H]+.
4.52. In vivo study of antihypertensive activity
4.49. (S)-2-(N-((50-Fluoro-20-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)biphenyl-4-yl)methyl)butyramido)-3-methylbutanoic acid
(2)
The new compounds were subjected to biological evaluation for
their effects on systolic blood pressure (SBP) and diastolic blood
pressure (DBP) of spontaneous hypertensive rats (SHRs) (6 weeks
old, purchased from Second military medical university, China).
Male SHRs were randomly divided into different experimental
groups of ten animals (negative control group, positive control
groups, compounds 1–8 low-dose groups and high-dose groups).
Each compound was suspended in a 0.5% solution of sodium car-
boxymethyl cellulose and administered orally at the dose of
15 mg/kg and 30 mg/kg separately. Valsartan and irbesartan
(15 mg/kg and 30 mg/kg) were taken as positive controls. The neg-
ative control group was administered the same volume of sodium
carboxymethyl cellulose solution. Blood pressure and heart rate
were monitored at 0–10 h after the administration by a biological
signal analysis system (MPA-2000, Alcott Biotech, China). Ten
determinations were made in every session of blood pressure mea-
surements and the means of ten values were taken as the systolic
blood pressure level and diastolic blood pressure level, respec-
tively. Results of the study were expressed as mean SD. A proba-
bility level of less than 0.05 was considered significant. The
completed animal research here adhered to the ‘Principles of Lab-
oratory Animal Care’ and was approved by IACUC.
A solution of 17 (748 mg, 1.59 mmol) in methanol (21 mL) was
added LiOH (234 mg) in 7 mL H2O. The mixture was refluxed for
4.5 h under nitrogen. After the reaction was completed, the solvent
was removed in vacuo, the residue was dissolved in 15 mL H2O,
then washed with ether. The water layer was added dropwise
1 mol/L hydrochloric acid until the white solid was precipitated.
The precipitate was filtered and was purified by chromatography
with petroleum CH2Cl2/CH3OH (15:1) as eluant. A white solid
230 mg was obtained. Yield: 31.7 %. NMR spectra are complicated
due to amide rotomers. 1HNMR (400 MHz, CDCl3) (CM: major rot-
amer; Cm: minor rotamer) d: 8.31 (brs, 1H), 7.85–8.00 (m, 1H, CM,
Cm), 7.20–7.37 (m, 6H, CM, Cm), 3.75–3.79 and 4.56–4.71 and
5.86–5.91 (m, 2H, CM, Cm), 4.16 (brs, 1H), 3.39 and 3.99 (d, 1H,
J = 11.2 Hz, CM, Cm), 2.69 (m, 1H, CM, Cm), 2.48–2.54 (m, 2H,
CM, Cm), 1.77 (m, 2H, CM, Cm), 0.89–1.05 (m, 9H, CM, Cm). ESI-
MS (m/z): 478.2[M+Na]+. 13C NMR (CDCl3, 125 MHz) d: 174.7,
172.4, 163.5, 161.4, 156.7, 140.0, 137.1, 135.2, 132.3, 131.8,
129.4, 128.8, 128.0, 117.8, 117.5, 117.4, 115.9, 115.6, 64.6, 36.1,
35.6, 26.8, 26.3, 19.8, 18.8, 18.7, 18.5, 14.0, 13.9,
4.50. Cell lines and cell culture
4.53. Toxicity studies
The vascular smooth muscle cells (VSMCs) were obtained from
thoracic aorta of SD rats and cultured by the tissue explants meth-
ods. 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 1 h at 37 °C. The homogenate was centrifuged at
10000 g for 5 min and then incubated with 1 ml of DMEM supple-
mented with 15% fetal bovine serum (FBS) at 37 °C in 95% air 5%
CO2. Cells were characterized morphologically as smooth muscle
The toxicity of compound 1b was determined in normal ICR rats
(6–8 weeks, Academia Sinica, China), which were weighed individ-
ually. LD50 was delivered via intragastric administration (ig) at
doses of 5000.0, 3823.6, 2924.0, 2236.1, 1710.0 and 1000.0 mg/
kg. Survival was assessed daily for two 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,
mobility, aggressiveness and respiratory movements. Finally, the
survivals were dissected and examined pathological changes of
organs.
by phase contrast microscopy and by immunostaining with
a-
actin.13
4.51. Radioligand binding assay
Acknowledgments
The new compounds, valsartan and irbesartan were dissolved in
DMSO and diluted to different concentration with PBS before the
This work was supported by Grant of Chinese Government
(30976611, 81102407, 81101298), Shanghai Government
(10DZ0502300, 10ZZ45), and Donghua University (11D10501,
11D10518, 105-10-0044030).
experiment. The final concentrations were 1 ꢃ 10ꢁ10
M to
1 ꢃ 10ꢁ4 M.14 125I-Ang II (Northern Biotechnology Company) was
dissolved in phosphate buffered saline (PBS) and diluted to

Y. Nie et al. / Bioorg. Med. Chem. 20 (2012) 2747–2761
2761
6. White, W. B.; Weber, M. A.; Sica, D., et al Hypertension 2011, 57, 413.
7. Bakris, G. L.; Sica, D.; Weber, M., et al J Clin Hypertens. 2011, 13, 81.
Supplementary data
8. Kim, D.; Mantlo, N. B.; Cheng, R. S. L., et al Bioorg. Med. Chem. Lett. 1994, 4, 41.
9. Bühlmayer, P.; Furet, P.; Leoluca Criscione, et al Bioorg. Med. Chem. Lett. 1994, 4,
29.
10. Kohara, Y.; Imamiya, E.; Kubo, K., et al Bioorg. Med. Chem. Lett. 1995, 5, 1903.
11. Kohara, Y.; Kubo, K.; Imamiya, E., et al J. Med. Chem. 1996, 39, 5228.
12. Ferrari, B.; Taillades, J.; Perreaut, P., et al Bioorg. Med. Chem. Lett. 1994, 4, 45.
13. Liu, X. P.; Liu, Q.; Zheng, Y. S., et al J. Shantou. Univ. Med. Coll. 2008, 21, 137.
14. Wang, Yu.; Wang, Wei.; Wang, Qiujuan., et al Biochem. Biophys. Res. Commun.
2007, 364, 118.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.02.003.
References and notes
1. De Gasparo, M.; Catt, K. J.; Inagami, T., et al Pharmacol. Rev. 2000, 52, 415.
2. Schmidt, B.; Schieffer, B. J. Med. Chem. 2003, 469, 2261.
3. Chung, O.; Unger, T. Basic Res. Cardiol. 1998, 93, 15.
4. Kirch, W.; Horn, B.; Schweizer, J. Eur. J. Clin. Invest. 2001, 31, 698.
5. Varagic, J.; Frohlich, E. D.; Susic, D., et al Am. J. Physiol. Heart Circ. Physiol. 2008,
294, 853.
15. Andrea, C.; Chiara, N.; Andrea, G., et al J. Med. Chem. 2008, 51, 2137.
16. Eric, N.; Gerard, C.; Joel, G., et al J. Med. Chem. 1994, 37, 2371.