Synthesis and biological evaluation of 4′-[(benzimidazole-1-yl) methyl]biphenyl-2-sulfonamide derivatives as dual angiotensin II/endothelin A receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.06.011

A series of 4′-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfonamide derivatives (Ia-Il) were synthesized and biologically evaluated. It was found that Ig, the most active compound, antagonized both Ang II AT₁ and endothelin ET_A receptors (AT₁ IC₅₀ = 8.5, ET_A IC₅₀ = 8.9 nM), and was more potent than losartan in RHRs with no significant effect on heart rate. The preliminary structure-activity relationships were also discussed in the present paper.

Full text of DOI:10.1016/j.bmc.2012.06.011

Bioorganic & Medicinal Chemistry 20 (2012) 4661–4667
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 40-[(benzimidazole-1-yl)methyl]biphenyl-
2-sulfonamide derivatives as dual angiotensin II/endothelin A receptor
antagonists
Renren Bai a, Zhen Wei a, Jie Liu a, Weijia Xie a, Hequan Yao a,b, Xiaoming Wu a,b, , Jieyun Jiang d,
⇑
Qiujuan Wang c, Jinyi Xu a,b,
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
c Department of Physiology, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
d Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
A series of 40-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfonamide derivatives (Ia–Il) were synthesized
and biologically evaluated. It was found that Ig, the most active compound, antagonized both Ang II AT1
and endothelin ETA receptors (AT1 IC50 = 8.5, ETA IC50 = 8.9 nM), and was more potent than losartan in
RHRs with no significant effect on heart rate. The preliminary structure–activity relationships were also
discussed in the present paper.
Article history:
Received 21 April 2012
Revised 5 June 2012
Accepted 6 June 2012
Available online 15 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Antihypertensive activity
DARAs
AT1 receptor
ETA receptor
Structure–activity relationships
1. Introduction
Indeed, L-746,072 (1) was first documented to possess an ability
to antagonize both AT1 and ETA receptors, with IC50 values of 24
Antihypertensive drugs with high efficacy and low side-effects
remain to be one of the largest unmet medical needs, especially
when hypertension is considered to be the portent of the future
debilitating cardiovascular disease.1,2 Most of the currently used
antihypertensive agents cannot be used as a single drug therapy
because of their limited efficacy,3 therefore, the development of
drugs with multiple therapeutic effects is most desirable. It is
well-known that angiotensin II subtype 1 (AT1) receptor antago-
nists are clinically useful for the management of hypertension
and heart failure,4,5 and endothelin subtype A (ETA) receptor antag-
onists show promising effects in the treatment of the similar indi-
cations.6 Interestingly, some of these two types of antagonists have
close similarity in structure. Hence, lots of attempt has been made
to hybridize both AT1 and ETA receptor antagonistic properties in
one molecule. It is anticipated that drugs with dual AT1 and ETA
receptor antagonistic action could be more effective than the cur-
rent standard therapies for the treatment of hypertension.
and 13 nM respectively in ETA and AT1 radioligand binding assays.7
Shortly after, BMS-248360 (2)1 and BMS-346567 (3)8 (Fig. 1) were
also proved to be potent dual-action receptor antagonists (DARAs).
Compound BMS-248360 as a potential DARA, which had potent
affinity for both AT1 (Ki = 10 nM) and ETA (Ki = 1.9 nM) receptors.
Additionally, a series of 40-[(imidazol-1-yl)-methyl] biphenyl-
sulfonamides have been reported to be such dual-action antago-
nists in the treatment of hypertension, heart failure, and other
cardiovascular disorders.6
Previously, we designed and synthesized a series of derivatives
of 2-alkylbenzimidazoles with N-phenyl pyrrolyl-2-tetrazole moi-
ety. Among them, compound 4 is an orally active AT1 receptor
antagonist (IC50 = 9.8 nM) that is more potent and efficacious than
losartan, which selectively inhibited Ang II-induced contractions of
rabbit aortic strips in a competitive manner and had no effect on
the contraction induced by norepinephrine (10 nM), KCl (10 nM),
and histamine (10 nM).9 It should be also noted that when we
started to prepare our manuscript, the novel AT1 receptor antago-
nist azilsartan medoxomil (TAK-491) with benzimidazole moiety
has been approved by FDA in 2011.10 Given that an acidic function-
ality such as N-isoxazolyl substituted sulfonamide is crucial for
⇑
Corresponding authors. Tel.: +86 025 83271445 (J.X.).
E-mail addresses: xmwu@cpu.edu.cn (X. Wu), jinyixu@china.com (J. Xu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.011

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R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
H3C
N
N
CH3
CH3
N
N
N
CH3
N
O
N
O
H3C
H3C
CH3
CH3
N
O
N
O
O
C2H5
N
O
O
O
O
O
CH3
CH3
O
S
S
H
N
O
N
N
O
O
H
H
S
CH3
CH3
O
O
L-746,072, 1
BMS-248360, 2
BMS-346567, 3
Figure 1. The structures of known DARAs.
R2
N
Heterocycles
O
N
N
Bu-n
N N
NH
N
R3
N
O
O
N
H3COOC
CH3
N
O
S
N
O
O
CH3
H
S
CH3
N
N
H
CH3
Ia-e
AT1 receptor antagonist, 4
ETA receptor antagonists
R2
N
N
R1
N
O
O
O
CH3
S
N
H
CH3
If-l
Figure 2. Strategy for the design and optimization of target DARAs.
DARAs 2 and 3, and that modifications of the classical biphenyl
tetrazole moiety in some AT1 receptor antagonists did not drasti-
cally decrease the potency,11–14 it may be rational to replace
N-phenyl pyrrolyl-2-tetrazole moiety in 4 by biphenyl-2-[3,4-
dimethyl-5-isoxazolyl]-sulfonamide of compound 2 in order to
develop single agents with the ability to antagonize both AT1 and
ETA receptors.
modification at the 6-position of benzimidazole of Ie to obtain
the series of compounds If–Il (Fig. 2).
Herein, we report the synthesis and biological evaluation of
40-[(benzimidazole-1-yl)-methyl]-biphenyl-2-sulfonamides deriv-
atives (Ia–Il).
2. Results and discussions
2.1. Chemistry
First, a series of 40-[(benzimidazole-1-yl)methyl]biphenyl-2-
sulfonamides derivatives (Ia–Ie) were synthesized. The pharmaco-
logical results suggested that compound Ie, with a butyl group at
the 2-position of benzimidazole, was the most potent one in this
series. Our efforts were followed by additional optimization and
The synthetic route used to synthesize the title compounds is
outlined in Schemes 1–3.
N
C
O
O
C
NH2
N
H2
C
a
O
b
c
H3C
Br
CN
O
H3C
OBu
H3C
H3C
CH3
CH3
8
5
6
7
N
N
N
B(OH)2
O
O
Br
O
O
O
O
O
S
O
N
CH3
CH3
CH3
S
N
H
S
d
e
N
CH3
CH3
CH3
MEM
MEM
9
10
11
Scheme 1. Reagents and conditions: (a) NaOBu, Xylene, reflux, 3 h, 55–67%; (b) NH2OHꢀHCl, NaOH/H2O, reflux, 3 h, 32.7%; (c) 4-bromobenzenesulfonyl chloride; Pyridine, rt,
over night, 90%; (d) MEMCl, NaH, THF, 0 °C to rt, 5 h, 70.6%; (e) B(OBu-n)3, n-BuLi, toluene/THF, ꢁ40 °C, 30 min, 38.6%.

R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
4663
N
N
NH2
NH2
R2
N
g
R2
R1
f
R1
R1
N
H
Br
12
13a-l
14a-l
Scheme 2. Reagents and conditions: (f) R2COOH, 4 N HCl, reflux, 2 h, 45.0%; (g) 1-bromo-4-(bromomethyl)benzene, NaH, DMF, 0 °C to rt, 5 h, 64.0%.
R2
R2
N
N
N
N
h
i
11 14a-l
R1
N
R1
N
O
O
O
N
O
CH3
O
O
CH3
S
S
N
H
CH3
MEM
CH3
Ia-l
15a-l
R1 = H, CH3, OCH3, Cl, Br, F, CF3, NO2
R2 = H, Me, Et, n-Pr, n-Bu
Scheme 3. Reagents and conditions: (h) (a) Pd(OAc)2, 2 N Na2CO3, Ph3P, N2, reflux, 1 h; (b) H2O, rt, 3 h, 60.0%; (i) EtOH, 6 N HCl, reflux, 2 h, 64%.
Ethyl cyanide (5) reacted with butyl acetate (6) in xylene to ob-
Table 1
tain 2-methyl-3-oxobutanenitrile (7). Treatment of 7 with oxam-
monium hydrochloride in sodium hydroxide solution afforded 3,
4-dimethylisoxazol-5-amine (8), which was then subjected to
chlorosulfonation with 2-bromobenzene-1-sulfonyl chloride at
room temperature to provide N-(3,4-dimethyl-5-isoxazolyl)-
2-bromobenzene-sulfonamide (9). Further reaction with MEM
chloride gave the protected benzenesulfonamide (10). The key
intermediate 2-borono-N-(3,4-dimethyl-5-isoxazolyl)-N-(methoxy-
ethoxy- methyl)benzenesulfonamide (11) was prepared by treating
10 with tributyl borate and n-BuLi in THF at ꢁ40 °C. Cyclization of
the 2-substituent o-phenylenediamines (12) with aliphatic carbox-
ylic acids in aqueous 4 N HCl provided imidazolines (13a–13l),
which were then alkylated with 1-bromo-4-(bromomethyl)ben-
zene to provide benzimidazole derivatives (14a–14l). The impor-
tant intermediates (11 and 14a–14l) in propanol were then
treated with triphenyl phosphine and palladium(II) acetate under
mild condition to give 15a–15l, followed by MEM deprotection,
to provide 40-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfona-
mides derivatives Ia–Il as target compounds.
SAR of 40-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfonamide derivatives (Ia–Il)
R2
N
N
R1
N
O
O
O
CH3
CH3
AT1 receptor IC50 (nM) ETA receptor IC50 (nM)
S
N
H
Compound R1
R2
Ia
Ib
Ic
Id
Ie
If
Ig
Ih
Ii
Ij
Ik
Il
H
H
H
H
H
Me
H
Me
Et
92 8.6
45 2.1
28 1.5
16 1.4
99 14
75 6.1
39 4.3
22 0.9
9.8 0.3
9.3 0.1
8.9 0.3
9.6 0.4
9.8 0.2
10 0.9
10 0.7
11 0.8
/
n-Pr
n-Bu 9.9 0.1
n-Bu 9.1 0.2
OMe n-Bu 8.5 0.2
Cl
Br
F
CF3
NO2
/
n-Bu 9.8 0.3
n-Bu 10 0.7
n-Bu 10.5 1.0
n-Bu 11 0.9
n-Bu 12 0.6
2.2. Pharmacological evaluation
2.2.1. In vitro Ang II AT1 and endothelin ETA receptors
antagonism
Losartan
Bosentan
/
/
95 11
/
All target compounds were evaluated for Ang II AT1 and endo-
thelin ETA receptors antagonism in vitro and the activity was ex-
pressed as IC50 values (Table 1). In Ang II AT1 and endothelin ETA
binding assays, the unsubstituted benzimidazole derivative Ia
was found to be the least active, which indicated that the optimi-
zation of benzimidazole is quite necessary. The activity of com-
pounds Ib–Ie with alkyl substitution at the 2-position increased
obviously with the enlargement of alkyl groups. IC50 values of
Ib–Ie decreased from 45 to 9.9 nM in AT1 receptor antagonism as-
says and from 75 to 9.8 nM in ETA receptor antagonism evaluation
respectively, which possibly arose from hydrophobic interactions
between different alkyl groups of Ib–Ie with AT1 and ETA receptors.
/
8.9 0.5
antagonist reference drug (losartan) and comparable to ETA recep-
tor antagonist reference drug (bosentan).
On the basis of combined strong dual receptor blockade of com-
pounds Ia–Ie, further optimization of our strategy was to introduce
different functional groups at the 6-position of the benzimidazole
moiety. Substitution of 6-H with –CH3, –OCH3, –Cl, –Br, –F, –CF3
and –NO2 groups obtained the corresponding compounds If–Il.
Generally, the title compounds of this series did not bring dramatic
changes in their antagonism. As shown in Table 1, Ig was found to
exhibit slightly better activity (AT1 IC50 = 8.5 nM, ETA IC50 = 8.9 nM)
than Ie.
Especially, compound Ie possessing
a n-butyl group clearly
demonstrated the most powerful effect (AT1 IC50 = 9.9 nM, ETA
IC50 = 9.8 nM), which was more powerful than AT1 receptor

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R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
Table 2
Effects on blood pressure in renal antihypertensive rats
Groups
Control
No. of animals
Dose (mg/kg)
Parameter
Time of observation (min)
0 h
2 h
4 h
6 h
8 h
10
SAP (mmHg)
DAP (mmHg)
HR (BPM)
185.50 11.2
193.70 5.0
190.22 7.8
186.45 10.1
184.24 12.8
8.20 6.2
149.90 7.0
4.72 3.4
147.12 5.3
0.95 1.1
146.54 9.2
ꢁ1.26 1.6
145.50 5.4
145.61 10.3
4.40 2.5
416.90 20.4
1.62 2.2
410.80 19.4
1.04 1.1
415.78 20.8
0.11 2.3
416.80 22.5
414.68 10.7
2.22 10.5
ꢁ3.88 15.1
1.10 11.3
2.12 20.6
Losartan
10
10
20
20
SAP (mmHg)
DAP (mmHg)
HR (BPM)
198.15 11.2
155.14 12.4
413.75 15.1
191.74 14.6
183.04 16.6
180.02 10.5
182.14 11.7
ꢁ6.41 3.7*
ꢁ15.11 6.5**
ꢁ18.13 9.8**
ꢁ16.01 9.8**
144.09 10.1
134.99 9.6
133.57 8.1
141.06 10.2
ꢁ11.05 2.7**
ꢁ20.15 7.8***
ꢁ21.57 7.8***
ꢁ14.08 6.0**
407.25 24.1
411.06 16.1
412.80 15.4
410.55 10.2
ꢁ6.50 19.7
ꢁ2.69 15.2
0.95 17.1
ꢁ3.2 20.0
Ig
SAP (mmHg)
DAP (mmHg)
HR (BPM)
192.88 10.7
152.68 12.5
412.40 18.6
181.33 10.6
168.92 12.1
168.42 10.5
172.03 12.1
ꢁ11.55 5.9**
ꢁ23.96 5.4***
ꢁ24.46 3.1***
ꢁ20.85 4.3***
138.62 10.5
130.58 8.4
127.66 9.2
133.68 9.1
ꢁ14.06 4.1**
ꢁ22.10 6.5***
ꢁ25.02 5.6***
ꢁ19.00 7.4**
408.14 14.9
407.28 22.1
415.62 27.5
417.97 29.2
ꢁ4.26 9.7
ꢁ5.12 10.0
3.22 20.1
5.57 20.8
Each value represents the mean SEM (n = 8).
*
Significance levels p <0.1.
Significance levels p <0.05.
*
***
Significance levels p <0.01 as compared with the respective control. Changes of BP (HR) (D) = BP (HR) after administration—P (HR) before administration.
Figure 3. (A) The acute antihypertensive activities of compound Ig and losartan in RHRs (SAP, systolic arterial pressure); (B) The acute antihypertensive activities of
compound Ig and losartan in RHRs (DAP, diastolic arterial pressure); (C) The changes of heart rate (HR) of compound Ig and losartan in RHRs.
2.2.2. In vivo antihypertensive activity of compound Ig
biphenylsulfonamide ETA receptor antagonist (BMS-248360), a
series of 40-[(benzimidazole-1-yl) methyl]biphenyl- 2-sulfonamides
derivatives (Ia–Ie) were designed and synthesized. Compound Ie
had an IC50 of 9.8 nM for functional antagonism of ETA receptor,
and displayed IC50 of 9.9 nM in AT1 receptor antagonism assays.
To analyze the influence of electron-donating and electron-
withdrawing groups on the aimed molecules, compounds If–Il
were further designed and obtained. The results revealed that
the derivatives of this series exhibited powerful antagonism on
both AT1 and ETA receptors, better or equal than either losartan
or bosentan respectively. The most prospective compound Ig was
found to have the most effective antagonism for Ang II AT1 and
endothelin ETA receptors, exhibiting more potent activity (AT1
IC50 = 8.5 nM, ETA IC50 = 8.9 nM) than losartan and equivalent
activity to bosentan.
Preliminary SAR suggested that the activities of the compounds
with alkyl substitution at the 2-position of benzimidazole in-
creased obviously with the enlargement of the alkyl groups. Espe-
cially, compounds possessing the n-butyl group clearly
demonstrated the most potent inhibitory effect. This suggests a
hydrophobic pocket of receptor into which an aliphatic chain of
the proper length, for example, n-butyl group, fits tightly.
Further substitution with electron-withdrawing groups (–Cl, –Br,
–F, –CF3, –NO2) and electron-donating groups (–CH3 and –OCH3) at
Biological evaluation in vitro demonstrated that compound Ig
was the most potent DARA among the titled compounds. As an
interesting new entity, compound Ig was selected for further eval-
uation of antihypertensive effects in vivo. Its influence on blood
pressure (BP) was analyzed in renal antihypertensive rats (RHRs)
under ethyl carbamate (EC) anesthesia (1.0 g kgꢁ1 ip) compared
to the reference drug losartan. After the intravenous administra-
tion at 20 mg kgꢁ1, Ig reduced the blood pressure significantly
(Table 2). This trend continued throughout the remaining time of
the study. The average SAP of the RHRs treated with Ig was re-
duced by almost 10–20 mmHg for about 8 h, which was obviously
superior to the SAP reduction achieved by losartan (Fig. 3). Mean-
while, the maximum antihypertensive effect on DAP of compound
Ig was prominently more effective than that of losartan. Heart
rates recorded taken from RHRs proved that Ig, just the same as
losartan, did not cause noticeable changes of HR. Preliminary bio-
logical evaluation in vivo showed that compound Ig was promising
enough for further investigation.
3. Conclusions
In summary, by merging together the key structural element
of AT1 receptor antagonist 4 with the key structural moiety of

R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
4665
the 6-position of benzimidazoles did not increased the activities
4.1.4. 2-Bromo-N-(3,4-dimethyl-5-isoxazolyl)-N-(methoxy-
notablely. Strongly electron-withdrawing ones (–F, –CF3, –NO2) re-
duced the antagonistic activity of both receptors slightly compared
to the weekly electron-withdrawing ones (–Cl, –Br). On the con-
trary, the electron-donating groups (–CH3 and –OCH3) are favor-
able in improving the activity slightly, especially compound Ig
bearing methoxyl moiety.
Biological evaluation in vivo suggested that Ig is more potent
and efficacious than losartan in RHRs, with no significant impact
on heart rate. These results suggested that further optimization
of this series of derivatives could yield highly-potent, orally active
dual receptor antagonists.
ethoxy-methyl)benzenesulfonamide (10)
Compound 9 (1.53 g, 0.046 mol) was dissolved in THF, then be-
low 0 °C NaH (0.445 g, 60% in mineral oil, 0.011 mol) was added in
batches to the solution of 9 and the reaction solution was stirred
under nitrogen for 15 min. Then MEMCl (0.87 g, 0.056 mol) was
added and stirred at room temperature for 5 h. The mixture was
concentrated, diluted with water (25 mL), and extracted with
EtOAc (3 ꢂ 20 mL). The combined organic extracts were concen-
trated and purified by flash column chromatography with n-hex-
ane/ethyl acetate (3:1, v/v) to afford a white solid 1.37 g in yield
of 70.6%, mp 63–65 °C.
4. Experimental
4.1. Chemistry
4.1.5. 2-Borono-N-(3,4-dimethyl-5-isoxazolyl)-N-(methoxy-
ethoxy-methyl)benzenesulfonamide (11)
At ꢁ40 °C and under nitrogen, n-BuLi (4.8 mL, 11.8 mmol) was
added dropwise to the mixture of toluene (13 mL), THF (8 mL),
tributyl borate (3.2 mL, 11.8 mmol) and compound 10 (3.30 g,
7.9 mmol). The reaction mixture was stirred for 30 min, then its
temperature increased slowly to ꢁ20 °C. 2 N hydrochloric acid
(8 mL) was added and the temperature was rised to room temper-
ature. The water layer was basified to pH 7 with sodium hydroxide
solution and extracted with THF (3 ꢂ 10 mL). The combined organ-
ic extracts were concentrated and purified by flash column chro-
matography with petroleum ether/EtOAc (1:3, v/v) to afford a red
mucoid material in yield of 38.6%. This material was used without
further purification.
Most chemicals and solvents were of analytical grade and, when
necessary, were purified and dried by standard methods. Melting
points were taken on an XT-4 micro melting point apparatus and
uncorrected. IR spectra were recorded in KBr on a Nicolet Impact
410 grating infrared spectrophotometer (vmax in cmꢁ1) and 1H
NMR spectra were recorded with 300 or 500 MHz.
Spectrometers in the indicated solvents (TMS as internal stan-
dard): the values of the chemical shifts are expressed in d values
(ppm) and the coupling constants (J) in Hz. High-resolution mass
spectra were recorded using Agilent QTOF 6520.
Purity of all tested compounds was P95%, as estimated by HPLC
analysis. The major peak of the compounds analyzed by HPLC ac-
counted for P95% of the combined total peak area when moni-
tored by a UV detector at 254 nm. Flash chromatography was
done on Merck silica gel 60 (230–400 mesh).
4.1.6. General procedure for the preparation of 1H-
benzimidazole (13a–13l)
An
appropriately
2-substituent
o-phenylenediamines
(50 mmol) and derivatives of methanoic acid (60 mmol) were
added to 4 N hydrochloric acid (60 mL). The resulting mixture
was heated to reflux for 2 h and basified with ammoniae aqua.
The mixture was then filtered and the filter cake was washed with
water and recrystallized with 95% alcohol to provide 13a–13l as
white solids in yields of 60–75%.
4.1.1. 2-Methyl-3-oxobutanenitrile (7)
To the solution of butyl acetate (116.1 g, 1 mol) in 150 mL xy-
lene, NaOBu (48.1 g, 0.5 mol) was added, followed by ethyl cyanide
(27.5 g, 0.5 mol). The resulting mixture was heated to reflux for 3 h,
and cooled to room temperature, adjusting pH to 2.5–3 with acid
hydrochloric acid and adding 100 mL water. After partitioned,
water layer was extracted with toluene (3 ꢂ 80 mL), and the com-
bined organic extracts were washed with water, dried over MgSO4
and concentrated under reduced pressure, followed by atmo-
spheric distillation at 145–146 °C to afford 27 g compound 7 as a
yellowish oil in yield of 55–67%.
4.1.7. General procedure for the preparation of 1-(4-bromo-
benzyl)-benzimidazole (14a–14l)
NaH (0.48 g, 60% in mineral oil, 12.0 mmol) was added in
batches to a solution of 13 in DMF (10 mL) at 0 °C, and the mixture
was stirred for 15 min.
A solution of 1-bromo-4-(bromo-
methyl)benzene (1.0 g, 4.0 mmol) in DMF (5 mL) was then added
and the mixture was stirred for 5 h at room temperature. The mix-
ture was poured into cold saturated NaCl solution, filtered, and the
filter cake was recrystallized with 95% alcohol to give 14a–14l as
white solid in yields of 45–65%.
4.1.2. 3,4-Dimethylisoxazol-5-amine (8)
To
a mixture of 2-methyl-3-oxobutanenitrile (7) (9.7 g,
0.1 mol), hydroxylamine hydrochloride (7 g, 0.1 mol) and water
(20 mL), a solution of NaOH in water (4 g, 10 mL) was added drop-
wise at 40 °C. Then the mixture was heated to reflux for 3 h, cooled
and partitioned. The organic layer stood overnight and was fil-
trated to give compound 8 as a white crystal (3.66 g), 32.7%, mp
123–125 °C.
4.1.8. General procedure for the preparation of 40-[(1H-
benzimidazole-1-yl)-methyl]-N-(3,4-dimethyl-5-isoxazolyl)-N-
(methoxyethoxymethyl)[1,10-biphenyl]-2-sulfonamide (15a–
15l)
Under nitrogen, the intermediates 11 (1 mmol) and 14
(1.6 mmol) were dissolved in propanol (10 mL), then triphenyl
phosphine (2 mg, 0.01 mmol), palladium(II) acetate (8 mg,
0.03 mmol) and 2 N sodium carbonate solution (1 mL) were added.
The mixture was heated to reflux for 1 h, and then reacted for an
additional 3 h at room temperature. EtOAc (5 mL) was added, the
water layer was extracted with EtOAc (3 ꢂ 20 mL). The combined
organic extracts were concentrated and purified by flash column
chromatography with n-hexane/ethyl acetate (1:3, v/v) to give
15a–15l as pale yellow oil in yields of 30–50%.
4.1.3. N-(3,4-Dimethyl-5-isoxazolyl)-2-bromobenzenesulfon-
amide (9)
3,4-Dimethylisoxazol-5-amine (1.32 g, 11.74 mmol) was added
to
a solution of 2-bromobenzene-1-sulfonyl chloride (3.0 g,
11.74 mmol) in pyridine and the mixture was stirred at room tem-
perature overnight. The resulting mixture was first mixed with ice
water (150 mL) and filtrated by vacuum. The pH of the filtrate was
then adjusted to 2 with 6 N hydrochloric acid and then filtrated
again. The residue was purified by flash column chromatography
with n-hexane/ethyl acetate/acetic acid (200:200:1, v/v/v) as elu-
ent to afford 9 as a white crystal in yield of 90%, mp 125–126 °C.

4666
R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
4.1.9. General procedure for the preparation of 40-[(1H-
benzimidazole-1-yl)-methyl]-N-(3,4-dimethyl-5-isoxazolyl)
[1,10-biphenyl]-2-sulfonamide (Ia–Il)
3H), 1.74-1.77 (m, 2H), 1.42 (s, 3H), 1.37–1.40 (m, 2H), 0.88–0.92
(t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C30H33N4O4S:
545.2217, found 545.2214.
Compound 15 (2.75 mmol) was dissolved in 95% EtOH (10 mL),
then 6 N hydrochloric acid (10 mL) was added. The mixture was
heated to reflux for 2 h and the pH was adjusted to 4 with hydro-
chloric acid. The resulting solution was concentrated and purified
by flash column chromatography with CH2Cl2/MeOH (20:1, v/v)
to give Ia–Il as white solids in yields of 30–80%.
4.1.9.8. 40-[(2-Butyl-6-chloro-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Ih). White solid, 59.8%, mp 256–258 °C. IR(KBr/cmꢁ1): 2948,
1610, 1470, 1328, 1168. 1H NMR (300 MHz, DMSO-d6): d 7.22–
8.19 (m, 11H), 5.60 (s, 2H), 2.86–2.89 (t, J = 7.4 Hz, 2H), 1.91 (s,
3H), 1.76-1.79 (m, 2H), 1.55 (s, 3H), 1.39-1.44 (m, 2H), 0.90-0.95
(t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C29H30ClN4O3S:
549.1722, found 549.1726.
4.1.9.1. 40-[(1H-Benzimidazole-1-yl)-methyl]-N-(3,4-dimethyl-
5-isoxazolyl)[1,1’-biphenyl]-2-sulfonamide (Ia). White solid,
64.0%, mp 212–214 °C. IR(KBr/cmꢁ1): 2353, 1650, 1496, 1336,
1165. 1H NMR (300 MHz, DMSO-d6): d 8.47 (s,1H), 7.18–8.00 (m,
12H), 5.55 (s, 2H), 2.01 (s, 3H), 1.55 (s, 3H). HR-MS (ESI, M+H) m/
z: calcd for C25H23N4O3S: 459.1485, found 459.1488.
4.1.9.9. 40-[(2-Butyl-6-bromo-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Ii). White solid, 53.5%, mp 253–255 °C. IR(KBr/cmꢁ1): 2928,
1615, 1460, 1320, 1164. 1H NMR (300 MHz, DMSO-d6): d 7.20–
8.21 (m, 11H), 5.55 (s, 2H), 2.86–2.89 (t, J = 7.4 Hz, 2H), 1.90 (s,
3H), 1.76–1.78 (m, 2H), 1.54 (s, 3H), 1.39–1.43 (m, 2H), 0.89-0.93
(t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C29H30BrN4O3S:
593.1217, found 593.1212.
4.1.9.2. 40-[(2-Methyl-1H-benzimidazole-1-yl)-methyl]-N-(3,4-
dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Ib). White solid, 46.5%, mp 214–215 °C. IR(KBr/cmꢁ1): 2924,
1616, 1467, 1341, 1168. 1H NMR (300 MHz, DMSO-d6): d 7.10–
8.01 (m, 12H), 5.53 (s, 2H), 2.63 (s, 3H), 1.98 (s, 3H), 1.54 (s, 3H).
HR-MS (ESI, M+H) m/z: calcd for C26H25N4O3S: 473.1642, found
473.1640.
4.1.9.10. 40-[(2-Butyl-6-fluoro-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Ij). White solid, 66.7%, mp 262–264 °C. IR(KBr/cmꢁ1): 3015,
1608, 1480, 1335, 1166. 1H NMR (300 MHz, DMSO-d6): d 7.00–
8.10 (m, 11H), 5.56 (s, 2H), 2.85–2.88 (t, J = 7.4 Hz, 2H), 1.92 (s,
3H), 1.78–1.80 (m, 2H), 1.56 (s, 3H), 1.38–1.42 (m, 2H), 0.90–0.94
(t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C29H30FN4O3S:
533.2017, found 533.2019.
4.1.9.3.
40-[(2-Ethyl-1H-benzimidazole-1-yl)-methyl]-N-(3,4-di
methyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide (Ic). White
solid, 72.3%, mp 221–223 °C. IR(KBr/cmꢁ1): 2946, 1658, 1464, 1337,
1167. 1H NMR (300 MHz, DMSO-d6): d 7.22–8.06 (m, 12H), 5.81 (s,
2H), 3.23–3.30 (m, 2H), 2.03 (s, 3H), 1.59 (s, 3H), 1.39–1.44 (t,
J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C27H27N4O3S:
487.1798, found 487.1795.
4.1.9.11. 40-[(2-Butyl-6-trifluoromethyl-1H-benzimidazole-1-yl)-
methyl]-N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfon-
amide (Ik). White solid, 58.9%, mp 270–272 °C. IR(KBr/cmꢁ1):
3010, 1636, 1475, 1332, 1170. 1H NMR (300 MHz, DMSO-d6): d
7.18–8.25 (m, 11H), 5.60 (s, 2H), 2.85–2.89 (t, J = 7.4 Hz, 2H),
1.96 (s, 3H), 1.73–1.76 (m, 2H), 1.54 (s, 3H), 1.37–1.40 (m, 2H),
0.88–0.92 (t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for
4.1.9.4. 40-[(2-Propyl-1H-benzimidazole-1-yl)-methyl]-N-(3,4-di
methyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide (Id). White
solid, 38.1%, mp 228–230 °C. IR(KBr/cmꢁ1): 2967, 1615, 1464,
1340, 1164. 1H NMR (300 MHz, DMSO-d6): d 7.07–8.00 (m, 12H),
5.55 (s, 2H), 2.84-2.89 (t, J = 7.4 Hz, 2H), 1.99 (s, 3H), 1.77–1.84
(m, 2H), 1.55 (s, 3H), 0.95-1.00 (t, J = 7.4 Hz, 3H). HR-MS (ESI,
M+H) m/z: calcd for C28H29N4O3S: 501.1955, found 501.1958.
C30H30F3N4O3S: 583.1985, found 583.1988.
4.1.9.12. 40-[(2-Butyl-6-nitro-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Il). White solid, 42.6%, mp 276–278 °C. IR(KBr/cmꢁ1): 2968,
1606, 1498, 1325, 1158. 1H NMR (300 MHz, DMSO-d6): d 7.22–
8.85 (m, 11H), 5.56 (s, 2H), 2.87–2.89 (t, J = 7.4 Hz, 2H), 1.98 (s,
3H), 1.78–1.81 (m, 2H), 1.55 (s, 3H), 1.40–1.43 (m, 2H), 0.90–0.98
(t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for C29H30N5O5S:
560.1962, found 560.1960.
4.1.9.5. 40-[(2-Butyl-1H-benzimidazole-1-yl)-methyl]-N-(3,4-di
methyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide (Ie). White
solid, 40.8%, mp 229–232 °C. IR(KBr/cmꢁ1): 2956, 1615, 1464,
1316, 1163. 1H NMR (300 MHz, DMSO-d6): d 6.98–8.00 (m, 12H),
5.50 (s, 2H), 2.86-2.89 (t, J = 7.4 Hz, 2H), 1.84 (s, 3H), 1.74-1.77
(m, 2H), 1.43 (s, 3H), 1.39-1.42 (m, 2H), 0.88–0.91 (t, J = 7.4 Hz,
3H). HR-MS (ESI, M+H) m/z: calcd for C29H31N4O3S: 515.2111,
found 515.2115.
4.2. Pharmacological evaluation
4.1.9.6. 40-[(2-Butyl-6-methyl-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(If). White solid, 54.1%, mp 245–247 °C. IR(KBr/cmꢁ1): 2960,
1616, 1468, 1320, 1165. 1H NMR (300 MHz, DMSO-d6): d 6.90–
8.05 (m, 11H), 5.60 (s, 2H), 2.86–2.88 (t, J = 7.4 Hz, 2H), 2.45 (s,
3H), 1.86 (s, 3H), 1.75-1.77 (m, 2H), 1.45 (s, 3H), 1.38–1.41 (m,
2H), 0.87–0.90 (t, J = 7.4 Hz, 3H). HR-MS (ESI, M+H) m/z: calcd for
4.2.1. Angiotensin II receptor (AT1) binding assay
Membrane fractions or bovine adrenal cortex were prepared by
modification of the method of Maeda et al.15 The freshly isolated
bovine adrenal cortex was homogenized in ice-cold medium con-
taining 10 mM sodium phosphate buffer (pH 7.4), 30 mM NaCl,
1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 1 lM
(p-amidinophenyl) methanesulfonyl fluoride HCl (p-APMSF), and
0.02% NaN3. The homogenate was layered on a 41% sucrose solu-
tion and centrifuged at 95000g for 60 min. The interfacial band be-
tween the supernatant and the sucrose portion was collected. The
membrane fraction was washed by centrifugation at 95000g for
20 min. The pellet obtained was used as the source of AT1 receptor.
Binding of [125I] AngII to membranes was performed at 22 °C
C30H33N4O3S: 529.2268, found 529.2271.
4.1.9.7. 40-[(2-Butyl-6-methoxy-1H-benzimidazole-1-yl)-methyl]-
N-(3,4-dimethyl-5-isoxazolyl)[1,10-biphenyl]-2-sulfonamide
(Ig). White solid, 60.3%, mp 230–231 °C. IR(KBr/cmꢁ1): 2952, 1620,
1465, 1332, 1165. 1H NMR (300 MHz, DMSO-d6): d 6.77–7.99 (m,
11H), 5.52 (s, 2H), 3.82 (s, 3H), 2.84-2.86 (t, J = 7.4 Hz, 2H), 1.82 (s,
for 120 min in 96-well plates. Each 200 lL of incubated solution

R. Bai et al. / Bioorg. Med. Chem. 20 (2012) 4661–4667
4667
contained the following (final concentration): 20 mM Tris HCl (pH
7.4), 120 mM NaCl, 5 mM MgCl2, 0.05% bovine serum albumin
(BSA), 1
M p-APMSF, 0.5 mM EDTA, 0.1 mM DTT, 0.1 nM [125I]
Ang II, the test compound and membrane preparations (10 ug of
protein/well). At the end of incubation, bound complex was
trapped on filters (GF/C) and washed with cold Tris buffer (pH
Acknowledgments
l
This study was financially supported by grant from ‘Eleventh
Five-Year’ Major Innovation Projects for New Drug Candidates
(No. 2009ZX09102-036), Jiangsu Provincial Science Fund (No.
BK2008344) and Project for Research and Innovation of Graduates
in Colleges and Universities of Jiangsu Province (CXZZ11-0798).
7.4; 3 ꢂ 250
lL). Filter disks were dried, punched out, and counted
in an r-counter. Specific binding was defined as total binding
minus nonspecific binding, which was estimated in the presence
References and notes
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