Synthesis and cytotoxic evaluation of N-(4-methoxy-1H-benzo[d]imidazol-7- yl)-arylsulfonamide and N-aryl-(4-methoxy-1H-benzo[d]imidazol)-7-sulfonamide analogs of combretastatin A-4

Article abstract of DOI:10.1080/10286020.2011.556091

Two series of novel benzoimidazole sulfonamides as combretastatin A-4 analogs were synthesized. The cytotoxicities of the title compounds were evaluated against five different cancer cell lines. Among the tested compounds, four compounds displayed cytotoxicities against the HCT8 cell line. Compound 6a has shown the strongest potency against the tested human tumor cell lines with an IC50 value ranging from submicromolar to micromolar level.

Full text of DOI:10.1080/10286020.2011.556091

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Synthesis and cytotoxic evaluation of
N-(4-methoxy-1H-benzo[d]imidazol-7-
yl)-arylsulfonamide and N-aryl-(4-
methoxy-1H-benzo[d]imidazol)-7-
sulfonamide analogs of combretastatin
A-4
Jie Zhou a , Yi Zhang a , Yi-Wen Cui a b , Zhan-Mei Li a b , Hong-Rui
Song b , Jin-Hua Dong b , Xiao-Guang Chen a & Bai-Ling Xu a
a Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing, 100050, China
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang, 110016, China
Available online: 30 Mar 2011
To cite this article: Jie Zhou, Yi Zhang, Yi-Wen Cui, Zhan-Mei Li, Hong-Rui Song, Jin-Hua Dong,
Xiao-Guang Chen & Bai-Ling Xu (2011): Synthesis and cytotoxic evaluation of N-(4-methoxy-1H-
benzo[d]imidazol-7-yl)-arylsulfonamide and N-aryl-(4-methoxy-1H-benzo[d]imidazol)-7-sulfonamide
analogs of combretastatin A-4, Journal of Asian Natural Products Research, 13:04, 330-340
To link to this article: http://dx.doi.org/10.1080/10286020.2011.556091
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Journal of Asian Natural Products Research
Vol. 13, No. 4, April 2011, 330–340
Synthesis and cytotoxic evaluation of N-(4-methoxy-1H-
benzo[d]imidazol-7-yl)-arylsulfonamide and N-aryl-(4-methoxy-1H-
benzo[d ]imidazol)-7-sulfonamide analogs of combretastatin A-4
Jie Zhoua†, Yi Zhanga†, Yi-Wen Cuiab, Zhan-Mei Liab, Hong-Rui Songb, Jin-Hua Dongb,
Xiao-Guang Chena and Bai-Ling Xua*
aInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China; bSchool of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China
(Received 2 December 2010; final version received 17 January 2011)
Two series of novel benzoimidazole sulfonamides as combretastatin A-4 analogs were
synthesized. The cytotoxicities of the title compounds were evaluated against five
different cancer cell lines. Among the tested compounds, four compounds displayed
cytotoxicities against the HCT8 cell line. Compound 6a has shown the strongest
potency against the tested human tumor cell lines with an IC50 value ranging from
submicromolar to micromolar level.
Keywords: combretastatin; sulfonamide; benzo[d ]imidazole; cytotoxicity
1. Introduction
aqueous solubility, higher lipophilicity,
and metabolic instability, which are
caused by the easy isomerization from
the active cis-isomer to the inactive trans-
isomer [7,8].
Combretastatin A-4 (CA-4) (Figure 1) is a
natural product containing a cis-stilbene
fragment and first isolated from the bark of
Combretum caffrum in 1982 by Pettit et al.
[1]. CA-4 exhibits strong cytotoxic activi-
ties against a wide variety of cancer cell
lines including multidrug-resistant cell
lines with an IC50 value ranging from
1028 to 10210 M [2,3]. It has been
demonstrated that CA-4 can result in the
microtubule depolymerization and mitotic
arrest of the cancer cells via the interaction
with colchicine-binding site on tubulin
[4,5]. Most interestingly, CA-4 has shown
tumor vascular disrupting activity at a
nontoxic dose and was referred as the
vascular disrupting agent [6]. However,
CA-4 has shown some disadvantages in
relation to physicochemical and pharma-
cokinetic properties, for example the lower
In view of the structural simplicity, the
strong in vitro potency, the validated
microtubule-depolymerization mechan-
ism, and the unique antiangiogenesis
activity, considerable efforts have been
taken in developing the novel CA-4
analogs with the improved pharmaco-
kinetic properties employing CA-4 as a
lead structure [9–11]. Disodium phos-
phate of CA-4 (CA-4P) (Figure 1), a
water-soluble prodrug, is now under Phase
II trial in the treatment of anaplastic
thyroid cancer1 [12]. AVE8062 (Figure 1),
a serine derivative of AC-7739 (Figure 1),
has been tested in the phase IIb/III clinical
trial2 [13].
*Corresponding author. Email: xubl@imm.ac.cn
†Both authors contributed equally to this work.
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.556091
http://www.informaworld.com

Journal of Asian Natural Products Research
331
O
O
O
A
O
O
P
B
ONa
ONa
O
O
OH
O
O
O
CA-4
CA-4P
O
O
O
O
O
O
O
NH2
N
H
OH
NH2HCl
O
O
AC-7739
AVE-8062
O
O
O
B
B
H
N
O
S
R1
R2
S
N
R1
NH
NH
A
A
O
H
N
O
N
R2
R3
R3
6a–6l
8a–8d
Figure 1. The structures of CA-4, CA-4 analogs and designed sulfonamide derivatives.
The structure–activity relationship
(SAR) studies on a number of CA-4
analogs have disclosed that the key
structural features of CA-4 derivatives
comprise a 3,4,5-trimethoxyl phenyl ring
as A ring, a 4-methoxyl group presented on
B ring, and the cis-orientation of A and B
rings. In this work, two series of novel CA-
4 analogs containing 4-methoxy-7-amino-
1H-benzo[d ]-imidazole as B ring and
sulfonamide as linker (6a–6 l, 8a–8d,
Figure 1) have been designed. The 4-
methoxy group was installed deliberately
on the benzoimidazole ring according to
the known SAR studies on B ring [9–11].
The sulfonamide linker has ever been
efficiently utilized in CA-4 analogs [14–
16]. The sulfonamide linker SO2NH in the
series 6a–6 l was reversed into NHSO2
relative to A and B rings in compounds
8a–8d. Besides the 3,4,5-trimethoxyl
phenyl ring, other substituted phenyl
groups were also exploited as A ring to
examine the SAR of this new series CA-4
Table 1. Structures of N-(4-methoxy-1H-benzo[d ]imidazol-7-yl)-arylsulfonamides (6a–6 l).
Compound
R1
R2
R3
Compound
R1
R2
R3
6a
6b
6c
6d
6e
6f
H
H
H
H
H
Cl
OCH3
CH3
OCH3
CH3
CH3
H
CH3
OCH3
Cl
NO2
Cl
6 g
6 h
6i
6j
6k
6 l
H
H
H
H
H
OCH3
OCF3
H
CH3
OCH3
Br
H
OCH3
F
F
CH3
OCH3
OH
OCH3

332
J. Zhou et al.
OH
O
O
NH2
NO2
NH2
NH2
NH2
a
b
89%
77%
NO2
2
1
O
O
O
N
N
c
d
O2N
N
+
N
H
83%
46%
N
H
N
H
NO2
3
4b
4a
O
S
Cl
O
R1
R2
O
O
O
S
R1
R2
N
e
R3
N
H
NH
O
N
97%
N
H
f
28–60%
NH2
R3
5
6a–6l
Scheme 1. Synthetic route of the N-(4-methoxy-1H-benzo[d ]imidazol-7yl)-arylsulfonamides
(6a–6 l). Reagents and conditions: (a) CH3I, K2CO3, DMF, rt; (b) 10% Pd/C, H2, rt; (c) HCOOH,
reflux; (d) NaNO3, TFA, 708C; (e) 10% Pd/C, H2, rt; (f) Pyridine, DCM, rt.
analogs. The chemical synthesis and
cytotoxicities of these novel CA-4 analogs
are described as follows.
in refluxing HCOOH. The nitration reac-
tion of benzoimidazole 3 with NaNO3 and
trifluoroacetic acid (TFA) at 708C furn-
ished two constitutional isomers 4-meth-
oxy-7-nitro-1H-benzo[d]imidazole (4a) in
46% yield and 4-methoxy-5-nitro-1H-
benzo[d]imidazole (4b) in 31% yield,
respectively. Subsequently, the isolated
compound 4a was reduced into key
intermediate 4-methoxy-7-amino-1H-ben-
zo[d]imidazole (5) in almost quantitative
yield on treatment with H2 catalyzed by
10% Pd-C. The coupling reaction of the
amino-substituted benzoimidazole 5 with a
variety of arylsulfonyl chloride in the
presence of pyridine delivered total 12
novel sulfonamides 6a–6 l in 28–60%
yield.
2. Results and discussion
Target arylsulfonamides 6a–6 l (Table 1)
were synthesized according to Scheme 1.
Treatment of 2-amino-3-nitrophenol with
methyl iodide and K2CO3 provided 2-
methoxy-6-nitroaniline (1) in 89% yield.
The catalytic hydrogenation of compound
1 was performed to give compound 2 in
77% yield. Conversion of o-phenylenedi-
amine (2) into 4-methoxy-1H-benzo[d]i-
midazole (3) was achieved in 83% yield
Table 2. Structures of N-aryl-(4-methoxy-1H-
benzo[d ]imidazol)-7-sulfonamides (8a–8d).
The other series of benzoimidazole-
substituted sulfonamides 8a–8d (Table 2)
were synthesized as outlined in Scheme 2.
Treatment of 4-methoxy-1H-benzo[d]imi-
dazole (3) with ClSO3H at 25 to 08C
furnished 4-methoxy-1H-benzo[d ]imida-
zole-7-sulfonic acid (7) in a yield of
Compound
R1
R2
R3
8a
8b
8c
8d
H
H
OCH3
F
OCH3
OCH3
OCH3
F
H
OCH3
OCH3
F

Journal of Asian Natural Products Research
333
NH2
R1
R2
O
O
O
H
N
O
S
R1
R2
N
a
N
R3
NH
O
N
66%
b,c
25–64%
N
H
N
H
SO2OH
R3
8a–8d
7
Scheme 2. Synthetic route of the N-aryl-(4-methoxy-1H-benzo[d ]imidazol)-7-sulfonamides
(8a–8d). Reagents and conditions: (a) ClSO3H, 25 to 08C; (b) SOCl2/reflux; (c) Et3N, DCM, rt
or pyridine, DCM, rt.
66%. Heating of compound 7 in refluxing
SOCl2 afforded the corresponding sulfonyl
chloride, which was then reacted with a
range of the substituted arylamines in the
presence of Et3N or pyridine at room
temperature to produce arylsulfonamides
8a–8d in 25–64% yield.
that two stable conformers exist with the
NH2 group eclipsing or staggering the SO2
group in benzenesulfonamide system [17].
In our cases, it seemed likely that the ratios
of two conformers are highly dependent on
chemical structure of target molecules, for
example with compounds 6c and 6j, a
mixture of two conformers was observed.
By contrast, in other synthesized com-
pounds the restricted conformers were not
detected by 1H NMR spectra.
It is worth mentioning that two
conformers originated from the restrict
rotation of ZSO2NHZ group were
observed in a ratio of 4:6 with compound
1
6c. The H NMR spectrum of compound
The cytotoxicities of all target sulfo-
namides were evaluated by MTT assay,
wherein five cancer cell lines were used:
HCT-8, Bel7402, BGC803, A549, and
A2708. The cytotoxicities are expressed as
IC50. CA-4 was used as reference com-
pound. The cytotoxic potency of the active
compounds is presented in Table 3.
6c was obtained at various temperatures,
25, 50 and 708C. At 258C, in one conformer
C2–H of benzoimidazole ring resonated at
8.05 ppm, and in other conformer the
signal of C2–H of benzoimidazole ring
appeared at 8.00 ppm. As the temperature
was raised to 508C, the two peaks of C2–H
from two conformers approached closely,
and finally at 708C two peaks of C2–H in
two conformers were coalescent. Simi-
larly, it was demonstrated by calculations
It was noteworthy that the substitution
pattern in compounds 6a–6 l is highly
preferred over that in reverse sulfonamides
8a–8d because compounds 8a–8d did not
Table 3. In vitro cytotoxic activities of compounds 6a, 6d, 6k, 6 l, and CA-4 against five human
cancer cell lines.
Cytotoxicity (IC50, mM)a
Compound
HCT8
Bel7402
BGC823
A549
A2708
6a
6d
6k
6 l
2.65
3.83
57.8
3.74
0.19
2.15
27.4
NAb
NA
0.77
NA
NA
NA
2.72
NA
NA
NA
0.49
1.61
NA
NA
NA
4.48
CA-4
0.16
0.012
Notes: a Compound dose (mM) required to inhibit cell growth by 50%.
b NA, not active.

334
J. Zhou et al.
show any activities at all against the tested
tumor cell lines. In comparison with
compounds 6a and 6 l, the complete loss
of activities of compounds 8a and 8c has
further demonstrated that the substitution
pattern of sulfonamides in compounds 6a
and 6 l plays a crucial role in their
interactions with the biological target.
As shown in Table 3, compounds 6a,
6d, 6k, and 6 l are active against HCT8
cancer cells with IC50 values ranging from
2.65 to 57.8 mM. Among the tested
compounds, compound 6a is the most
potent and showed cytotoxicities against
all tested tumor cell lines at lower
micromolar level. Compound 6a exhibited
a stronger potency against BGC803
(IC50 ¼ 0.77 mM) than that of other four
tumor cell lines. In addition, the cytotox-
icity of compound 6a against A2708
(IC50 ¼ 1.61 mM) is comparable to that
of CA-4 (IC50 ¼ 4.48 mM).
obtained on a Varian Mercury 300
spectrometer. Coupling constants (J) are
reported in hertz, and chemical shifts are
reported in parts per million (d) relative to
CDCl3 (7.26 ppm for 1H) or DMSO-d6
(2.49 ppm for 1H) or Acetone-d6
1
(2.04 ppm for H). Molecular weights of
compounds were determined by an Agi-
lent Technologies LC/MSD TOF spec-
trometer, and values are expressed as
[M þ H]þ.
4.2 The preparation procedure of the
intermediates
4.2.1 2-Methoxy-6-nitroaniline (1)
The chemical synthesis of compound 1
was according to the procedure described
by Zhang et al. [18]. The title compound
was obtained as salmon pink powder
1
(89%). Mp 68–698C; H NMR (CDCl3,
300 MHz, d ppm): d 7.73 (1H, d,
J ¼ 9.0 Hz), 6.88 (1H, d, J ¼ 7.8 Hz),
6.60 (1H, dd, J1 ¼ 9.0 Hz, J2 ¼ 7.8 Hz),
6.42 (2H, br s), 3.92 (3H, s); HR-MS
(ESI) m/z: 169.0607 [M þ 1]þ (calcd for
C7H9N2O3, 169.0608).
3. Conclusions
A variety of novel sulfonamides incorpor-
ating 1H-benzo[d]imidazol fragment were
synthesized. The preliminary study on the
SAR disclosed that installment of 1H-
benzo[d ]imidazol motif on nitrogen atom
of sulfonamide is beneficial to the
cytotoxicities of the target compounds. In
this series, compound 6a with a para-
methoxyl group on the phenyl ring is the
most potent chemical entity against five
cancer cell lines with cytotoxic IC50 value
ranging from submicromolar to low
micromolar level. The investigation on
other novel benzoimidazole CA-4 analogs
is currently in progress.
4.2.2 3-Methoxy-O-phenylenediamine
(2)
The chemical synthesis of compound 2
was according to the procedure described
by Doherty et al. [19]. The title compound
was obtained as off-white powder (77%).
Mp 77–788C; 1H NMR (CDCl3, 300 MHz,
d ppm): d 6.68 (1H, t, J ¼ 7.8 Hz), 6.42
(1H, d, J ¼ 7.8 Hz), 6.40 (1H, d,
J ¼ 7.8 Hz), 3.84 (3H, s), 3.31 (4H, br s);
HR-MS (ESI) m/z: 138.0870 [M þ 1]þ
(calcd for C7H9N2O, 138.0866).
4. Experimental
4.1 General experimental procedures
4.2.3 4-Methoxy-1H-benzo[d]imidazole
(3)
Unless noted otherwise, all reagents and
solvents were used as purchased without
further purification. Melting points (m.p.)
were determined on a Yanaco apparatus
and are uncorrected. NMR spectra were
The chemical synthesis of compound 3
was according to the procedure described
by Lane and Williams [20]. The title
compound was obtained as white powder

Journal of Asian Natural Products Research
335
(83.9%). Mp 165–1678C; 1H NMR
(acetone-d6, 300 MHz, d ppm): d 8.08
(1H, s), 7.21 (1H, d, J ¼ 7.8 Hz), 7.11 (1H,
t, J ¼ 7.8 Hz), 6.73 (1H, d, J ¼ 7.8 Hz),
3.97 (3H, s); HR-MS (ESI) m/z: 149.0707
[M þ 1]þ (calcd for C8H9N2O, 149.0709).
10% Pd/C (16 mg) in ethanol (10 ml) was
hydrogenated at room temperature and
atmospheric pressure for 5 h. The reaction
mixture was filtered and the filtrate was
evaporated to afford the title compound 5
(65 mg, 97% yield) as purple powder. Mp
150–1538C; 1H NMR (DMSO-d6,
300 MHz, d ppm): d 7.95 (1H, s), 6.46
(1H, d, J ¼ 8.1 Hz), 6.25 (1H, d,
J ¼ 8.1 Hz), 3.79 (3H, s); HR-MS (ESI)
m/z: 164.0816 [M þ 1]þ (calcd for
C8H10N3O, 164.0818).
4.2.4 4-Methoxy-7-nitro-1H-benzo[d]-
imidazole (4a) and 4-methoxy-5-nitro-1H-
benzo[d]imidazole (4b)
The mixture of 4-methoxy-1H-benzo[d]-
imidazole (3, 148 mg, 1 mmol) and sodium
nitrate (127 mg, 1.5 mmol) in TFA (3 ml)
was heated at 708C for 16 h. The reaction
mixture was cooled to room temperature
and poured into ice/water (20 ml). Precipi-
tation occurred at pH < 8 when the
mixture was treated with 10% NaOH
aqueous solution. The resulting mixture
was extracted with ethyl acetate
(10 ml £ 3). The solid in the water layer
was filtered and washed with water,
affording the desired compound 4a
(90 mg, yield 46.6%) as amber powder.
Mp 265–2678C; 1H NMR (acetone-d6,
300 MHz, d ppm): d 13.12 (1H, br s), 8.28
(1H, s), 8.17 (1H, d, J ¼ 8.4 Hz), 6.94 (1H,
d, J ¼ 8.4 Hz), 4.09 (3H, s); HR-MS (ESI)
m/z: 194.0562 [M þ 1]þ (calcd for
C8H8N3O3, 194.0560). The ethyl acetate
phase was concentrated to give a crude
product, which was purified by column
chromatography with CH2Cl2 –MeOH
(v/v ¼ 10:1). The side product 4b
(60 mg) was afforded as yellow powder
with 31% yield. Mp 161–1628C; 1H NMR
(acetone-d6, 300 MHz, d ppm): d 11.91
(1H, br s), 8.42 (1H, s), 7.72 (1H, d,
J ¼ 8.7 Hz), 7.29 (1H, d, J ¼ 8.7 Hz), 4.53
(s, 3H); HR-MS (ESI) m/z: 194.0563
[M þ 1]þ (calcd for C8H8N3O3,
194.0560).
4.2.6 4-Methoxy-1H-benzo[d]imidazole-
7-sulfonic acid (7)
4-Methoxy-1H-benzo[d]imidazole (3,
500 mg, 3.37 mmol) was added in portions
into chlorosulfonic acid (1.8 ml, 27 mmol)
at 258C. The mixture was stirred at 08C
for 4 h. After the reaction was quenched
with water, the solid was collected by
filtration and washed with water to give the
crude product, which was purified by
recrystallization with ethanol. The desired
product 7 (512 mg) was obtained as white
powder with 66.7% yield. Mp . 3008C;
1H NMR (DMSO-d6, 300 MHz, d ppm): d
9.47 (1H, s), 7.66 (1H, d, J ¼ 8.4 Hz), 7.10
(1H, d, J ¼ 8.4 Hz), 4.03 (3H, s); HR-MS
(ESI) m/z: 229.0277 [M þ 1]þ (calcd for
C8H9N2O4S, 229.0278).
4.3 General procedure for the prepara-
tion of N-(4-methoxy-1H-benzo[d]-
imidazol-7-yl)-arylsulfonamides (6a–6 l)
The reaction mixture of 4-methoxy-7-
amino-1H-benzo[d]imidazole (5, 163 mg,
1 mmol), sulfonyl chloride (1 mmol), and
pyridine (0.39 ml, 5 mmol) in CH2Cl2
(10 ml) was stirred at room temperature.
After the reaction completed, the precipi-
tate was filtered and washed with CH2Cl2
and water. The crude product was
recrystallized from the mixture of metha-
nol, dichloromethane, and petroleum
ether.
4.2.5 4-Methoxy-7-amino-1H-benzo[d]-
imidazole (5)
The mixture of 4-methoxy-7-nitro-1H-ben-
zo[d]imidazole (4a, 80mg, 0.46mmol) and

336
J. Zhou et al.
4.3.1 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-4-methoxybenzenesulfona-
mide (6a)
(1.8H, s), 3.74 (1.2H, s), 3.70 (3H, s); HR-
MS (ESI) m/z: 364.0959 [M þ 1]þ (calcd
for C16H18N3O5S, 364.0962).
Following the general procedure, starting
from 4-methoxybenzenesulfonyl chloride
(206 mg, 1 mmol), the title compound 6a
(200 mg, 60% yield) was obtained as off-
4.3.4 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-chloro-4-methylbenzene-
sulfonamide (6d)
1
white powder. Mp 191–1938C; H NMR
Following the general procedure, starting
from 3-chloro-4-methylbenzenesulfonyl
chloride (224 mg, 1 mmol), the title
compound 6d (200 mg, 56.9% yield) was
obtained as off-white powder. Mp 250–
(DMSO-d6, 300 MHz, d ppm): d 9.86 (1H,
s), 8.61 (1H, s), 7.62 (2H, d, J ¼ 8.7 Hz),
7.01 (2H, d, J ¼ 8.7 Hz), 6.70 (2H, s), 3.88
(3H, s), 3.78 (3H, s); HR-MS (ESI) m/z:
334.0864
C15H16N3O4S, 334.0856).
[M þ 1]þ
(calcd
for
1
2528C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 10.07 (1H, br s), 8.45 (1H, s),
7.73 (1H, s), 7.45–7.51 (2H, m), 6.70 (2H,
s), 3.88 (3H, s), 2.34 (3H, s); HR-MS
(ESI) m/z: 352.0519 [M þ 1]þ (calcd for
C15H15ClN3O3S, 352.0517).
4.3.2 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3,4- dimethylbenzene-
sulfonamide (6b)
Following the general procedure starting
from 3,4-dimethylbenzenesulfonyl chlor-
ide (204 mg, 1 mmol), the title compound
6b (100 mg, 30.3% yield) was obtained as
off-white powder. Mp 240–2438C; 1H
NMR (DMSO-d6, 300 MHz, d ppm): d
9.96 (1H, br s), 8.74 (1H, s), 7.52 (1H, s),
7.41 (1H, d, J ¼ 8.1 Hz), 7.24 (1H, d,
J ¼ 8.1 Hz), 6.73 (2H, s), 3.88 (3H, s),
2.23 (3H, s), 2.21 (3H, s); HR-MS (ESI)
m/z: 332.1061 [M þ 1]þ (calcd for
C16H18N3O3S, 332.1063).
4.3.5 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-nitro-4-methylbenzene-
sulfonamide (6e)
Following the general procedure, starting
from 3-nitro-4-methylbenzenesulfonyl
chloride (234 mg, 1 mmol), the title
compound 6e (188 mg, 51.9% yield) was
obtained as off-white powder. Mp 253–
1
2558C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 12.78 (0.5 H, br s), 12.33 (0.5 H,
br s), 10.02 (1H, br s), 8.29 (1H, s), 8.06
(1H, s), 7.85 (1H, br s), 7.60 (1H, d,
J ¼ 7.2 Hz), 6.64–6.93 (2H, m), 3.86 (3H,
s), 2.53 (3H, s); HR-MS (ESI) m/z:
4.3.3 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3,4-dimethoxybenzene-
sulfonamide (6c)
363.0757
C15H15N4O5S, 363.0758).
[M þ 1]þ
(calcd
for
Following the general procedure, starting
from
3,4-dimethoxybenzenesulfonyl
chloride (236 mg, 1 mmol), the title
compound 6c (200 mg, 55% yield) was
obtained as white powder. Mp 227–
4.3.6 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3,5-dichloro-4-hydroxyl-
benzene-sulfonamide (6f)
1
2298C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 12.75 (0.4H, s), 12.17 (0.6H, s),
9.75 (0.4H, br s), 9.56 (0.6H, s), 8.05
(0.4H, s), 8.00 (0.6H, s), 7.39 (0.6H, s),
7.32 (0.4H, d, J ¼ 8.7 Hz), 7.17–7.19 (m,
1H), 6.99 (0.6H, d, J ¼ 8.7 Hz), 6.93–6.98
(1H, m), 6.65 (0.4H, d, J ¼ 8.1 Hz), 6.52
(1H, q, J ¼ 8.1 Hz), 3.84 (3H, s), 3.77
Following the general procedure, starting
from 3,5-dichloro-4-hydroxylbenzenesul-
fonyl chloride (260 mg, 1 mmol), the title
compound 6f (110 mg, 28.4% yield) was
obtained as gray powder. Mp 283–2858C;
1H NMR (DMSO-d6, 300 MHz, d ppm): d
12.11 (1H, br s), 9.95 (1H, br s), 8.14 (1H,

Journal of Asian Natural Products Research
337
s), 7.61 (2H, s), 6.74 (1H, d, J ¼ 8.4 Hz),
6.65 (1H, d, J ¼ 8.4 Hz), 3.87 (3H, s);
HR-MS (ESI) m/z: 387.9918 [M þ 1]þ
(calcd for C14H12Cl2N3O4S, 387.9920).
compound 6i (180 mg, 53.7% yield) was
obtained as off-white powder. Mp 234–
1
2368C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 12.78 (0.4 H, br s), 12.27 (0.6 H,
br s), 9.88 (1H, br s), 8.03 (1H, s), 7.41
(3H, br s), 6.57–6.82 (2H, m), 3.86 (3H,
s), 2.24 (3H, s); HR-MS (ESI) m/z:
4.3.7 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-4-trifluoromethoxybenzene-
sulfonamide (6 g)
336.0815
C15H15FN3O3S, 336.0813).
[M þ 1]þ
(calcd
for
Following the general procedure, starting
from 4-trifluoromethoxybenzenesulfonyl
chloride (260 mg, 1 mmol), the title
compound 6 g (140 mg, 36.1% yield) was
obtained as off-white powder. Mp 234–
4.3.10 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-fluoro-4-methoxybenzene-
sulfonamide (6j)
1
2358C; H NMR (DMSO-d6, 300 MHz, d
Following the general procedure, starting
from 3-fluoro-4-methoxybenzenesulfonyl
chloride (226 mg, 1 mmol), the title
compound 6j (120 mg, 34.1% yield) was
obtained as off-white powder. Mp 221–
ppm): d 12.86 (0.5 H, br s), 12.25 (0.5 H,
br s), 9.91 (1H, br s), 7.99 (1H, s), 7.81
(2H, d, J ¼ 7.5 Hz), 7.47 (2H, d,
J ¼ 7.5 Hz), 6.59–6.89 (2H, m), 3.86
(3H, s); HR-MS (ESI) m/z: 388.0570
[M þ 1]þ (calcd for C15H13F3N3O4S,
388.0573).
1
2238C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 12.76 (0.4 H, br s), 12.23 (0.6 H,
br s), 9.92 (0.4H, br s), 9.70 (0.6H, s), 8.04
(0.4H, s), 8.01 (0.6H, s), 7.62 (0.4H, d,
J ¼ 11.1 Hz), 7.51 (0.4H, d, J ¼ 9.3 Hz),
7.48 (0.6H, d, J ¼ 11.1 Hz), 7.41 (0.6H, d,
J ¼ 9.3 Hz), 7.25 (0.6H, t, J ¼ 8.4 Hz),
7.19 (0.4H, t, J ¼ 8.4 Hz), 6.93 (0.6H, d,
J ¼ 8.4 Hz), 6.66 (0.4H, d, J ¼ 8.4 Hz),
6.50 (1H, s), 3.83–3.87 (6H, m); HR-MS
(ESI) m/z: 352.0758 [M þ 1]þ (calcd for
C15H15FN3O4S, 352.0762).
4.3.8 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-methoxybenzenesulfon-
amide (6 h)
Following the general procedure, starting
from 3-methoxybenzenesulfonyl chloride
(206 mg, 1 mmol), the title compound 6 h
(130 mg, 39% yield) was obtained as off-
1
white powder. Mp 178–1808C; H NMR
(DMSO-d6, 300 MHz, d ppm): d 12.62
(1H, br s), 9.86 (1H, br s), 8.08 (1H, s),
7.38 (1H, t, J ¼ 8.1 Hz), 7.25–7.28 (2H,
m), 7.11 (1H, dd, J1 ¼ 8.1 Hz,
J2 ¼ 1.8 Hz), 6.69 (1H, d, J ¼ 8.7 Hz),
6.57 (1H, d, J ¼ 8.7 Hz), 3.85 (3H, s), 3.73
(3H, s); HR-MS (ESI) m/z: 334.0860
[M þ 1]þ (calcd for C15H16N3O4S,
334.0856).
4.3.11 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-methyl-4-bromobenzene-
sulfonamide (6k)
Following the general procedure, starting
from 3-methyl-4-bromobenzenesulfonyl
chloride (269 mg, 1 mmol), the title
compound 6k (190 mg, 50% yield) was
obtained as off-white powder. Mp 263–
1
2668C; H NMR (DMSO-d6, 300 MHz, d
4.3.9 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3-fluoro-4-methylbenzene-
sulfonamide (6i)
ppm): d 12.46 (1H, br s), 9.93 (1H, br s),
8.04 (s, 1H), 7.69 (2H, d, J ¼ 8.1 Hz), 7.43
(1H, d, J ¼ 8.1 Hz), 6.68 (1H, br s), 6.58
(1H, d, J ¼ 8.1 Hz), 3.86 (3H, s), 2.33 (3H,
s); HR-MS (ESI) m/z: 396.0008 [M þ 1]þ
(calcd for C15H15BrN3O3S, 396.0012).
Following the general procedure, starting
from 3-fluoro-4-methylbenzenesulfonyl
chloride (208 mg, 1 mmol), the title

338
J. Zhou et al.
4.3.12 N-(4-Methoxy-1H-benzo[d]-
imidazol-7-yl)-3,4,5-trimethoxybenzene-
sulfonamide (6 l)
anhydrous Na2SO4. The organic phase was
concentrated to give the crude product,
which was purified by column chromatog-
raphy with CH2Cl2–MeOH (v/v ¼ 20:1).
The reaction mixture of 4-methoxy-7-
amino-1H-benzo[d]imidazole 5 (124 mg,
0.76 mmol), 3,4,5-trimethoxybenzenesul-
fonyl chloride (200 mg, 0.7 mmol), and
pyridine (0.15 ml, 1.9 mmol) in CH2Cl2
(10 ml) and DMF (0.5 ml) was stirred at
room temperature. After the reaction
completed, the reaction mixture was
diluted with water (30 ml) and extracted
with ethyl acetate (30 ml £ 3). The com-
bined extracts were washed with water and
brine, and then dried over anhydrous
Na2SO4, filtered, and concentrated to
give the crude product, which was purified
by column chromatography on silica gel
with CH2Cl2–MeOH (v/v ¼ 10:1). The
title compound 6 l (80 mg, 26.8% yield)
was obtained as white powder. Mp 183–
4.4.1 N-(4-Methoxyphenyl)-(4-methoxy-
1H-benzo[d]imidazole)-7-sulfonamide (8a)
Following the general procedure, starting
from 4-methoxybenzenamine (108 mg,
0.88 mmol), the title compound 8a
(140 mg, 63.9% yield) was obtained as
1
brown powder. Mp 181–1838C; H NMR
(acetone-d6, 300 MHz, d ppm): d 8.56 (1H,
br s), 8.18 (1H, br s), 7.49 (1H, d,
J ¼ 8.4 Hz), 6.98–7.02 (2H, m), 6.79
(1H, d, J ¼ 8.4 Hz), 6.70 (1H, d,
J ¼ 8.4 Hz), 4.04 (3H, s), 3.67 (3H, s);
HR-MS (ESI) m/z: 334.0862 [M þ 1]þ
(calcd for C15H16N3O4S, 334.0856).
1
1858C; H NMR (DMSO-d6, 300 MHz, d
4.4.2 N-(3,4-Dimethoxyphenyl)-(4-
methoxy-1H-benzo[d]imidazole)-7-
sulfonamide (8b)
ppm): d 8.05 (1H, s), 7.01 (2H, br s), 6.79
(1H, br s), 6.62 (1H, d, J ¼ 7.2 Hz), 3.87
(3H, s), 3.72 (6H, s), 3.67 (3H, s); HR-MS
(ESI) m/z: 394.1071 [M þ 1]þ (calcd for
C17H20N3O6S, 394.1067).
Following the general procedure, starting
from 3,4-dimethoxybenzenamine
(134.6 mg, 0.88 mmol), the title compound
8b (122 mg, 51% yield) was obtained as
1
brown powder. Mp 198–2008C; H NMR
(acetone-d6, 300 MHz, d ppm): d 8.58 (1H,
br s), 8.19 (1H, br s), 7.54 (1H, d,
J ¼ 8.4 Hz), 6.81 (1H, d, J ¼ 8.4 Hz),
6.73 (1H, d, J ¼ 2.4 Hz), 6.68 (1H, d,
J ¼ 8.4 Hz), 6.57 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 2.4 Hz), 4.04 (3H, s), 3.66 (3H, s),
3.60 (3H, s); HR-MS (ESI) m/z: 364.0957
[M þ 1]þ (calcd for C16H18N3O5S,
364.0962).
4.4 General procedure for the prepara-
tion of N-aryl-(4-methoxy-1H-benzo-
[d]imidazole)-7-sulfonamides (8a–8d)
4-Methoxy-1H-benzo[d]imidazole-7-sul-
fonic acid (7, 150 mg, 0.66 mmol) in
thionyl chloride (5 ml, 68.5 mmol) and
DMF (5 drops) was refluxed for 4 h. The
reaction mixture was concentrated and the
crude 4-methoxy-1H-benzo[d]imidazole-
7-sulfonyl chloride was directly used to
the next step without further purification.
The arylamine (0.88 mmol) and Et3N
(0.88 mmol) were added to the solution
of sulfonyl chloride in CH2Cl2 (15 ml) and
DMF (1 ml) at room temperature. After the
reaction completed, the solvent was
evaporated under reduced pressure and
the residue was taken up with EtOAc
(60 ml). The organic solution was washed
with water and brine, then dried over
4.4.3 N-(3,4,5-Trimethoxyphenyl)-(4-
methoxy-1H-benzo[d]imidazole)-7-
sulfonamide (8c)
Following the general procedure, starting
from3,4,5-trimethoxybenzenamine(120 mg,
0.66 mmol), the title compound 8c (44 mg,
25.4% yield) was obtained as brown
powder. Mp 91–938C; 1H NMR

Journal of Asian Natural Products Research
(acetone-d6, 300 MHz, d ppm): d 8.76 (1H, 5.2 Cytotoxicity assay
339
br s), 8.21 (1H, br s), 7.63 (1H, d,
J ¼ 8.4 Hz), 6.84 (1H, d, J ¼ 8.4 Hz),
6.43 (2H, s), 4.04 (3H, s), 3.61 (6H, s),
3.56 (3H, s); HR-MS (ESI) m/z: 394.1069
[M þ 1]þ (calcd for C17H20N3O6S,
394.1069).
The in vitro cytotoxic activities of tested
compounds were determined by MTT [3-
(4,5-dimethylthiazo-2-yl)-2,5-diphenylt-
etrazolium bromide, Sigma, St Louis, MO,
USA] assays. Briefly, cells were seeded
into 96-well plates at a density of 1–
2 £ 103/well (depending on the cell
growth rate). Twenty-four hours later,
triplicate wells were treated with media
and agents. After 72 h incubation at 378C
in 5% CO2, the medium containing drug
was removed and replaced by fresh
medium. The cells in each well were
then incubated in culture medium with
100 ml of 0.5 mg/ml MTT solution for 4 h.
After the medium was removed, 150 ml of
DMSO was added to each well. The plates
were gently agitated until the color of the
reaction was uniform and the OD570 was
determined using a microplate reader
(Wellscan MK3, Labsystems Dragon,
Helsinki, Finland). Microsoftw Excel
2003 was used for data analysis. Media-
only treated cells served as the indicator of
100% cell viability. The 50% inhibitory
concentration (IC50) was defined as the
concentration that reduced the absorbance
of the untreated wells by 50% of the
vehicle in the MTT assay.
4.4.4 N-(3,4,5-Trifluorophenyl)-(4-
methoxy-1H-benzo[d]imidazole)-7-
sulfonamide (8d)
To a solution of 4-methoxy-1H-benzo[-
d]imidazole-7-sulfonyl chloride (250 mg,
1.02 mmol) in CH2Cl2 (8 ml) was added
3,4,5-trifluorobenzenamine
(179 mg,
1.22 mmol) and pyridine (0.3 ml,
3.05 mmol). The reaction mixture was
stirred at room temperature for 5 h. A crude
product was obtained by filtration and was
recrystallized from methanol. The title
compound 8d (90 mg, 24.7% yield) was
obtained as white powder. Mp 225–
1
2278C; H NMR (DMSO-d6, 300 MHz, d
ppm): d 8.34 (1H, s), 7.72 (1H, d,
J ¼ 8.7 Hz), 7.01 (1H, d, J ¼ 6.6 Hz),
6.98 (1H, d, J ¼ 6.6 Hz), 6.92 (1H, d,
J ¼ 8.7 Hz), 4.01 (3H, s); HR-MS (ESI)
m/z: 358.0472 [M þ 1]þ (calcd for
C14H11F3N3O3S, 358.0468).
5. Biological evaluation
Acknowledgements
5.1 Cell culture
This work was supported by the Special Funds
for National Public Benefit Research Institutes
(No. 2010CHX03) and National Program of
New Drug Innovation and Production (No.
2009ZX09301-003-1-1).
The human lung adenocarcinoma cell line
A549, the human hepatocellular carci-
noma cell line Bel7402, the human
stomach adenocarcinoma cell line
BGC823, the human colon carcinoma
cell line HCT8, and the human ovarian
carcinoma cell line A2780 were cultured at
378C in a 5% CO2 and 95% air atmosphere
using RPMI-1640 with 10% FBS and
penicillin (100 U/ml). All the cells were
purchased from the American Type
Culture Collection and the Cell Culture
Center of Institute of Basic Medical
Science, Chinese Academy of Medical
Sciences.
Notes
1. Oxigene company web site: www.oxigene.
com (March 2010).
2. Aventis company web site: www.sanofi-
aventis.com.
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