Design, synthesis, and evaluation of 5-sulfamoyl benzimidazole derivatives as novel angiotensin II receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2008.10.056

A series of 5-alkylsulfamoyl benzimidazole derivatives have been designed and synthesized as novel angiotensin II (Ang II) receptor antagonists. The compounds have been evaluated for in vitro Ang II antagonism and for in vivo antihypertensive activity on isolated rat aortic ring and desoxycortisone acetate induced hypertensive rats, respectively. The activity is found related to size of alkyl group. The maximum activity is observed with a compact and bulky alkyl group like tert-butyl and cyclohexyl. The compounds 4g and 4h have shown promising both in vitro and in vivo activities. A receptor binding model is also proposed on the basis on the basis of structure-activity relationship in this study.

Full text of DOI:10.1016/j.bmc.2008.10.056

Bioorganic & Medicinal Chemistry 16 (2008) 10210–10215
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of 5-sulfamoyl benzimidazole derivatives
as novel angiotensin II receptor antagonists
*
Navneet Kaur, Amardeep Kaur, Yogita Bansal, Dhvanit I. Shah, Gulshan Bansal , Manjeet Singh
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab 147 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-alkylsulfamoyl benzimidazole derivatives have been designed and synthesized as novel
angiotensin II (Ang II) receptor antagonists. The compounds have been evaluated for in vitro Ang II
antagonism and for in vivo antihypertensive activity on isolated rat aortic ring and desoxycortisone
acetate induced hypertensive rats, respectively. The activity is found related to size of alkyl group.
The maximum activity is observed with a compact and bulky alkyl group like tert-butyl and cyclo-
hexyl. The compounds 4g and 4h have shown promising both in vitro and in vivo activities. A recep-
tor binding model is also proposed on the basis on the basis of structure–activity relationship in this
study.
Received 22 September 2008
Revised 24 October 2008
Accepted 25 October 2008
Available online 29 October 2008
Keywords:
Benzimidazole
Angiotensin II antagonists
Sulfamoyl
Ó 2008 Elsevier Ltd. All rights reserved.
Antihypertensive
Binding profile
1. Introduction
proposed (Fig. 2) in the previous report10 where n-butyl chain
interacts with pocket L1 lined by Phe182, Phe171, Ala163, and
Angiotensin II (Ang II) AT1 receptor antagonists are widely
used in treatment of hypertension and maintenance of homeo-
stasis.1,2 Losartan is the protypical Ang II antagonist and numer-
ous modifications in chemical structure of losartan have
generated large artillery of Ang II antagonists3–5 like candesar-
tan, zolasartan, irbesartan, and telmisartan (Fig. 1). In general,
all these ‘sartans’ are composed of an appropriately substituted
heterocyclic nucleus coupled to an acidic group (carboxylic or
tetrazole) bearing biphenyl system through a methylene linker.
Candesartan is an Ang II antagonist having benzimidazole nu-
cleus substituted with carboxyl function at 7-position.6,7 Substi-
tution at 6-position of benzimidazole nucleus has also produced
derivatives having excellent Ang II antagonistic activity.8 How-
ever, the 5-position of benzimidazole nucleus has remained rel-
atively unexplored in search of potent Ang II antagonists. The 5-
nitrobenzimidazole derivative 1 (Fig. 1) has been designed and
synthesized earlier in our laboratory and it is found more active
than candesartan.9 Another series of 5-alkylcarboxamido (–
NHCOR) benzimidazole derivatives has also been designed and
synthesized in our laboratory10 from which 5-acetamido analog
2 (Fig. 1) is found equipotent to candesartan. Based on these
earlier results, a previous study on triazolone based AT1 antag-
onist 311 and other similar studies12–15 a binding profile was
Ser103 and the biphenyl system interacts with pocket L2 lined
by Val108 through van der Waal interactions while N-3 of benz-
imidazole nucleus and terminal carboxyl group interact through
H-bonding with Tyr113 and Tyr184. The nitro group of 1 and
acetamido group of 2 can interact with pocket L4 lined by
Tyr292, His256, Tyr253, and leu112 through van der Waal and/or
H-bonding interactions. However, insignificant increase in activ-
ity of 2 with respect to 110 has suggested that interactions of –
NO2 group alone with receptor surface are much stronger than
those of whole of the –NHCOCH3 group. It indicated that –
NO2 group is required for strong binding interaction and it is
hypothesized that further extension at nitrogen atom of –NO2
group with substituents (like alkylamino groups) may strength-
en the binding interactions through lipophilic and/or H-bonding
interactions with pocket L3 or L4. Hence, in continuing endeav-
ors to search novel and potent Ang II antagonists, the present
study reports 5-substituted benzimidazole derivatives 4 (Fig. 1)
designed by isosteric replacement of –NO2 group with sulfonyl
(–SO2) group and extending the latter with alkylamino group
(–NHR). The designing of these compounds was based on
hypothesis that while the –SO2 mimics the –NO2 group, the –
NH– can interact with an H-bond acceptor group and the alkyl
group (R) may interact with L3 or L4 to form a stronger drug–
receptor complex. The in vitro Ang II antagonism and in vivo
antihypertensive activity of the compounds were evaluated
using losartan, candesartan and 1 as reference compounds to
study the structure–activity relationship.
* Corresponding author. Tel.: +91 175 3046255; fax: +91 175 283073.
E-mail addresses: gulshanbansal@rediffmail.com, gulshanbansal@gmail.com (G.
Bansal).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.056

N. Kaur et al. / Bioorg. Med. Chem. 16 (2008) 10210–10215
10211
Figure 1. Angiotensin II receptor antagonists.
under controlled temperature conditions to form target sulfon-
amide products 4a–h. The intermediate II was also hydrolyzed
with water to give sulfonic acid derivative 5. All compounds were
purified by crystallization and purity was ascertained by thin-layer
chromatography. Structures of the compounds were established
through IR (Table 1), 1H NMR (Table 2) and mass spectral analyses.
2.2. Pharmacological evaluation
2.2.1. In vitro Ang II receptor antagonism
The target compounds were evaluated for in vitro Ang II antag-
onism and activity was expressed as pA2 values (Table 3). The pA10
values were also determined to establish the mode of antago-
nism.17 The unsubstituted sulfonamide derivative 4a was found
to be the least active and equipotent to sulfonic acid derivative 5.
Both 4a and 5 were significantly less active than the lead com-
pound 1. It indicated that –NO2 group interacts with the receptor
more strongly than the isosteric –SO2 group. The activity of alkyl
substituted sulfonamide analogs increased with increasing size of
alkyl group at the sulfonamide moiety except for compound 4f
where activity was found to decrease. The compound 4g emerged
as maximally active and even more active than losartan. This var-
ied activity due to different alkyl groups suggested that a compact
alkyl group (like tert-butyl and cyclohexyl) at sulfonamide moiety
may interact with receptor site by filling L3 or L4 but a long alkyl
group (like n-butyl) weakens the binding interactions, probably
due to overfilling of the pocket. However, contrary to the hypoth-
esis, activity of the most active derivative 4g was comparable to
that of the lead compound 1. It suggested that interactions of the
tert-butyl group with receptor site are sufficient to compensate
the decreased binding affinity due to replacement of –NO2 with –
SO2 group as indicated by decreased activities of 4a and 5. Further,
comparison of 3D structures of compounds 2, 3, and 4g in their en-
ergy minimized conformations using ChemOffice 6.0 revealed that
Figure 2. Possible binding profile of 5-substituted benzimidazole derivatives.
2. Results and discussion
2.1. Chemistry
The synthetic scheme employed to synthesize the designed
compounds is outlined in Figure 3. The compound I was prepared
through a series of reactions as reported earlier9,10 which was then
subjected to chlorosulfonation with chlorosulfonic acid at temper-
ature of 10–15 °C. The –SO2Cl was substituted selectively at 5-po-
sition of benzimidazole nucleus similar to selective nitration at
indole.16 Moreover, the substitution of chlorosulfonyl group was
not expected to occur on terminal phenyl ring of biphenyl system
due to deactivation of the latter by electron withdrawing –COOH
group and the same was also not seemed possible on spacer phenyl
ring of biphenyl system due to electron withdrawing effects and
steric hindrances of the adjoining benzimidazole nucleus and deac-
tivated terminal phenyl ring. The chlorosulfonyl intermediate II
was coupled with varied amines (ammonia or alkylamines)

10212
N. Kaur et al. / Bioorg. Med. Chem. 16 (2008) 10210–10215
Figure 3. Synthetic scheme (i) chlorosulfonic acid, 10–15 °C stirring 30 min followed by 3.5 h; (ii) 10–15 °C, amine drop wise, continuous stirring, 30 min, dil HCl; (iii) rt,
water drop wise, continuous stirring.
tert-butyl group of 4g is disposed in an orientation different from
positioning of methyl group of 2 but similar to phenyl ring of the
benzoic acid moiety of 3. Also, difference in pA2 and pA10 values
of the compounds 4a–4f indicated the compounds to be competi-
tive inhibitors while the compounds 4g and 4h were found to be
non-competitive ones similar to 1 and candesartan. The changed
mode of antagonism of compounds 4a–4f with respect to 1 indi-
cated that binding pattern of –NO2 group is different from that of
–SO2 group. It also suggested that the tert-butyl and cyclohexyl
groups probably interact with some different sites in the receptor
pocket which is responsible for altered mode of antagonism as well
as for compensation of decreased binding affinity of –SO2 group.
Hence, based on these findings it is proposed that while SO2 inter-
acts with L4, the tert-butyl and cyclohexyl group may be interact-
ing with L3 similar to reported binding profile of 3.11
effect of the cyclohexyl substituted analog (4h) was further in-
creased marginally over 4g and it could be attributed probably to
increased distribution of the molecule as result of increased lipo-
philicity of cyclohexyl group.
3. Conclusion
A series of 5-alkylsulfamoyl substituted benzimidazole deriva-
tives are designed and synthesized as Ang II antagonists and are
evaluated for in vitro Ang II antagonistic activity as well as
in vivo antihypertensive activity to study the structure–activity
relationship. The antagonistic activity is found dependent upon
size and bulk of alkyl group while antihypertensive activity is
found largely corresponding to in vitro activity and also dependent
upon the overall lipophilicity of the molecule. The tert-butylsulfa-
moyl analog 4g has emerged as maximally active compound dur-
ing in vitro studies but the cyclohexylsulfamoyl analog 4h has
maximum decrease in MABP in hypertensive rats. Based on the re-
sults a binding profile for the target compounds is proposed where
the tert-butyl or cyclohexyl groups can interact with L3 pocket
similar to binding profile of non-competitive triazolone based
Ang II antagonist.
2.2.2. In vivo antihypertensive activity
The in vivo antihypertensive activity was evaluated on desoxy-
cortisone acetate (DOCA) induced hypertensive rats18 and activity
was expressed as decrease in mean arterial blood pressure
(MABP) taking losartan, candesartan, and 1 as reference com-
pounds (Table 3). The MABP in sham control and DOCA induced
hypertensive group were 107 4.8 and 187 6.3 mmHg, respec-
tively. The activity was found to increase marginally with increase
in alkyl group up to iso-propyl derivative (4e). Thereafter, it
decreased for 4f in consonant with in vitro activity data. The de-
crease in MABP by 4g was significant and this reduction in MABP
was even more than all reference compounds. It may be attributed
to both optimum binding interactions of the pharmacophoric
groups and hydrophilic–lipophilic balance of the molecule. How-
ever, in contrast to the in vitro activity trend, the antihypertensive
4. Experimental
4.1. Chemistry
The melting points were recorded in open sulfuric acid bath and
uncorrected. 1H NMR spectra were recorded on Bruker AC 300
NMR Spectrometer (300 MHz), mass spectra were recorded on Vg
Micro Mass 7070F spectrometer and GC–MS (Gcq) Spectrometer
Table 1
IR spectral data of the target compounds.
Vibrational band
Wave number (cmꢀ1
)
4a
4b
4c
4d
4e
4f
4g
4h
5
Free O–H str.
O–H and N–H str.
C@O str.
S@O str.
N–H wagging
C–S str.
—
—
3739
3700–2200
1708
1329, 1126
826
918
—
—
3746
3700–2200
1710
1333, 1132
834
622
—
—
3750
3700–2200
1711
1370, 1161
834
615
3650
3700–2200
1713
1370, 1170
846
630
—
—
—
—
—
3600–2500
1711
1320, 1160
830
621
—
3700–2200
1708
1330, 1123
829
619
—
3600–2800
1713
1320, 1160
830
616
1395, 1377
—
3600–2800
1713
1317, 1170
830
616
—
3600–2400
1711
1315, 1172
—
614
—
—
C–H bend (gem-dimethyl)
Cyclohexyl bend
1382, 1365
—
—
—
1454

N. Kaur et al. / Bioorg. Med. Chem. 16 (2008) 10210–10215
10213
Table 2
1H NMR spectral data of the target compounds.
Compound d (number of protons, multiplicity, J in Hz, proton assignment)
4a
8.12–7.92 (2H, m, ArH); 7.87 (1H, m, ArH); 7.68–7.46 (6H, m, ArH, SONH2); 7.35–7.29 (2H, m, ArH); 7.14–7.05 (3H, m, ArH, COOH); 4.01 (2H, s, CH2); 2.97 (2H, t,
7.5, CH2CH2CH2CH3); 1.97 (2H, qv, 7.5, CH2CH2CH2CH3); 1.45 (2H, sx, 7.5, CH2CH2CH2CH3) and 0.97 (3H, t, 7.5, CH2CH2CH2CH3)
4b
8.03–7.96 (2H, m, ArH); 7.86 (1H, s, br, COOH); 7.76–7.74 (1H, m, ArH); 7.61–7.50 (4H, m, ArH); 7.48–7.42 (2H, m, ArH); 7.36–7.29 (2H, m, ArH); 7.17 (1H, s, br,
SONH); 4.04 (2H, s, CH2); 2.58 (3H, s); 2.25 (2H, t, 7.5, CH2CH2CH2CH3); 1.63 (2H, qv, 7.5, CH2CH2CH2CH3); 1.35 (2H, sx, 7.5, CH2CH2CH2CH3) and 0.98 (3H, t, 7.5,
CH2CH2CH2CH3)
4c
8.25–7.70 (5H, m, br, ArH, COOH, SONH); 7.62–7.29 (8H, m, ArH); 4.04 (2H, s, CH2); 3.03 (2H, t, 7.5, CH2CH2CH2CH3); 2.94 (2H, q, 7, CH2CH3); 2.59 (2H, qv, 7.5,
CH2CH2CH2CH3); 2.02 (2H, sx, 7.5, CH2CH2CH2CH3); 1.28 (3H, t, 7, CH2CH3) and 0.98 (3H, t, 7.5, CH2CH2CH2CH3)
4d
7.97–7.70 (5H, m, ArH, COOH, SONH); 7.64–7.54 (4H, m, ArH); 7.52–7.44 (2H, m, ArH); 7.33 (2H, m, ArH); 4.05 (2H, s, CH2); 3.06 (2H, t, 7.5, CH2CH2CH2CH3); 3.03
(2H, t, 7.2, CH2CH2CH3); 2.62 (2H, sx, 7.2, CH2CH2CH3); 1.74 (2H, qv, 7.5, CH2CH2CH2CH3); 1.69 (3H, t, 7.2, CH2CH2CH3); 1.64 (2H, sx, 7.5, CH2CH2CH2CH3) and 0.90
(3H, t, 7.5, CH2CH2CH2CH3)
4e
4f
4g
4h
5
8.05–7.90 (3H, br, m, COOH, NH, ArH); 7.76–7.74 (1H, m, ArH); 7.73–7.26 (9H, m, ArH); 4.0 (2H, d, 6, CH2); 3.70 (1H, sp, 7.5, CH(CH3)2); 3.24 (2H, t, 7,
CH2CH2CH2CH3); 1.43 (6H, d, 7.5, CH(CH3)2); 1.95 (2H, qv, 7, CH2CH2CH2CH3); 1.45 (2H, sx, 7, CH2CH2CH2CH3) and 0.99 (3H, t, 7, CH2CH2CH2CH3).
8.06–8.18 (1H, br, s, COOH); 7.75–7.62 (3H, m, ArH and SO2NH); 7.55–7.31 (9H, m, ArH); 4.04 (2H, d, 6, CH2); 3.21 (4H, m, CH2 CH2 CH2 CH3); 1.71 (4H, m,
CH2CH2CH2CH3); 1.41 (4H, m, CH2CH2CH2CH3) and 0.96 (6H, m, CH2CH2CH2CH3)
8.0–7.32 (13H, br, m, COOH, SO2NH and ArH); 4.0 (2H, d, 6, CH2); 3.02 (2H, t, 7, CH2CH2CH2CH3); 2.1 (9H, s, C(CH3)3); 1.93 (2H, qv, 7, CH2CH2CH2CH3); 1.47 (2H, sx,
7, CH2CH2CH2CH3) and 0.99 (3H, t, 7, CH2CH2CH2CH3)
8.1–7.58 (8H, br, m, COOH, SO2NH and ArH); 7.64–7.31 (5H, br, m, ArH); 4.02 (2H, d, 6, CH2); 4.4 (1H, m, CH); 3.25 (2H, t, 7, CH2CH2CH2CH3); 2.27–1.17 (10H, m,
(CH2)5); 1.95 (2H, qv, 7, CH2CH2CH2CH3); 1.43 (2H, sx, 7, CH2CH2CH2CH3) and 0.96 (3H, t, 7, CH2CH2CH2CH3)
8.12–7.90 (1H, m, ArH); 7.69–7.74 (1H, m, ArH); 7.63–7.58 (2H, m, ArH, COOH); 7.56–7.51 (4H, m, ArH); 7.48–7.39 (3H, m, ArH, SO3H); 7.35–7.28 (2H, m, ArH);
4.25 (2H, s, CH2); 2.95 (2H, t, 7.5, CH2CH2CH2CH3); 1.67 (2H, qv, 7.5, CH2CH2CH2CH3); 1.35 (2H, sx, 7.5, CH2CH2CH2CH3) and 0.97 (3H, t, 7.5, CH2CH2CH2CH3)
reaction mixture was used as such without product isolation to
synthesize the target compounds.
Table 3
In vitro and in vivo activities of target and reference compounds.
4.1.2. General procedure for synthesis of 5-alkylsulfamoyl
derivatives (4a–4h)
pA2 pA10 Mode of antagonism Maximum decrease in MABPa,b
a
a
Compound
The amine reactant (45 mmol) was added drop wise to the
cooled (10–15 °C) reaction mixture of product II (Caution: vigorous
reaction) with continuous stirring. The temperature was not al-
lowed to exceed 15 °C during the addition. After the addition was
complete, stirring continued for another 25–30 min at the same
temperature. The reaction mixture was acidified with dil HCl and
solid was filtered at pump to afford the product as amorphous
powder which was recrystallized from ethanol and water.
4a
4b
4c
4d
4e
4f
4g
4h
6.7
6.9
7.1
7.1
7.2
7.0
8.4
7.9
6.6
8.5
8.0
6.1
6.3
6.2
6.4
6.3
6.2
5.4
5.7
5.8
6.7
7.1
6.7
C
C
C
C
C
C
NC
NC
C
NC
C
51.4 6.1
52.5 3.4
49.9 4.8
50.2 5.9
51.7 3.1
47.6 6.4
62.4 4.5c,d,e
64.4 3.9c,d,e
44.7 6.1
55.9 3.4
58.3 5.6
56.2 6.3
5
1
Losartan
Candesartan 8.5
4.1.2.1. 40-[(2-Butyl-5-sulfamoyl-1H-benzimidazol-1-yl)methyl]
biphenyl-2-carboxylic acid (4a). Amine reactant: Ammonia
solution (25%), Yield 63%, mp 150–152 °C. Anal. Calcd for C25H25
N3O4S: C, 64.59; H, 5.38; N, 9.12. Found: C, 64.78; H, 5.44; N,
9.06. MS: 463 (M).
NC
a
n = 6.
b
Dose is 3.0 mg kgꢀ1 except for losartan and candesartan (5.0 mg kgꢀ1).
p < 0.05 versus 1.
p < 0.05 versus losartan.
c
d
e
p < 0.05 versus candesartan.
4.1.2.2. 40-[(2-Butyl-5-methylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4b). Amine reactant:
Methylamine, Yield 51%, mp 140–142 °C. Anal. Calcd for C26H27
N3O4S: C, 65.08; H, 5.76; N, 8.89. Found: C, 65.39; H, 5.70; N, 8.80.
MS: 477 (M).
and FT-IR spectra were recorded on FT-IR Perkin-Elmer 1710 ser-
ies. In 1H NMR, chemical shifts were reported in d values using tet-
ramethylsilane as internal standard with multiplicities (br, broad;
s, singlet; d, doublet; t, triplet; q, quartet; qv, quintet; sx, sextet;
sp, septet; m, multiplet; dd, double doublet) and number of pro-
tons in DMSO-d6 . The coupling constants (J) were expressed in
Hz. IR spectra were recorded as KBr pellets. The elemental analyses
were performed on Heraus CHN–O rapid elemental analyzer.
When necessary, solvents and reagents were dried prior to use
over KOH or anhydrous Na2SO4 or fused CaCl2.
4.1.2.3. 40-[(2-Butyl-5-ethylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4c). Amine reactant:
Ethylamine, Yield 53%, mp 146–148 °C. Anal. Calcd for C27H29
N3O4S: C, 65.73; H, 5.89; N, 8.61. Found: C, 65.97; H, 5.95; N,
8.55. MS: 491 (M).
4.1.2.4. 40-[(2-Butyl-5-propylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4d). Amine reactant:
Propylamine, Yield 42%, mp 141–143 °C. Anal. Calcd for C28H31
N3O4S: C, 66.42; H, 6.26; N, 8.26. Found: C, 66.51; H, 6.18; N,
8.31. MS: 505 (M).
4.1.1. 40-[(2-Butyl-5-chlorosulfonyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (II)
Chlorosulfonic acid (1.2 ml, 18 mmol) was cooled to 10–15 °C in
a dry two necked 100 ml round bottomed flask and the compound I
(1 g, 2.6 mmol) was added with continuous stirring over a period of
half an hour. Stirring was further continued for 3–3.5 h maintain-
ing the temperature at 10–15 °C. Formation of sulfonylchloride
derivative II was ascertained by TLC. Chloroform/methanol
(98:2); Rf 0.78 and cyclohexane/ethylacetate (25:75); Rf 0.58. The
4.1.2.5. 40-[(2-Butyl-5-isopropylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4e). Amine reactant: iso-
Propylamine, Yield 52%, mp 155–156 °C. Anal. Calcd for C28H31

10214
N. Kaur et al. / Bioorg. Med. Chem. 16 (2008) 10210–10215
N3O4S: C, 66.39; H, 6.24; N, 8.37. Found: C, 66.51; H, 6.18; N, 8.31.
MS: 505 (M).
depolarizing Krebs–Henseleit solution to replace the equivalent
isotonic amount of NaCl. The aortic ring was exposed to the result-
ing depolarizing Krebs–Henseliet solution for 10 min and the prep-
aration was washed for 1 h with Krebs–Henseleit solution
relaxation of the preparation to the basal tone.
4.1.2.6. 40-[(2-Butyl-5-butylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4f). Amine reactant: n-
Butylamine, Yield 54%, mp 189–191 °C. Anal. Calcd for C29H33
N3O4S: C, 66.88; H, 6.34; N, 7.99. Found: C, 67.03; H, 6.40; N,
8.09. MS: 519 (M).
4.2.1.3. Experimental design. A CRC for Ang II was recorded with
increasing concentration of Ang II at an interval of 0.5 log units
(1 ꢂ 10ꢀ10
,
3 ꢂ 10ꢀ10
,
1 ꢂ 10ꢀ9
,
3 ꢂ 10ꢀ9
,
1 ꢂ 10ꢀ8
,
3 ꢂ 10ꢀ8
,
4.1.2.7. 40-[(2-Butyl-5-tert-butylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4g). Amine reactant:
tert-Butylamine, Yield 47%, mp 148–150 °C. Anal. Calcd for C29H33
N3O4S: C, 66.92; H, 6.32; N, 8.04. Found: C, 67.05; H, 6.38; N, 8.12.
MS: 519 (M).
1 ꢂ 10ꢀ7, 3 ꢂ 10ꢀ7, 1 ꢂ 10ꢀ6, 3 ꢂ 10ꢀ6, and 1 ꢂ 10ꢀ5). The prepara-
tion was washed repeatedly and it was sensitized before recording
the next CRC in presence of losartan. Losartan (1 nmol) was added,
preparation was allowed to equilibrate for 15 min and a CRC for
Ang II was recorded. Similarly CRC was recorded in absence and in
presence of losartan (3 nmol and 10 nmol), candesartan, 1, 4, and 5
(1, 3, and 10 nmol). Results were expressed as percentage of the
maximal tension developed in response to Ang II. The pA2 and
pA10 value of losartan, candesartan, 1, 4, and 5 were calculated using
Schild’s plot.17 Linear regression was employed to plot DRC between
developed tension and negative log molar concentration of Ang II.
Data were statistically analyzed using paired Student’s t-test.
p < 0.05 was considered to be statistically significant.
4.1.2.8. 40-[(2-Butyl-5-cyclohexylsulfamoyl-1H-benzimidazol-1-
yl)methyl]biphenyl-2-carboxylic acid (4h). Amine reactant:
Cyclohexylamine, Yield 43%, mp 170–172 °C. Anal. Calcd for C31H35
N3O4S: C, 67.97; H, 6.51; N, 7.78. Found: C, 68.23; H, 6.46; N, 7.70.
MS: 545 (M).
4.1.3. 40-[(2-Butyl-5-sulfo-1H-benzimidazol-1-yl)methyl]biphe-
nyl-2-carboxylic acid (5)
Cold water (10–15 °C) was added drop wise to the reaction mix-
ture of product II (Caution: vigorous reaction) with continuous stir-
ring. After the addition was complete, stirring continued for
another 25–30 min at the same temperature. The reaction mixture
was acidified with dil HCl and solid was filtered at pump to afford
the product as amorphous powder which was recrystallized from
ethanol and water. Yield 56%, mp 156–158 °C. Anal. Calcd for
C25H25N3O4S: C, 64.59; H, 5.38; N, 9.12. Found: C, 64.78; H, 5.44;
N, 9.06. MS: 464 (M).
4.2.2. In vivo antihypertensive activity
4.2.2.1. Experimental hypertension. The rats were uninephroc-
tomized and DOCA (40 mg kgꢀ1, sc) was administered twice a
week up to six weeks to produce hypertension. DOCA rats received
1.0% NaCl and 0.2% KCl in their drinking water. Sham rats received
tap water. The rats were anaesthetized (sodium thiopental
30 mg kgꢀ1, ip), heparanized (200 IU heparin, ip) and their trachea
were cannulated to facilitate respiration. The carotid artery was
isolated and cannulated to pressure transducer attached to BIOPAC
systems (BIOPAC, CA, USA) for measurement of MABP.
4.2. Pharmacological evaluation
4.2.2.2. Experimental design. The in vivo dose standardization
was performed using the lead compound 1. Different doses (0.1,
0.3, 1.0, 3.0, 10.0, and 30.0 mg kgꢀ1, ip) of 1 were administered to
rats and MABP was measured after 6 h of administration. It was
noted to produce plateau effect at 1 mg kgꢀ1 dose. In light of this,
compounds 4 and 5 were administered to rats at doses of 0.1,
1.0, 3.0, and 10.0 mg kgꢀ1 (ip) and MABP was measured after 6 h
of their administration. Losartan and candesartan (5 mg kgꢀ1, ip)
were used as reference standards in the study. Each value (n = 6)
represents mean SEM. Data were statistically analyzed by per-
forming one-way ANOVA followed by Tukey’s multiple range test.
p < 0.05 was considered to be statistically significant.
The experimental protocol involving use of animals for the
study was approved by the duly constituted Institutional Animal
Ethical Committee. The animals were taken care of in accordance
with the guidelines of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Ministry of Envi-
ronment and Forests, Government of India (Reg. No. CPCSEA/107/
1999).
4.2.1. In vitro Ang II antagonism
4.2.1.1. Isolated aortic ring preparation. The Ang II antagonism
activity of target and reference compounds was evaluated on iso-
lated aortic ring of rats. The rat was sacrificed by cervical disloca-
tion followed by exsanguinations. The thoracic aorta was carefully
dissected out, placed in ice-cold aerated Krebs–Henseliet solution
(NaCl, 118 mmol; KCl, 4.7 mmol; CaCl2, 5 mmol; MgSO4ꢁ7H20,
1.2 mmol; NaHCO3, 25 mmol; KH2PO4, 1.2 mmol, and glucose,
11.1 mmol), cleared from the adhered connective tissue and was
cut into 3 mm wide segments. Vascular endothelium from each
segment was removed by gently rubbing the inside of the ring with
tip of a blunt forceps. The ring was mounted in a 10 ml organ bath
containing Krebs–Henseleit solution maintained at 37 °C, pH 7.4.
The preparation was bubbled with oxygen. The ring was allowed
to equilibrate for one and half hour under 1 g tension. The prepa-
ration was washed with Krebs–Henseliet solution once in every
15 min. Isometric contractions were recorded using force trans-
ducer and BIOPAC four channel recorder (BIOPAC Systems Inc.,
CA, USA) to plot the cumulative response curve (CRC).
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The ring was sensi-

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