Synthesis and biological evaluation of new fluorine substituted derivatives as angiotensin II receptor antagonists with anti-hypertension and anti-tumor effects

Article abstract of DOI:10.1016/j.bmc.2012.09.065

The synthesis and pharmaceutical activity of new potent non-tetrazole angiotensin II (Ang II) receptor antagonists were described. These compounds were fluorine substituted derivatives of Losartan, Valsartan and Irbesartan with carboxylic acid group as re

Full text of DOI:10.1016/j.bmc.2012.09.065

Bioorganic & Medicinal Chemistry 20 (2012) 7101–7111
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new fluorine substituted derivatives
as angiotensin II receptor antagonists with anti-hypertension and
anti-tumor effects
⇑
Ya-jing Da , Wei-dong Yuan , Ting Xin, Yong-yan Nie, Ying Ye, Yi-Jia Yan, Li-sha Liang, 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:
The synthesis and pharmaceutical activity of new potent non-tetrazole angiotensin II (Ang II) receptor
antagonists were described. These compounds were fluorine substituted derivatives of Losartan,
Valsartan and Irbesartan with carboxylic acid group as replacements to the known potent tetrazole
moiety at the 20-biphenyl position. Their activities were evaluated by Ang II receptor binding assay as
well as by in vivo assay. All of the synthesized compounds showed nanomolar affinity for the AT1 recep-
tor subtype. The vivo biological evaluation showed that compounds 1a, 2 and 4 produced a dose-
dependent antihypertensive effect both in spontaneously hypertensive rats (SHR) and renal hypertensive
rats (RHR). Compound 4 especially showed an efficient and long-lasting effect in reducing blood pressure
which can last more than 24 h at dose of 10 mg/kg in SHR, which was much better than control Losartan
and Valsartan. Compound 4 can also inhibit the prostate cancer in vitro and in vivo. So compound 4 was
selected for in-depth investigation as potent, novel and long-lasting non-tetrazole anti-hypertension and
anti-tumor drug candidate.
Received 10 August 2012
Revised 26 September 2012
Accepted 27 September 2012
Available online 12 October 2012
Keywords:
Angiotensin II receptor antagonist
Synthesis
Anti-hypertension
Anti-tumor
Prostate cancer
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
some disadvantages in chemical synthesis and biometabolism.
For example, the synthesis of tetrazole derivatives could be dan-
Hypertension is recognized as one of the leading risk factors for
human morbidity and mortality. On a worldwide basis hyperten-
sion has been ranked third as a cause of disability.1 Angiotensin
II (Ang II) is a vasoconstrictive peptide hormone formed within
the renin–angiotensin system (RAS) which plays an important role
in regulating cardiovascular homeostasis.2–4 Research efforts for
the control of hypertension have focused on competing Ang II
binding to AT1 receptors. Ang II receptor antagonists have been
proved to lower blood pressure effectively,5 to treat congestive
heart failure and reduce the cardiac and vascular remodeling asso-
ciated with cardiovascular disease.6 They are better tolerated than
other classes of drugs.7,8
A number of Ang II type 1 (AT1) receptor antagonists, namely
the angiotensin II type 1 receptor blockers (ARBs) with different
acidic moiety have been investigated by David,9 and other scien-
tists. It was reported that tetrazole-containing compounds had
the greatest binding affinities and oral activities. Most of ARBs
have acidic group tetrazole ring, such as Valsartan, Losartan,
Candesartan and Ibersartan. However the tetrazole group had
gerous due to the use of toxic and explosive azide compounds such
as sodium azide or trialkytin azide. In this report we designed and
synthesized a series of new compounds with carboxylic acid moi-
ety to replace tetrazolium.
Frequently, the introduction of fluorine atoms or fluorine-con-
taining substituents into a drug molecule decreases toxicity and in-
creases stability of the compound, the biological activity being
unchanged or even enhanced. Besides in the past decades, much
progress has been achieved in the development of organo-fluorine
chemistry, including the synthesis of new fluorinating agents, dis-
covery of new reactions and development of new technologies. As
a
result, fluorine containing-organic compounds of various
classes became more available.10 Considering pKa for o-, m- and
p-fluorobenzoic were 3.47, 3.86 and 4.13, respectively, while the
pKa for benzoic acid was 4.2, fluorine atoms was introduced as
electron withdrawing moiety to the biphenyl part to enhance the
ionization of carboxylic acid into negative charge for better binding
with the positive charge of AT1. It could be suggested that com-
pounds with fluorine substituent on the biphenyl group would
exhibited better antihypertensive activity.
Although the RAS has a crucial role as vasopressor system that
maintains blood pressure, fluid homeostasis and electrolyte bal-
ance, it is now recognized to have much broader functions in the
⇑
Corresponding author. Tel./fax: +86 21 67792743.
E-mail address: zlchen1967@yahoo.com (Z.-I. Chen).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.065

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Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
Figure 1A. Energy-minimized conformation of compound 3 (179.8151 kJ/mol), Irbesartan (219.8643 kJ/mol) and their overlay conformation.
Figure 1B. Energy-minimized conformation of Losartan (60.0774 kJ/mol), compound 1a (ꢀ29.0096 kJ/mol) and their overlay conformation.
body with important actions on growth factors and cell growth.
Tissue-based or local RAS have been identified in the testis, epidid-
ymis, ovary uterus, brain, heart, peripheral blood vessels and kid-
ney. In recent years, all components of the classical RAS have
been reported in the prostate11 and some other tumors. Some evi-
dence suggests that Ang II directly stimulates cell growth via the
AT1 receptor and that its blockade inhibits tumor growth. AT1
receptor blockade can reduce tumor volume, vascular density, mi-
totic index and cell proliferation. These results suggest a possibility
that AT1 receptor blockades are potential therapeutic drugs for the
treatment of prostate cancer and other prostate diseases.
In this study, six compounds 1a, 1b,1c, 2, 3 and 4 were designed
and synthesized. The dominant conformations of compound 1a, 3,
Irbesartan and Losartan were shown as Figure 1A and B using com-
puter software Spartan 8. We found that the minimal energy con-
formations of the compounds 1a and 3 were lower than Irbesartan
and Losartan. The conformation of compoud 3 fits perfectly to
Irbesartan and compound 1a fits perfectly to Losartan. These com-
pounds were tested for their affinity for the AT1 receptor as mea-
sured by their ability to displace [125I] Ang II from its specific
binding sites in the rat vascular smooth muscle cells (VSMC). The
anti-hypertension effects of them were evaluated in spontaneously
hypertensive rats (SHR) in vivo after oral administration. Selected
compounds were further tested in vivo in renal hypertensive rats
(RHR) in vivo. Moreover, compound 4 were selected to evaluate
the anti-proliferative activity in vitro and antitumor activity
in vivo in prostate cancer.
starting materials were esterified with CH3OH catalyzed by
H2SO4 to give 5a–c. Compounds 6a–c were obtained through the
Suzuki coupling reaction of 5a–c with p-tolylboronic acid catalyzed
by PPh3 and Pd (OAc)2.
Compounds 6a–c were brominated with NBS and AIBN to pro-
duce compounds 7a–c, and then reacted with 2-butyl-4-chloro-
1H-imidazole-5-carboxaldehyde in the presence of K2CO3 to give
compounds 8a–c. Compounds 8a–c were oxidated with NaClO to
produce compounds 9a–c. Compounds were obtained through
the hydrolysis of 9a–c with sodium hydroxide solution.
Compound 2 was obtained from 8b which was reduced by
NaBH4 and then hydrolyzed with sodium hydroxide solution in
Scheme 2.
Scheme 3 was employed to synthesize compound 3. Methyl
4-fluoro-40-methylbiphenyl-2-carboxylate 6b was obtained as
the method in Scheme 1. Compound 6b was hydrolyzed to give
4-fluoro-40-methylbiphenyl-2-carboxylic acid 11 which was re-
acted with oxalylchloride and t-C4H9OK to give tert-butyl
4-fluoro-40-methylbiphenyl-2-carboxylate 12 in 44.2% yield.
Compound 12 was brominated to produce tert-butyl 40-(bromo-
methyl)-4-fluorobiphenyl-2-carboxylate 13. Tert-butyl-40-((2-bu-
tyl-4-oxo-1,3-iazaspiro[4.4]non-1-en-3-yl)methyl)-4-fluorobiphe-
nyl-2-carboxylate 14 was obtained from 13 and 2-butyl-4-
spirocyclopentane-2-imidazolin-5-one hydrochloride in the
presence of NaH. Compound
hydrolysis of 14 with TFA in CH2Cl2.
Valsartan derivative 4 was synthesized in Scheme 4. A mixture of
7b, -Valine methyl ester hydrochloride and DIPEA was refluxed to
3 was obtained through the
L
produce (S)-methyl 4-fluoro-40-((1-methoxy-3-methyl-1-oxob-
utan-2-ylamino)-methyl)-biphenyl-2-carboxylate 15 which was
acylated with n-butyryl chloride under the presence of DIPEA to give
(S)-methyl 4-fluoro-40-((N-(1-methoxy-3-methyl-1-oxobutan-2-
yl)butyramido)methyl)biphenyl-2-carboxylate 16. The compound
3 was obtained through the hydrolysis of 16 with TFA.
2. Results and discussion
2.1. Chemistry
The synthetic route described in Scheme 1 was employed to
synthesize the target compounds 1a–c. Compounds 4a–c as

Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
7103
Br
Br
Br
COOH
R1
COOCH3
b
c
a
COOCH3
R1
COOCH3
R1
R2
R2
R1
R2
R3
R3
R2
R3
R3
7a-c
5a-c
6a-c
4a-c
Cl
Cl
COOH
N
Cl
COOH
N
N
N
N
CHO
N
f
e
d
COOH
R1
COOCH3
R1
COOCH3
R1
R2
R2
R2
R3
R3
(a) R1 R3 =H, R2=F
(b) R1 R2 =H, R3=F
(c) R2 R3 = F, R1=H
R3
1a-c
9a-c
8a-c
Scheme 1. Preparation of compounds 1a–c.
Cl
N
Cl
CH2OH
N
CHO
N
N
Br
a
b
COOCH3
COOCH3
COOCH3
F
F
F
10
7b
8b
Cl
CH2OH
N
N
c
COOH
F
2
Scheme 2. Preparation of compound 2.
2.2. Biological evaluation
2.2.1. Radioligand binding assay
Angiotensin II interacted with a single population of binding
sites in the rat vascular smooth muscle cells (VSMC) AT1 receptors.
Radioligand binding assay showed that all of these compounds
have nanomolar affinity for the AT1 receptor subtype (Table 1).
The specific binding of 125I-Ang II was inhibited in a concentra-
tion-dependent manner by compounds 1a, 1b, 1c, 2, 3, 4 and
Irbesartan, Losartan, Valsartan as exemplified by compounds 1a

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Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
Br
c
b
a
O
O
COOH
COOCH3
O
O
F
F
F
F
13
6b
12
11
N
N
O
O
N
N
e
d
O
COOH
O
F
3
F
14
Scheme 3. Preparation of compound 3.
O
HN
COOCH3
Br
N
COOCH3
a
b
COOCH3
COOCH3
COOCH3
F
F
F
7b
16
15
O
N
COOH
c
COOH
F
4
Scheme 4. Preparation of compound 4.
and 4 in Figure 2. The IC50 values of compound 4, Losartan, Irbesar-
tan and Valsartan were 0.58 0.10, 1.64 0.22, 3.11 0.24 and
2.64 0.75 nM, respectively. The Ki values of compound 4,
Losartan, Irbesartan and Valsartan were 0.42 0.54, 1.41 0.12,
2.75 0.97 and 2.08 0.17, respectively. These results suggested
that compound 4 exhibited more affinity to AT1 receptors than
Losartan, Irbesartan and Valsartan because their IC50 and Ki values
were significantly lower compared with the positive control
groups (P <0.05). So compound 4 might had better antihyperten-
sive activity in vivo.
2.2.2. Antihypertensive effects in rats
In spontaneously hypertensive rats (SHR), the effects of com-
pounds 1a, 1b, 1c, 2, 3 and 4 (5,10 mg/kg) and Losartan (10 mg/

Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
7105
Table 1
IC50 and Ki values of tested compounds
O
N
N
N
Cl
N
N
CH2OH
O
COOH
N
N
N
N
N
N
N
N
N
N
H
N
N
H
H
Losartan
Irbesartan
mp(°C)
Valsartan
Compound
R1
R2
R3
Formula
22H20CIFN2O4
C22H20CIFN2O4
IC50 SEM (nM)
Ki (nM)
1a
1b
1c
2
3
4
Losartan
Irbesartan
Valsartan
H
H
H
H
H
F
F
F
H
F
208–211
196–199
218–221
206–209
214–217
215–218
C
13.57 3.90
26.12 0.15
13.48 0.03
4.11 1.20
9.98 0.03
0.584 0.10
1.64 0.22
3.11 0.24
2.64 0.75
9.17 2.30
17.65 0.15
9.11 0.11
3.78 1.20
6.75 2.60
0.42 0.54
1.41 0.12
2.75 0.97
2.08 0.17
C
C
C
C
22H19CIF2N2O4
22H22CIFN2O3
25H27FN2O3
23H26FNO5
F
H
than Losartan and Valsartan whose antihypertensive effects main-
tained for 7 and 9 h, respectively, and the maximal reduction was
27, 28 mm Hg, respectively at 10 mg/kg.
1a
4
125
100
75
50
25
0
All these compounds did not influence heart rate of the rats.
Because compounds 1a, 2 and 4 had significant antihyperten-
sive effects in SHR, renal hypertensive rats (RHR) was used to test
these three compounds at dose 5, 10 mg/kg compared with
Losartan at 10 mg/kg and Valsartan at 10 mg/kg. In RHR, we ob-
served almost the same effects as in SHR (Fig. 3B). Compound 4
at 5 mg/kg showed the equal effect to that of Losartan and Valsar-
tan at 10 mg/kg. And at dose of 10 mg/kg, compound 4 had much
better and longer antihypertensive effect. Compounds 1a and 2
at 10 mg/kg showed the similar effect to that of Losartan and Val-
sartan at the same dose.
valsartan
irbesartan
losartan
These results in SHR and in RHR showed that compound 4 was
superior to Losartan and Valsartan, which were in accord with the
data of the former receptor binding assay.
-13 -12 -11 -10
-9
-8
-7
-6
-5
compound, log(M)
Figure 2. Inhibitory effects of compounds 1a, 4, Losartan, Irbesartan and Valsartan
(10ꢀ6–10ꢀ12 M) on the specific binding of 125I-Ang II to AT1 receptors in VSMCs.
2.2.3. Antiproliferative activity in LNCap cells
Compound 4 was selected to examine the activity on carcinoma
of prostate respecting its favorable antihypertensive activity. The
effect of compound 4 on cell viability was assessed by MTT assay.
Treatment of LNCaP cells with Ang II at the concentration of
1 ꢁ 10ꢀ4 M resulted in an increase in the viability of cells. A signif-
icant reduction in the viability as compared to DMSO-treated con-
trols was observed after 24 h of treatment with Losartan and
compound 4. Increase in treatment times to 48, 72 and 96 h further
reduced the viability of cells (Fig. 4). However, Losartan and com-
pound 4 could not influence the growth of LNCaP cells without
treatment with Ang II.
kg), Valsartan (10 mg/kg) on the mean arterial pressure (MAP)
in vivo after oral administration were shown in Figure 3A. The re-
sults indicated that under the dosage of 5 mg/kg or 10 mg/kg, com-
pound 1b, 1c and
3 could not decreased blood pressure
significantly compared with the negative control group. Com-
pounds 1a, 2 and 4 could cause significant decrease on MAP in a
dose dependent manner. The maximal response of compound 1a
(5,10 mg/kg) was observed at 3 h after dosing, it lowered 23 mm
Hg and 32 mm Hg of MAP, respectively, and the significant
(p <0.05) antihypertensive effect of it was sustained for at least
8 h. Compound 2 (5,10 mg/kg) lowered MAP to 18, 25 mm Hg,
respectively. The maximal reduction and the time of duration are
almost the same as compound 1a. The antihypertensive effects of
compounds 1a and 2 at 10 mg/kg were almost equal to Losartan
and Valsartan at the same dose.
When compound 4 at 5 mg/kg and 10 mg/kg was administered
orally, the MAP was lowered 27 mmHg and 42 mmHg, respec-
tively. The maximal response was obtained at 3–5 h after dosing.
With 10 mg/kg, the reductive effect of compound 4 was observed
even at 24 h after drug administration, which was much better
2.2.4. Antitumor activity in nude mice
Based on the in vitro results, we further tested the antitumor
activity of compound 4 in vivo, LNCap cells were established as
xenografts in nude mice (n = 10). When the tumors reached about
5 mm in diameter, the animals were given compound 4 at 5.0 or
10 mg/kg/day. Losartan at 5.0 or 10 mg/kg/day were used as posi-
tive control group. The negative control group was received water
containing sodium hypochlorite (10 ppm).12 As shown in Figure 5,
at 4 weeks, control group had developed large tumor of

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Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
losartan 10mg/kg
valsartan 10mg/kg
control
compound 1b 5mg/kg
compound 1b 10mg/kg
losartan 10mg/kg
valsartan 10mg/kg
control
compound 1a 5mg/kg
compound 1a 10mg/kg
10
0
10
0
-10
-20
-30
-40
*
-10
-20
-30
-40
*
*
*
* *
*
*
*
*
*
*
*
*
*
*
*
-1 0
1 2 3 4 5 6 7 8 9 10 1112 24
-1 0
1 2 3 4 5 6 7 8 9 10 1112 24
Time(h)
Time(h)
losartan 10mg/kg
losartan 10mg/kg
valsartan 10mg/kg
compound 1c 5mg/kg
compound 1c 10mg/kg
compound 2 5mg/kg
compound 2 10mg/kg
valsartan 10mg/kg
control
control
10
0
10
0
-10
-20
-30
-40
-10
-20
-30
-40
*
*
*
*
*
*
*
*
*
*
*
*
*
*
* *
*
-1
0 1 2 3 4 5 6 7 8 9 10 1112 24
-1 0
1
2
3
4
5
6
7 8 9 10 1112 24
Time(h)
Time(h)
losartan 10mg/kg
valsartan 10mg/kg
losartan 10mg/kg
valsartan 10mg/kg
compound 4 5mg/kg
compound 4 10mg/kg
compound 3 5mg/kg
compound 3 10mg/kg
control
control
10
10
0
0
-10
-20
-30
-40
-10
-20
-30
-40
-50
-60
*
*
*
*
*
*
*
*
*
*
**
**
**
**
**
**
**
**
**
-1 0
1 2 3 4 5 6 7 8 9 10 1112 24
-1 0
1
2
3
4
5
6
7
8
9 10 1112 24
Time(h)
Time(h)
Figure 3A. Effects of compounds 1a, 1b, 1c, 2, 3, 4, Losartan and Valsartan on arterial pressure (MAP) in spontaneously hypertensive rats. ⁄ ⁄⁄Significant difference from the
,
control, p <0.05 and p <0.01, respectively.
28.34 2.05 relative volume compared with those at 0 week. Mice
treated with compound 4 at 5.0 or 10 mg/kg/day was showed more
inhibition of tumor relative volume at 4 weeks by 17.44 1.78 and
12.39 1.23, respectively, compared with tumor relative volume at
4 weeks by 21.44 2.182 and 15.75 1.89, respectively, treated
with Losartan at 5.0 or 10 mg/kg/day.
values and Ki values showed that compound 4 had the strongest
inhibitory ability against Ang II. In anti-hypertensive assays
in vivo, compounds 1a, 2 and 4 showed efficient and long effect
in decreasing blood pressure in spontaneously hypertensive rats
and renal hypertensive rats. These results were corresponding to
the data in radioligand binding assay. Both in spontaneously
hypertensive rats and in renal hypertensive rats, compound 4
showed an efficient and long-lasting effect in reducing blood pres-
sure, it could last more than 24 h which was much better than
Losartan and Valsartan. The anti-prostate cancer test showed that
compound 4 could inhibit the prostate cancer in vitro and in vivo.
This led to the discovery of compound 4 as a potent, non-
tetrazole AT1 receptor antagonist. This orally active compound pro-
duced a marked and long lasting decrease in blood pressure in SHR
models and RHR models, which was proved to be superior to Losar-
tan and Valsartan. It has also anti-prostate cancer effects. On the
3. Conclusions
In this paper the tetrazole moiety of Ang II type 1 receptor
antagonists has been successfully replaced by carboxylic acid
group at the 20-biphenyl position with fluorine substitution at 40
or 50-biphenyl position of Losartan, Valsartan and Irbesartan as a
novel class of angiotensin II receptor antagonists
Radioligand binding assay showed that all of these compounds
have nanomolar affinity for the AT1 receptor subtype. The IC50

Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
7107
losartan 10mg/kg
compound 1a 5mg/kg
compound 1a 10mg/kg
losartan 10mg/kg
valsartan 10mg/kg
control
compound 2 5mg/kg
compound 2 10mg/kg
valsartan 10mg/kg
control
10
0
10
0
-10
-20
-30
-40
-10
-20
-30
-40
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
-1 0 1 2 3 4 5 6 7 8 9 10 1112 24
Time (h)
-1 0 1 2 3 4 5 6 7 8 9 10 1112 24
Time (h)
losartan 10mg/kg
valsartan 10mg/kg
compound 4 5mg/kg
compound 4 10mg/kg
control
10
0
-10
-20
-30
-40
-50
-60
*
*
*
*
*
*
*
*
*
*
*
*
**
**
**
**
**
**
**
-1 0 1 2 3 4 5 6 7 8 9 10 1112 24
Time (h)
Figure 3B. Effects of compounds 1a, 2, 4, Losartan and Valsartan on arterial pressure (MAP) in renal hypertensive rats. ⁄ ⁄⁄Significant difference from the control, p <0.05 and
,
p <0.01, respectively.
basis of this profile, compound 4 was chose as novel anti-hyperten-
sion and anti-tumor drug candidates and deserved for further
investigation.
petroleum ether–ethyl acetate (80/1) as eluant to afford colorless li-
quid. Yield: 88.3%. This compound was prepared according to the
general procedure reported by Baker.13 The spectral data were con-
sistent with that reported in the literature.
4. Experimental section
4.1. Chemistry
4.1.2. Methyl 2-bromo-5-fluorobenzoate (5b)
Compound 5b was prepared according to the procedure of 5a.
Yield: 84.7%. 1H NMR (CDCl3, 400 MHz) d: 7.74–6.92(3H, m,
Ph-H), 3.80(3H, s, Ph-COOCH3). ESI-MS (m/z): 234.0 [M+1]+.
All chemicals used were of reagent grade. Yields refer to puri-
fied products and are not optimized. 1H NMR spectra were mea-
sured on a Bruker 400 MHz spectrometer. ESI-MS spectra were
recorded on a Micromass triple quadrupole mass spectrometer.
Column chromatography was performed using silic gel H (300–
400). All melting points were measured using an Eletrothermal
9200 apparatus and are uncorrected. Solvents were dried accord-
ing to standard procedures. Solutions were dried over MgSO4 be-
fore evaporation under reduced pressure.
4.1.3. Methyl 2-bromo-4, 5-difluorobenzoate (5c)
Compound 5c was prepared as the procedure of 5a. Yield:
80.3%. ESI-MS (m/z): 252.0 [M+1]+.
4.1.4. Methyl 5-fluoro-40-methylbiphenyl-2-carboxylate (6a)
To a solution of 5a (2.00 g, 8.58 mmol), Palladium acetate
(0.008 g, 0.04 mmol), PPh3 (0.47 g, 1.69 mmol) and p-tolylboronic
acid (10 mL) was added 6 mL of 2 M K2CO3. The mixture was re-
fluxed under nitrogen for 5 h, then diluted with ethyl acetate,
and washed with water (50 mL ꢁ 3) and brine (50 mL ꢁ 3). The or-
ganic layer was dried over MgSO4. The solvent was removed under
reduced pressure. The residue was purified by flash column chro-
matography with petroleum ether–ethyl acetate (100/1) as eluant.
The product was obtained as a yellow oil. Yield: 82.6%. 1H NMR
(CDCl3, 400 MHz) d: 7.53(1H, dd, J1 = 8.4 Hz, J2 = 2.8 Hz, Ph-H),
7.35(1H,dd, J1 = 8.4 Hz, J2 = 5.6 Hz, Ph-H), 7.25–7.16(5H, m, Ph-H),
4.1.1. Methyl 2-bromo-4-fluorobenzoate (5a)
To
a
solution of 2-bromo-4-fluorobenzoic acid (2.17 g,
10.00 mmol) in methanol (10 mL) was added sulfuric acid
(1.63 mL, 30 mmol) , the reaction mixture was refluxed for 4 h. The
solvent was removed in vacuo. The mixture was diluted with diethyl
ether, and washed water (50 mL ꢁ 3) and brine (50 mL ꢁ 3), dried
over MgSO4. The solvent was removed under reduced pressure.
The residue was purified by flash column chromatography with

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Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
over MgSO4, and the solvent was removed under reduced pressure.
control
losartan
compound 4
Ang II
Ang II + losartan
Ang II + compound 4
The residue was purified by flash column chromatography with
petroleum ether–ethyl acetate (100/1) as eluant to afford a yellow
oil. Yield: 67.8%. 1H NMR(CDCl3, 400 MHz) d: 7.53(1H, dd,
J1 = 8.4 Hz, J2 = 2.8 Hz, Ph-H),7.44(2H, m, Ph-H), 7.32–7.15(4H, m,
Ph-H), 4.56(2H, s, Ph-CH2–Br), 3.69(3H, s, Ph-COOCH3); 13C
NMR(CDCl3, 400 MHz) d: 169.74, 165.26, 162.79, 142.88, 140.45,
139.27, 134.82, 134.61, 131.23, 128.66, 120.87, 120.68, 119.11,
54.54, 35.62. ESI-MS (m/z): 323.1 [M+1]+.
140
*
130
120
110
100
90
*
4.1.8. Methyl 40-(bromomethyl)-4-fluorobiphenyl-2-
carboxylate (7b)
Compound 7b was prepared as the procedure of 7a. Yield:
67.8%. 1H NMR(CDCl3, 400 MHz) d: 7.92(1H, m, J = 6.0 Hz, Ph-H),
7.44(2H, d, J = 2.0 Hz, Ph-H), 7.27(2H, d, J = 2.0 Hz, Ph-H),
7.16–7.03(2H, m, Ph-H), 4.56(2H, s, Ph-CH2–Br), 3.67(3H, s, Ph-
COOCH3). ESI-MS (m/z): 323.1 [M+1]+.
*
*
*
80
*
*
70
96
12
24
48
hour
72
Figure 4. Cell viability effect of losartan and compound 4 in prostate carcinoma cell
lines (LNCaP) and normal prostate cells measured by MTT. ⁄Significant difference
from the control, P <0.05.
4.1.9. Methyl 40-(bromomethyl)-4,5-difluorobiphenyl-2-
carboxylate (7c)
Compound 7c was prepared according to the procedure of 7a.
Yield: 87.9%. 1H NMR (CDCl3, 400 MHz) d: 7.75(1H, dd,
J1 = 10.0 Hz, J2 = 8.4 Hz, Ph-H), 7.44(2H, d, J = 2.0 Hz, Ph-H),
7.25(2H, dd, J1 = 5.4 Hz, J2 = 1.6 Hz, Ph-H), 7.16(2H, dd,
J1 = 10.4 Hz, J2 = 7.6 Hz, Ph-H), 4.54(2H, s, Ph-CH2–Br), 3.67(3H, s,
Ph-COOCH3). ESI-MS (m/z): 341.1 [M+H]+.
35
control
compound 4:5mg/kg/day
30
25
20
15
10
5
compound 4:10mg/kg/day
losartan:5mg/kg/day
losartan:10mg/kg/day
4.1.10. Methyl 40-((2-butyl-4-chloro-5-formyl-4,5-dihydro-1H-
imidazol-1-yl)methyl)-5-fluorobiphenyl-2-carboxylate (8a)
To a solution of 7a (2.262 g, 7 mmol), 2-butyl-4-chloro-1H-
imidazole-5-carboxaldehyde (1.437 g, 7.7 mmol) and 25 mL of ace-
tonitrile was added potassium carbonate (1.449 g, 10.5 mmol). The
mixture was stirred at 50 °C under nitrogen for 6 h. The mixture
was diluted with ethylacetate. The organic layer was washed with
water (30 mL ꢁ 3) and brine (30 mL ꢁ 3), dried over MgSO4. After
filtration, the solvent was removed under reduced pressure. The
residue was purified by flash column chromatography with petro-
leum ether–ethyl acetate (3/1) as eluant to afford a white solid.
Yield: 54.5%. 1H NMR(CDCl3, 400 MHz) d: 9.94(1H, s,
C-CHO),7.58(1H, dd, J1 = 9.2 Hz, J2 = 2.8 Hz, Ph-H), 7.32ꢂ7.22(4H,
m, Ph-H), 7.08(2H, d, J = 8.4 Hz, Ph-H), 5.23(2H, s, Ph-CH2–N),
3.67(3H, s, Ph-COOCH3), 2.67(2H, t, J = 8.0 Hz, CH2CH2CH2CH3),
1.71(2H, m, J = 8.0 Hz, CH2CH2CH2CH3), 1.37(2H, m, J = 8.0 Hz,
CH2CH2CH2CH3),0.90(3H, t, J = 8.0 Hz, CH2CH2CH2CH3); 13C NMR
(CDCl3, 400 MHz) d: 182.97, 169.50, 165.32,162.85, 152.70,
143.07, 140.18, 136.69, 135.71, 134.85, 131.63, 128.31, 127.27,
120.87, 119.36, 54.50, 49.22, 32.01, 31.53, 29.86, 24.69, 15.99,
12.31; ESI-MS(m/z): 431.2 [M+1]+.
*
**
*
**
0
0
1W
2W
3W
4W
Date
Figure 5. Antitumor activity of compound 4 and Losartan (5,10 mg/kg/day) in nude
mice, tumor growth of LNCap xenografts was measured at indicated times.
⁄ ⁄⁄Significant difference from the control, p <0.05 and p <0.01, respectively.
,
3.69(3H, s, Ph-COOCH3)2.41(3H, s, Ph-CH3). ESI-MS (m/z): 245.2
[M+1]+.
4.1.5. Methyl 4-fluoro-40-methylbiphenyl-2-carboxylate (6b)
Compound 6b was prepared according to the procedure of 6a.
Yield: 82.6%. 1H NMR (CDCl3, 400 MHz) d: 7.86(1H, m, Ph-H),
7.21(4H, m, Ph-H), 7.13–7.08 (2H, m, Ph-H), 3.70(3H, s, Ph-
COOCH3), 3.43(3H, s, Ph-CH3). ESI-MS (m/z): 245.2 [M+1]+.
4.1.11. Methyl40-((2-butyl-4-chloro-5-formyl-4,5-dihydro-1H-
imidazol-1-yl)methyl)-4-fluorobiphenyl-2-carboxylate (8b)
Compound 8b was prepared as the procedure of 8a. Yield:
34.1%. 1H NMR (CDCl3, 400 MHz) d: 9.92(1H, s, C-CHO), 7.91(1H,
dd, J1 = 8.4 Hz, J2 = 5.6 Hz, Ph-H), 7.28(2H, t, J = 8.0 Hz, Ph-H),
7.11(3H, dd, J1 = 11.2 Hz, J2 = 2.4 Hz, Ph-H, Ph-H),7.00(1H, dd,
J1 = 9.2 Hz, J2 = 2.4 Hz, Ph-H), 5.23(2H, s, Ph-CH2–N), 3.64(3H, s,
Ph-COOCH3), 2.67(2H, t, J = 7.6 Hz, CH2CH2CH2CH3), 1.71(2H, m,
J = 7.6 Hz, CH2CH2CH2CH3), 1.37(2H, m, J = 7.6 Hz, CH2CH2CH2CH3),
0.90(3H, t, J = 7.6 Hz, CH2CH2 CH2CH3). ESI-MS(m/z): 431.2 [M+1]+.
4.1.6. Methyl 4,5-difluoro-40-methylbiphenyl-2-carboxylate (6c)
Compound 6a was prepared according to the procedure of 6a.
Yield: 82.6%. 1H NMR (CDCl3, 400 MHz) d: 7.74(1H, dd,
J1 = 10.8 Hz, J2 = 2.8 Hz, Ph-H), 7.28–7.18(5H, m, Ph-H), 3.73(3H, s,
Ph-COOCH3), 2.46(3H, s, Ph-CH3). ESI-MS (m/z): 263.2 [M+1]+.
4.1.7. Methyl 40-(bromomethyl)-5-fluorobiphenyl-2-
carboxylate (7a)
A solution of 6a (1.57 g, 6.43 mmol), NBS 1.26 g, 7.08 mmol),
AIBN (0.16 g, 0.97 mmol) and cyclohexane(30 mL) was refluxed
for 3 h. The mixture was diluted with dichloromethane (30 mL),
and washed with brine (30 mL ꢁ 3). The organic layer was dried
4.1.12. Methyl 40-((2-butyl-4-chloro-5-formyl-4,5-dihydro-1H-
imidazol-1-yl)methyl)-4,5-difluorobiphenyl-2-carboxylate (8c)
Compound 8c was prepared according to the procedure of 8a.
Yield: 43.2%. 1H NMR (CDCl3, 400 MHz) d: 9.90(1H, s, C-CHO),
7.72(1H, dd, J1 = 10.4 Hz, J2 = 8.0 Hz, Ph-H), 7.25(2H, t, J = 7.6 Hz,
Ph-H), 7.12–7.06(3H, m, Ph-H), 5.23(2H, s, Ph-CH2–N), 3.64(3H, s,

Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
7109
Ph-COOCH3), 2.65(2H,t, J = 7.6 Hz, CH2CH2CH2CH3), 1.68(2H, m,
4.1.17. 2-Butyl-1-((20-carboxy-40-fluorobiphenyl-4-yl)methyl)-
J = 7.6 Hz, CH2CH2CH2CH3), 1.35(2H, m, J = 7.6 Hz, CH2CH2CH2CH3),
4-chloro-1H-imidazole-5-carboxylic acid (1b)
0.88(3H, t, J = 7.6 Hz, CH2CH2CH2CH3); ESI-MS(m/z): 449.2 [M+1]+.
Compound 1b was prepared as the procedure of 1a. mp:
196–199 °C Yield: 95.3%. Anal. Calcd for C22H20CIFN2O4: C, 61.33;
H, 4.68; N, 6.50. Found: C, 61.30; H, 4.69; N, 6.52. 1H NMR (DMSO,
400 MHz,) d: 12.68(s, 1H, COOH), 7.81(1H, dd, J1 = 8.8 Hz,
J2 = 6.0 Hz, Ph-H), 7.34(2H, d, J = 8.0 Hz, Ph-H), 7.30–7.26(1H, m,
Ph-H), 7.19(1H, dd, J1 = 9.6 Hz, J2 = 2.4 Hz, Ph-H), 7.09(2H, d,
J = 8.0 Hz, Ph-H), 5.31(2H, s, Ph-CH2-N), 2.62(2H, t, J = 7.6 Hz,
CH2CH2CH2CH3), 1.55(2H, m, J = 7.6 Hz, CH2CH2CH2CH3), 1.28(2H,
4.1.13. 2-Butyl-4-chloro-1-((50-fluoro-20-(methoxycarbonyl)
biphenyl-4-yl)methyl)-1H-imidazole-5-carboxylic acid (9a)
To a solution of 8a (0.368 g, 0.86 mmol) and n-butanol (10 mL)
was added dropwise to a solution of sodium hypochlorite (0.670 g,
7.37 mmol), sodium dihydrogen phosphate (0.677 g, 5.65 mol) and
distilled water (10 mL). The mixture was stirred for 10 h, and di-
luted with ethyl acetate. The organic layer was washed with water
(30 mL ꢁ 3) and brine (30 mL ꢁ 3), dried over MgSO4. The solvent
was removed under reduced pressure. After recrystallization, a
white solid was obtained. mp: 203–205 °C. Yield: 98.5%. 1H NMR
(CDCl3, 400 MHz) d: 7.57(1H, dd, J1 = 8.8 Hz, J2 = 2.8 Hz, Ph-H),
7.32–7.22(4H, m, Ph-H), 7.08(2H, d, J = 8.0 Hz, Ph-H), 5.23(2H, s,
Ph-CH2-N), 3.66(3H, s, Ph-COOCH3), 2.73(2H, t, J = 8.0 Hz,
CH2CH2CH2CH3), 1.65(2H, m, J = 8.0 Hz, CH2CH2CH2CH3), 1.36(2H,
m,
J = 7.6 Hz,
CH2CH2CH2CH3),
0.82(3H,
t,
J = 7.6 Hz,
CH2CH2CH2CH3); 13C NMR (DMSO, 400 MHz) d: 170.93, 165.43,
164.98, 162.54, 151.20, 142.10, 139.54, 137.52, 136.96, 133.44,
131.67, 129.37, 128.75, 125.19, 120.61, 118.63, 49.13, 42.87,
31.46, 39.25, 24.40, 16.35; ESI-MS(m/z): 431.1 [M+1]+.
4.1.18. 2-Butyl-1-((20-carboxy-40,50-difluorobiphenyl-4-yl)
methyl)-4-chloro-1H-imidazole-5-carboxylic acid (1c)
m,
J = 8.0 Hz,
CH2CH2CH2CH3),
0.86(3H,
t,
J = 8.0 Hz,
Compound 1c was prepared according to procedure of 1a. Yield:
95.3%. mp: 218–221 °C. Anal. Calcd for C22H19CIF2N2O4: C, 58.87;
H, 4.27; N, 6.24. Found: C, 58.88; H, 4.29; N, 6.25. 1H NMR (DMSO,
400 MHz) d: 12.81(1H, s, COOH), 7.78(1H, dd, J1 = 10.8 Hz,
J2 = 8.0 Hz, Ph-H), 7.45(1H, dd, J1 = 11.2 Hz, J2 = 7.6 Hz, Ph-H),
7.32(2H, d, J = 8.0 Hz, Ph-H), 7.07(2H, d, J =8.0 Hz, Ph-H), 5.30(2H,
s, Ph-CH2-N), 2.61(2H, t, J = 7.6 Hz, CH2CH2CH2CH3), 1.55(2H, m,
J = 7.6 Hz, CH2CH2CH2CH3), 1.27(2H, m, J = 7.6 Hz, CH2CH2CH2CH3),
0.82(3H, t, J = 7.6 Hz, CH2CH2CH2CH3); ESI-MS(m/z): 449.1 [M+1]+.
CH2CH2CH2CH3); 13C NMR(CDCl3, 400 MHz) d : 182.11, 169.57,
165.30, 162.83, 151.39, 144.12, 142.97, 140.23, 135.88, 134.81,
134.42, 131.57, 128.29, 120.95, 120.43, 119.43,54.52, 49.56,
31.82, 29.63, 24.63, 15.99, 12.42; ESI-MS(m/z): 445.0 [M+1]+.
4.1.14. 2-Butyl-4-chloro-1-((40-fluoro-20-(methoxycarbonyl)
biphenyl-4-yl)methyl)-1H-imidazole-5-carboxylic acid (9b)
Compound 9b was prepared according to the procedure of 9a.
mp: 194–196 °C. Yield: 96.0%. 1H NMR (CDCl3, 400 MHz) d:
7.91(1H, dd, J1 = 8.4 Hz, J2 = 5.6 Hz, Ph-H), 7.28(2H, t,
J = 8.0 Hz,Ph-H), 7.11(3H, dd, J1 = 11.2 Hz, J2 = 2.4 Hz, Ph-H),
7.00(1H, dd, J1 = 9.2 Hz, J2 = 2.4 Hz, Ph-H),5.23(2H, s, Ph-CH2–N),
3.64(3H, s, Ph-COOCH3), 2.67(2H, t, J = 7.6 Hz, CH2CH2CH2CH3),
1.71(2H, m, J = 7.6 Hz, CH2CH2CH2CH3), 1.37(2H, m, J = 7.6 Hz,
4.1.19. Methyl 40-((2-butyl-4-chloro-5-(hydroxymethyl)-1H–
imidazol-1-yl)methyl)–4-fluorobiphenyl–2-carboxylate (10)
To a solution of 8b (661 mg, 1.54 mmol) in methanol (30 mL)
was added NaBH4 at 0 °C. The mixture was stirred at room temper-
ature for 1 h. The reaction mixture was poured into ice water and
the organic layers were washed with brine (30 mL ꢁ 3), dried with
MgSO4 and evaporated. The residue was recrystallized with ethyl
acetate-petroleum ether to give a white solid. Yield: 94.6%. 1H
NMR (400 MHz, CDCl3) d: 7.55(1H, dd, J1 = 9.2 Hz, J2 = 2.8 Hz,
Ph-H), 7.32–7.21(4H, m, Ph-H), 7.08(2H, d, J = 8.0 Hz, Ph-H),
5.14(2H, s, Ph-CH2-N), 4.62(2H, s,CH2OH), 3.65(3H, s,–OCH3),
2.63(2H, t, J = 7.6 Hz,CH2CH2CH2CH3), 1.63(2H, m, J = 7.6 Hz,
CH2CH2CH2CH3), 1.33(2H,m, J = 7.6 Hz, CH2CH2CH2CH3), 0.88(3H,
t, J = 7.6 Hz, CH2CH2CH2CH3); 13C NMR (400 MHz,CDCl3) d:
169.67, 150.58, 142.50, 140.36, 140.33, 137.50, 137.09, 134.86,
134.80, 134.49, 134.42, 131.35, 128.34, 120.88, 120.67, 119.11,
161.57, 58.49, 49.17, 32.08, 29.82, 24.72, 16.04; ESI-MS(m/z):
431.3 [M+H]+.
CH2CH2CH2CH3),
0.90(3H,
t,
J = 7.6 Hz,
CH2CH2CH2CH3).
ESI-MS(m/z): 445.0 [M+1]+.
4.1.15. 2-Butyl-4-chloro-1-((40,50-difluoro-20-(methoxycarbonyl)
biphenyl-4-yl)methyl)-1H-imidazole-5-carboxylic acid (9c)
Compound 9c was prepared according to the procedure of 9a.
mp: 208–211 °C. Yield: 98.2%. 1H NMR (CDCl3, 400 MHz) d:
7.75(dd, 1H, J1 = 10.4 Hz, J2 = 8.0 Hz, Ph-H), 7.27(2H, t, J = 8.0 Hz,
Ph-H), 7.15ꢂ7.07(3H, m, Ph-H), 5.23(2H, s, Ph-CH2–N), 3.66(3H, s,
Ph-COOCH3), 2.71(2H,t, J = 7.2 Hz, CH2CH2CH2CH3), 1.67(2H, m,
J = 7.2 Hz, CH2CH2CH2CH3), 1.36(2H, m, J = 7.2 Hz, CH2CH2CH2CH3),
0.91(3H, t, J = 7.2 Hz, CH2CH2CH2CH3); ESI-MS(m/z): 463.0
[M+1]+.
4.1.16. 2-Butyl-1-((20-carboxy-50-fluorobiphenyl-4-yl)methyl)-
4-chloro-1H-imidazole-5-carboxylic acid (1a)
4.1.20. 40-((2-Butyl-4-chloro-5-hydroxy-1H-imidazol-1-
yl)methyl)-4-fluorobiphenyl-2-carboxylic acid (2)
A solution of 9a (49 mg, 0.11 mmol) and 10 mL of 2 M NaOH in
methanol (10 mL) was refluxed for 5 h. The mixture was diluted
with ethyl acetate, and washed with water (50 mL ꢁ 3) and
brine(50 mL ꢁ 3), dried over MgSO4. The solvent was removed
under reduced pressure. The residue was recrystallized with petro-
leum ether and ethyl acetate to give a white solid. mp: 208–211 °C.
Yield: 95.5%. Anal. Calcd for C22H20CIFN2O4: C, 61.33; H, 4.68; N,
6.50. Found: C, 61.35; H, 4.70; N, 6.47. 1H NMR (DMSO,
400 MHz) d: 7.49(1H, dd, J1 = 9.2 Hz, J2 = 1.6 Hz, Ph-H), 7.45–
7.36(2H, m, Ph-H), 7.32(2H, d, J = 8.0 Hz, Ph-H), 7.05(2H, d,
J = 12.8 Hz, Ph-H), 5.30(2H, s, Ph-CH2-N), 2.63(2H, t, J = 7.6 Hz,
CH2CH2CH2CH3), 1.55(2H, m, J = 7.6 Hz, CH2CH2CH2CH3), 1.28(2H,
A solution of 10 (998 mg, 2.24 mmol) and 15 mL of 2 M NaOH in
methanol (30 mL) was refluxed for 6 h. The solvent was removed in
vacuo. The mixture was extracted with ethylacetate. The organic
layer was washed with water (30 mL ꢁ 3) and brine (30 mLꢁ3),
dried over MgSO4. The solvent was removed under reduced pres-
sure. The crude product was recrystallized with petroleum ether
and ethyl acetate to give a white solid. mp: 206-209 °C. Yield:
41.6%. Anal. Calcd for C22H22CIFN2O3: C, 63.39; H, 5.32; N, 6.72.
Found: C, 63.36; H, 5.33; N, 6.74. 1H NMR (CDCl3, 400 MHz,) d:
13.40(s, 2H, COOH), 7.53(1H, dd, J1 = 12.76 Hz, J2 = 7.88 Hz,Ph-H),
7.39(2H, d, J = 8.04 Hz, Ph-H), 7.31(1H, t, J = 8.96 Hz, Ph-H),
7.23(1H, d, J = 7.68 Hz, Ph-H), 7.07(d, J = 8.04 Hz, Ph-H), 5.62(2H, s,
Ph-CH2), 2.60(2H, t, J = 7.52 Hz, CH2CH2CH2CH3), 1.52(2H, m,
J = 7.64 Hz, CH2CH2CH2CH3), 1.25(2H, m, J = 7.64 Hz, CH2CH2CH2
CH3), 0.80(3H, t, J = 7.64 Hz, CH2CH2CH2CH3); 13C NMR (DMSO,
400 MHz) d: 170.93, 165.43, 162.54, 151.21, 142.10, 139.54,
137.52, 136.89, 135.45, 131.67, 129.37, 125.19, 120.61, 120.41,
m,
J = 7.6 Hz,
CH2CH2CH2CH3),
0.82(3H,
t,
J = 7.6 Hz,
CH2CH2CH2CH3); 13C NMR (DMSO, 400 MHz) d: 170.92, 165.44,
164.99, 162.55, 151.21, 142.10, 139.52, 137.52, 136.93, 135.45,
131.67, 129.37, 128.76, 125.19, 120.41, 118.63, 118.40,49.14,
31.47, 29.25, 24.41, 16.35; ESI-MS(m/z): 431.1 [M+1]+.

7110
Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
118.63, 49.13, 42.66, 41.83, 31.47, 29.25, 24.41, 16.35; ESI-MS(m/z):
4.1.25. 40-((2-Butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-
yl)methyl)-4-fluorobiphenyl-2-carboxylic acid (3)
417.1 [M+H]+.
A solution of 14(0.3 g, 0.627 mmol), TFA (2 mL) and dichloro-
methane (2 mL) was stirred for 2 h at 25 °C. The solvent was re-
moved in vacuo. The residue was triturated with diethyl ether
and filtered to afford a white powder. mp: 214–217 °C. Yield:
99.3%. Anal. Calcd for C25H27FN2O3: C, 71.07; H, 6.44; N, 6.63.
Found: C, 71.24; H, 6.46; N, 6.60. 1H NMR (DMSO, 400 MHz) d:
9.08(1H, s, Ph-COOH), 7.66(1H, dd, J1 = 8.8 Hz, J2 = 2.8 Hz, Ph-H),
7.33–7.23(6H, m, Ph-H), 4.81(2H, s, Ph-CH2–Br), 2.67(2H, t,
J = 7.6 Hz, N@CCH2CH2CH2CH3), 2.15–1.99(6H, m, CH2CH2CH2CH2),
1.56(2H, m, J = 7.6 Hz, N@CCH2CH2CH2CH3), 1.29(2H, m, J = 7.6 Hz,
N@CCH2CH2CH2CH3), 0.86(3H, t, J = 7.6 Hz, N@CCH2CH2CH2CH3);
13C NMR (DMSO, 400 MHz) d: 170.91, 170.89, 164.99, 162.55,
161.23, 142.10, 139.59, 139.56, 137.47, 136.92, 136.85, 135.38,
131.63, 129.33, 120.66, 120.45, 118.67, 118.44, 76.09, 45.82,
30.08, 29.12, 28.08, 24.24, 16.21; ESI-MS(m/z): 423.1 [M+1]+.
4.1.21. 4-Fluoro-40-methylbiphenyl-2-carboxylic acid (11)
A solution of 6a (2.687 g, 11 mmol) and 16 mL of 2 M NaOH in
methanol (30 mL) was refluxed for 6 h. The solvent was removed in
vacuo. The residue was added with H2O and the aqueous layer was
washed with diethyl ether and acidified with 1 M HCl to pH = 3.
The resulting precipitate was collected by filtration and dissolved
in EtOAc and dried with MgSO4. The solvent was removed under
reduced pressure and the residue was recrystallised with
EtOAc–petroleum ether. The product was obtained as white solid.
mp: 154–157 °C. Yield: 66.5%. 1H NMR (400 MHz, CDCl3) d: 7.72–
7.67 (3H, m, Ph-H), 7.38–7.22(4H, m, Ph-H), 2.42(3H, s, Ph-CH3);
ESI-MS (m/z): 231.0 [M+1]+.
4.1.22. Tert-butyl 4-fluoro-40-methylbiphenyl-2-carboxylate
(12)
4.1.26. (S)-Methyl 4-fluoro-40-((1-methoxy-3-methyl-1-
To a solution of 11 (1.884 g, 8.19 mmol) in dichloromethane
was added oxalyl chloride (0.86 mL,10 mmol) at 0 °C. After addi-
tion, the reaction mixture was warmed to 25 °C and stirred for
3 h. The excess oxalyl chloride was removed under reduced pres-
sure. Then potassium tert-butanolate (0.917 g, 8.19 mmol) was
added into the above residue in THF at 0 °C in portions. The reac-
tion mixture was stirred at room temperature for 1 h. The mixture
was poured into ice water and extracted with EtOAc .The combined
organic layers were washed with brine (10 mL ꢁ 3), dried with
MgSO4. After filtration, the solvent was removed under reduced
pressure. The residue was purified by flash column chromatogra-
phy with petroleum ether-ethyl acetate (125/1) as eluant. The
product was obtained as colourless oil. Yield: 44.2%. 1H NMR
(CDCl3, 400 MHz) d: 7.49(1H, dd, J1 = 8.8 Hz, J2 = 2.8 Hz, Ph-H),
7.32(1H, dd, J1 = 8.4 Hz, J2 = 3.2 Hz, Ph-H), 7.16ꢂ7.27 (5H, m,
Ph-H), 2.46(3H, s, Ph-CH3), 1.32(9H, s, Ph-COOC4H9); ESI-MS(m/
z): 287.3 [M+1]+
oxobutan-2-ylamino)-methyl)-biphenyl-2-carboxylate (15)
A solution of 7b (1.844 g, 11 mmol), L-Valine methyl ester
hydrochloride (2.9 g, 10 mmol), DIPEA (1.939 g, 15 mmol) and
dichloromethane (40 mL) was refluxed for 10 h under nitrogen.
The solvent was removed in vacuo. The residue was diluted with
H2O and extracted with ethyl acetate. The organic layer was
washed with water (10 mLꢁ3) and brine (10 mL ꢁ 3), dried over
MgSO4. The solvent was removed under reduced pressure. The res-
idue was purified by flash column chromatography with petroleum
ether–ethyl acetate (25/1) as eluant. Yield: 53.5%. 1H NMR (CDCl3,
400 MHz) d: 7.53(1H, dd, J1 = 8.8 Hz, J2 = 2.4 Hz, Ph-H), 7.39–
7.33(3H, m, Ph-H), 7.25–7.21(3H, m, Ph-H), 3.89(1H, d,
J = 13.2 Hz, Ph-CH2–N), 3.85(1H, d, J2 = 13.2 Hz, Ph-CH2–
N),3.75(3H, s, O@C–O–CH3), 3.67(3H, s, Ph-C@O–OCH3), 3.06(1H,
d, J = 6.0 Hz, HN–CH(C@O)CH(CH3)2), 1.96(1H, m, J = 6.8 Hz,
HN–CH(C@O)CH(CH3)2),
1.84(1H,s,
HN–CH(C@O)CH(CH3)2),
0.98(6H, dd, J1 = 17.6 Hz, J2 = 10.4 Hz, HN–CH(C@O)CH(CH3)2);
ESI-MS(m/z): 374.2 [M+1]+.
4.1.23. Tert-butyl 40-(bromomethyl)-4-fluorobiphenyl-2-
carboxylate (13)
4.1.27. (S)-Methyl 4-fluoro-40-((N-(1-methoxy-3-methyl-1-
oxobutan-2-yl)butyramido)methyl)biphenyl-2-carboxylate (16)
Compound 13 was prepared according to the procedure of 7a.
Yield: 65.7%. 1H NMR (CDCl3, 400 MHz) d: 7.52(1H, dd,
J1 = 9.2 Hz, J2 = 2.8 Hz, Ph-H),7.43(2H, d, J = 4.4 Hz, Ph-H), 7.31–
7.22(3H, m,Ph-H), 7.22–7.15(1H, m, Ph-H), 4.57(2H, s, Ph-CH2–
To
a solution of 15 (1.935 g, 5.3 mmol), DIPEA (1 mL,
15.7 mmol) and dichloromethane (15 mL) was added dropwise n-
butyryl chloride(0.81 mL, 7.8 mmol) at 0 °C. The mixture was stir-
red for 6 h at 28 °C. The resulting mixture was poured into ice
water, the organic layer was washed with diluted hydrochloric
Br), 1.25(3H, s, Ph-COOC4H9); ESI-MS(m/z): 365.2 [M+1]+
.
4.1.24. Tert-butyl40-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-
en-3-yl)methyl)-4-fluorobiphenyl-2-carboxylate (14)
acid,
sodium
bicarbonate
solution
(15 mL ꢁ 3)
and
brine(30 mL ꢁ 3), dried by MgSO4, and concentrated in vacuo.
The product was purified by chromatography with petroleum
ether–ethyl acetate (15/1) as eluant. The product was obtained as
colorless oil. Yield: 76.2%. 1H NMR (CDCl3, 400 MHz) d: 7.40–7.29
(7H, m, Ph-H), 4.97–4.66 (1H, two d, J1 = 15.2 Hz, J2 = 17.6 Hz, –
NCH2–), 4.90(1H, two d, J = 10.4 Hz, –N–CH–), 4.60–4.25 (2H, two
d, J1 = 15.2 Hz, J2 = 17.6 Hz, –NCH2–), 3.60–3.50 (3H, two s, -
OCH3), 3.39–3.34 (3H, two s, –PhCOOCH3), 2.34–2.19 (3H, m, –
CH2CH2CH3 and –CH(CH3)2), 1.79–1.61 (2H, m, –CH2CH2CH3),
0.99–0.72 (9H, m). ESI-MS (m/z): 459.2 [M+1]+.
A solution of 2-butyl-4-spirocyclopentane-2-imidazolin-5-one
hydrochloride (0.580 g, 2.63 mmol) and sodium hydride (0.250 g,
6.25 mmol) in THF (30 mL) under nitrogen was stirred for 0.5 h
at 50 °C. After cooling, a solution of 13 (0.804 g, 2.2 mmol) in the
THF (10 mL) was added dropwise. The resulting mixture was stir-
red at 50 °C for 3 h. The mixture was poured into 30 mL of ice-
water and extracted with EtOAc. The organic layers were washed
with brine (10 mL ꢁ 3), dried with MgSO4. After filtration, the
solvent was removed under reduced pressure. The residue was
purified by flash column chromatography with petroleum ether–
ethyl acetate (3/1) as eluant. A white solid 330 mg was obtained.
Yield: 30.6%. 1H NMR (CDCl3, 400 MHz) d: 7.47(1H, dd,
J1 = 9.2 Hz, J2 = 2.8 Hz, Ph-H), 7.27ꢂ7.14 (6H, m, Ph-H), 4.72(2H, s,
4.1.28. (S)-40-((N-(1-Carboxy-2-
methylpropyl)butyramido)methyl)-4-fluorobiphenyl–2-
carboxylic acid (4)
Compound 4 was prepared according to the procedure of 3. Mp:
215–218 °C. Yield: 99.5%. Anal. Calcd for C23H26FNO5: C, 66.49; H,
6.31; N, 3.37. Found: C, 66.48; H, 6.30; N, 3.39. 1H NMR (CDCl3,
400 MHz) d: 7.56(1H, dd, J1 = 8.0 Hz, J2 = 2.4 Hz, Ph-H), 7.28–7.10
Ph-CH2–Br),
2.35(2H,
t,
J = 7.6 Hz,
N@CCH2CH2CH2CH3),
2.05–1.92(8H, m, CH2CH2CH2CH2), 1.61(2H, m, J = 7.6 Hz,
N@CCH2CH2CH2CH3), 1.35(2H, m, J = 7.6 Hz, N@CCH2CH2CH2CH3),
1.25(3H, s, Ph-COOC4H9), 0.89(3H, t, J = 7.6 Hz, N@CCH2CH2
CH2CH3); ESI-MS(m/z): 479.2 [M+1]+.

Y. Da et al. / Bioorg. Med. Chem. 20 (2012) 7101–7111
7111
(6H, m, Ph-H), 4.94–4.74 (2H, two d, J1 = 16.0 Hz, J2 = 17.6
4.4. In vitro study of anticancer activity
Hz, –NCH2–), 4.54–4.31 (2H, two d, J1 = 15.2 Hz, J2 = 17.6
Hz, –NCH2–), 4.30–4.03 (1H, two d, J = 10.4 Hz, –N–CH –),
2.63–2.32 (3H, m, –CH2CH2CH3 and–CH(CH3)2), 1.76–1.61(2H, m,
–CH2CH2CH3), 0.98–0.86(3H, t, –CH2CH2CH3); 0.84–0.80(6H,
d, –CH(CH3)2). 13C NMR (DMSO, 400 MHz) d: 178.68, 175.82,
172.48, 165.09, 162.62, 142.06, 140.93, 140.88, 139.16, 137.50,
134.91, 134.22, 130.59, 129.57, 121.06, 119.64, 68.56, 53.61,
38.00, 29.84, 22.20, 21.11, 16.11; ESI-MS(m/z): 416.2 [M+1]+.
The MTT assays were performed as previously described.18
Briefly, cells (1 ꢁ 105 cells) were seeded in 96-well culture plates
and cultured overnight at 37 °C, 5% CO2 atmosphere. LNCaP cells
were incubated with Ang II (1 ꢁ 10 ꢀ7 M), Ang II (1 ꢁ 10
ꢀ7 M) + Losartan (10
l
M), Ang II (1 ꢁ 10ꢀ7 M) + compound
4
(10 M), Losartan (10 lM), compound 4 (10 lM), respectively.
l
The control group received drug-free medium with 0.05% v/v
DMSO. Subsequently, MTT (5 mg/mL) was added to each well after
24, 48, 72 and 96 h separately. After incubation at 37 °C for 4 h, the
4.2. Radioligand binding assay
medium was discarded and 150 lL DMSO was added into each
The vascular smooth muscle cells (VSMCs) were obtained from
thoracic aorta of SD rats and cultured by the tissue explants meth-
ods.14 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
10000ꢁg for 5 min. They were then incubated with 1 mL of DMEM
supplemented with 15% fetal bovine serum (FBS) at 37 °C in 95% air
5% CO2. Cells at passage 3–7 were used for the experiments.
well. The optical density was measured by a microplate reader at
a test wavelength of 570 nm.
4.5. In vivo study of antitumor activity
The antitumor activity of compound 4 was determined in athy-
mic nude mice bearing LNCap tumors. LNCap cells (107) were in-
jected into the flank region of athymic nude mice (4–6 weeks
old), and treatment was started on day 8 when the tumor mea-
sured 5 mm in diameter.12 Each mouse was received one of two
Each 250
l
L
incubate contained the following: 0.1 nM
different doses of compound 4 (5.0 or 10.0 mg/kg/day) and
[125]Angiotensin II (Northern Biotechnology Company, China) and
concentration of test compounds. The final concentrations were
1 ꢁ 10ꢀ6–1 ꢁ 10ꢀ12 M. They were incubated in 24-well plates with
VSMCs for 60 min at 37 °C. Nonspecific binding was measured in
Losartan (5.0 or 10.0 mg/kg/day) was taken as positive group.
The control group was received water containing sodium hypo-
chlorite (10 ppm). Each group consisted of 10 animals. Tumors
were measured with a caliper every 7 days. The volume of the tu-
mor was calculated using the formula: tumor volume (mm3) =
the presence of 1 lM Ang II and represented 5–10% of total bind-
ing. After the reaction, removed the liquid immediately, washed
five times with PBS, and digested cells with 0.1 M NaOH for
2
length ꢁ (width) ꢁ 0.5.
10 min. The radioactivity was counted with a
c-counter (Wallac
Acknowledgments
1470 Wizard, PerkinElmer, Finland). The IC50 value (concentration
for 50% displacement of the specifically bound [125]Angiotensin II)
was determined by regression analysis of displacement curves.15
The inhibition constant (Ki value) was calculated from the formula
Ki = IC50/(1 + [L]/kd),16 where [L] was the concentration of radioli-
gand present in tubes.
This work was supported by Chinese National Natural Science
Foundation (No.30973611; 81102407; 81101298), Foundation of
Shanghai government (No.10DZ0502300; 10ZZ45), Foundation of
Donghua University (No. 11D10501; 11D10518; 12D10515;
105-10-0044030; LK0902; BC201128) and Foundation of Yiwu
government (2011-G1-15)
4.3. In vivo study of anti-hypertensive effect
For preparing renal hypertensive rats, the left renal arteries of
Sprague-Dawley rats (250–350 g, Second Military Medical
University, China) were completely ligated under sodium pento-
barbital (40 mg/kg) anesthesia. Thereafter, the rats with SBP higher
than 160 mmHg were selected and used as renal hypertensive rats.
Spontaneous hypertensive rats (250–300 g) came from Second
Military Medical University, China too. Than both spontaneous
and renal hypertensive rats were randomly divided into different
experimental groups of 10 animals (negative control group, posi-
tive control groups, compound low-dose groups and high-dose
groups). Each compound was suspended in a 0.5% solution of so-
dium carboxymethyl cellulose and administered orally at the dose
of 5 and 10 mg/kg separately. Losartan (10 mg/kg) and Valsartan
(10 mg/kg) (SanXin Zhujiang Chemical Engineering Company)
were taken as positive control groups. The negative control group
was administered the same volume of sodium carboxymethyl cel-
lulose solution. To measure the blood pressure, the animals were
anesthetized with sodium pentobarbital (40 mg/kg, ip). The right
carotid artery and jugular vein were cannulated for arterial pres-
sure measurement and drug administration, respectively. The arte-
rial catheter was connected to a pressure transducer and displayed
on a computer and analyzed with a biological signal analysis
system (MPA-2000, Alcott Biotech, China). The parameters were
measured continuously for more than 10 h, and again at 24 h.17
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.065.
Reference and notes
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