Synthesis and biological evaluation of benzimidazole-4,7-diones that inhibit vascular smooth muscle cell proliferation

Article abstract of DOI:10.1016/j.bmcl.2004.04.051

A series of 6-arylamino-5-chloro-benzimidazole-4,7-diones were synthesized and tested for their inhibitory activity on the rat aortic smooth muscle cell (RAoSMC) proliferation. Among them, 6-arylamino-5-chloro-2-methyl-benzimidazole- 4,7-diones exhibited potent antiproliferative activity. Benzimidazole-4,7-dione 2c activated SAPK/JNK signaling pathway in the RAoSMCs.

Full text of DOI:10.1016/j.bmcl.2004.04.051

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3563–3566
Synthesis and biological evaluation of benzimidazole-4,7-diones
that inhibit vascular smooth muscle cell proliferation
Sung-Yu Hong,b,c Kwang-Hoe Chung,b,c Hea-Jung You,a Ik Hwa Choi,a Mi Jin Chae,a
Ja-Young Han,a Ok-Jai Jung,a Soo-Jung Kangb and Chung-Kyu Ryua,*
aCollege of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, South Korea
bNational Research Lab for Cardiovascular Nanotechnology, BioBud Co. Ltd, Seodaemun-ku, Seoul 120-110, South Korea
cCardiovascular Research Institute and BK21 Projects for Medical Sciences, Yonsei University College of Medicine,
Seoul 120-752, South Korea
Received 4 March 2004; accepted 8 April 2004
Abstract—A series of 6-arylamino-5-chloro-benzimidazole-4,7-diones were synthesized and tested for their inhibitory activity on the
rat aortic smooth muscle cell (RAoSMC) proliferation. Among them, 6-arylamino-5-chloro-2-methyl-benzimidazole-4,7-diones
exhibited potent antiproliferative activity. Benzimidazole-4,7-dione 2c activated SAPK/JNK signaling pathway in the RAoSMCs.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
O
H
N
H
N
R2
The abnormal proliferation and migration of vascular
smooth muscle cells (SMCs) play an important role in
the pathology of coronary artery atherosclerosis and
restenosis following angioplasty.1 Arterial injury results
in the migration of SMCs into the intimal layer of the
arterial wall, where they proliferate and synthesize
extracellular matrix components. Several growth factors
induce the proliferation and migration of arterial
SMCs.2 Platelet-derived growth factor (PDGF) is one of
the most potent promoters of the proliferation and
migration of SMCs.3
N
X
R1
O
R1= H, F, .. R2= H, alkyl or aryl
X = H or Cl
1
O
O
R1
O
H
N
H
N
H
N
H
N
R2
R3
R1
R2
R4
N
N
Cl
X
O
R1, R2, R3 = H, F, Cl ...
2: R4=CH3
R1, R2 = H, F, Cl, ..
4: X = Cl
Compounds containing the heterocyclic quinone group
represent an important class of biologically active mol-
ecules.4 However, the inhibitory activity of quinone
classes on the proliferation of SMC has not been
reported to the best of our knowledge. Therefore, we
synthesized and tested various quinone derivatives to
elucidate their contribution to the antiproliferative
effects on PDGF-stimulated SMC proliferation. Among
the quinones tested, benzimidazole-4,7-dione derivatives
1 (Fig. 1) showed the potent antiproliferative activity.
There have been several reports5–10 on some benzimid-
3: R4=CF3
5: X = Br
Figure 1. Benzimidazole-4,7-dione derivatives.
azole-4,7-dione derivatives, which exhibited antiproli-
ferative effect5 on human lymphoblastic leukemia and
non-Hodgkin lymphoma, cytotoxic activity6;7 against
cancer cell lines, inhibitory effect8 on protozoal purine
nucleoside phosphorylase and antifungal activity.9
We describe herein our preliminary results on the syn-
thesis of 6-arylamino-benzimidazole-4,7-diones 2–5 and
their antiproliferative activity on the rat aortic SMCs
(RAoSMCs).
* Corresponding author. Tel.: +82-2-3277-3027; fax: +82-2-3277-3051;
e-mail: ckryu@mm.ewha.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.051

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S.-Y. Hong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3563–3566
Table 1. Structures and IC50 values of the benzimidazole-4,7-diones
for inhibition of SMC proliferation
Additional data for the mechanism of SMC antiproli-
ferative activity of compound 2c is provided. Among the
signal transduction pathways, mitogen-activated protein
kinase (MAPK) pathways have a critical role in the
proliferation of the SMCs.11;12 Activation of MAPK
pathways is observed on treating SMCs with growth
factors, tumor necrosis factor, interleukin-1, and upon
stress exposure. A central function of the MAPK
pathways is the activation of gene expression, mediated
through phosphorylation of transcription factors. A
stress-activated protein kinase/Jun-N-terminal kinase
(SAPK/JNK) is a kind of MAPK. The SAPK/JNK is
required for cell cycle arrest, apotosis, and growth of the
SMCs.12 In order to investigate the effect of benzimid-
azole-4,7-dione 2c on the intracellular signaling of
SMCs, we examined whether the compound 2c activates
the SAPK/JNK.
O
R1
H
N
H
N
R2
R3
R4
N
Cl
O
b
Compds R1
R2
R3
R4
SMCa IC50
(lM)
2a
2b
2c
2d
2e
2f
H
H
H
H
H
Cl
Cl
F
Br
H
H
H
H
Cl
H
H
Cl
F
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CF3
CF3
1.0
0.8
CH3
H
0.8
2.8
Cl
F
3.0
0.6
H
2g
2h
2i
Cl
F
0.8
1.2
H
F
H
0.9
1.1
2j
F
2k
2l
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
I
0.6
1.0
2. Chemistry
3a
3b
3c
3d
3e
3f
CH3
CF3
H
40.0
20.0
50.0
15.0
25.0
12.0
>100
1.0
The method used to synthesize the 6-arylamino-5-
chloro-2-methyl-benzimidazole-4,7-diones 2 is shown in
Scheme 1. 4,7-Dimethoxy-2-methyl-benzimidazole (6a)
was prepared according to the known method.10 Cycli-
zation of 2,3-diamino-1,4-dimethoxybenzene with
CH3COOH gave the compound 6a. 5,6-Dichloro-2-
methyl-benzimidazole-4,7-dione (7a) was synthesized by
oxidizing the compound 6a with HNO3/HCl variation
resulting in 43% yields. The benzimidazole-4,7-diones
2a–l (Table 1) were prepared by nucleophilic substitu-
tion on the compound 7a with appropriate arylamines.
Most of these substitutions went as expected and had
overall high yields of 76–94%.
Cl
I
F
6a
MPA
a The SMCs were isolated from rat thoracic aorta.
b The inhibitory activity against the PDGF-induced proliferation of the
SMCs.
Demethylating the compound 8 with HBr gave 4,7-di-
hydroxybenzimidazole hydrobromide (10), which was
oxidized to the 5,6-dibromo-benzimidazole-4,7-dione
(11)14 in a HBr/NaBrO3 solution resulting in a 48%
yield. The 6-arylamino-5-bromo-benzimidazole-4,7-di-
ones 5a–f (Table 2) were synthesized by nucleophilic
substitution on the compound 11 with arylamines in
good yields.
In a similar manner, 6-arylamino-5-chloro-2-trifluo-
romethyl-benzimidazole-4,7-diones
3 were prepared
from 4,7-dimethoxy-2-trifluoromethyl-benzimidazole
(6b),13 which was oxidized to compound 7b. The benz-
imidazole-4,7-diones 3a–f (Table 1) were synthesized by
the substitution on the compound 7b with the arylam-
ines in good yields.
3. Antiproliferative activity
6-Arylamino-5-chloro-benzimidazole-4,7-diones 4 were
prepared from the 5,6-dichloro-benzimidazole-4,7-dione
(9) (Scheme 2). Compound 9 was prepared by oxidizing
4,7-dimethoxybenzimidazole (8)10 with the HNO3/HCl
variation. The benzimidazole-4,7-diones 4a–i (Table 2)
were synthesized by the substitution on the compound 9
with the arylamines in good yields.
The synthesized benzimidazole-4,7-diones 2–5 were tes-
ted in vitro for their antiproliferative activity on the
RAoSMCs. Inhibition of proliferation of these cells was
determined by WST colorimetric assay.15;16 The IC50
values were determined by comparison to mycophenolic
acid (MPA)17 as a standard agent. As indicated in the
OCH3
O
R1
O
H
N
H
H
N
H
Cl
Cl
R2
R3
N
N
a
b
R
R4
R
N
N
N
Cl
OCH3
O
O
7a : R=CH3
7b : R=CF3
6a : R=CH3
6b : R=CF3
R1, R2, R3 = H, F, Cl ...
2a-l : R4=CH3
3a-f : R4=CF3
Scheme 1. Synthesis of 2-alkyl-6-arylamino-benzimidazole-4,7-diones. Reagents and conditions: (a) c-HCl/c-HNO3/reflux/0.5 h/ 7a 43%; 7b 56%;
(b) arylamine (1 equiv)/EtOH/reflux/5 h/76–94%.

S.-Y. Hong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3563–3566
3565
OCH3
OCH3
O
O
H
N
H
N
H
N
H
N
Cl
Cl
R1
R2
b
a
N
N
N
Cl
O
O
9
8
R1, R2 = H, F, Cl ...
4a-i
c
OH
OH
O
O
O
H
H
N
H
N
H
N
Br
Br
N
R1
R2
b
d
HBr
N
N
N
Br
O
10
11
R1, R2 = H, F, Cl ...
5a-f
Scheme 2. Synthesis of 6-arylamino-benzimidazole-4,7-diones. Reagents and conditions: (a) c-HCl/c-HNO3/reflux/0.5 h/52%; (b) arylamine (1 equiv)/
EtOH/reflux/5 h/70–91%; (c) c-HBr/reflux/6h/39%; (d) HBr/NaBrO3/reflux/1 h/48%.
Table 2. Structures and IC50 values of the benzimidazole-4,7-diones
for inhibition of SMC proliferation
for the development as inhibitors of the SMC prolifer-
ations.
O
H
N
H
N
R1
R2
In terms of structure–activity relationship, the 6-aryl-
amino-2-methyl-benzimidazole-4,7-diones 2 showed, in
general, more potent antiproliferative activity than
benzimidazole-4,7-dione series 3–5. The 2-methyl
substituted compounds 2 exhibited the greatest activity,
indicating a correlation that may offer insight into the
mode of action of these compounds. The 2-trifluoro-
methyl moiety of compounds 3 did not appeared to
contribute partially toward biological potency. The
structure–activity relationship may not exist between
properties of substituents (R: F, Cl, Br,. . .) for 6-aryl-
amino moieties of benzimidazole-4,7-diones 2–5.
N
X
O
Compds
X
R1
R2
SMC IC50
(lM)
4a
4b
4c
4d
4e
4f
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
H
H
H
H
I
F
1.2
1.3
I
CH3
CF3
H
1.4
2.5
1.3
1.4
Cl
Br
H
F
H
4g
4h
4i
H
1.6
5.5
H
F
1.1
1.0
In addition, nonquinonoid compounds 6a and 8 exhib-
ited no antiproliferative activity. Thus, quinone moiety
of benzimidazole-4,7-diones may be essential for the
antiproliferative activity.
5a
5b
5c
5d
5e
5f
H
H
H
H
I
F
CH3
CF3
Cl
H
1.0
3.1
4.0
2.5
H
H
1.1
4. Effect of compound 2c on SAPK/JNK
8
>100
In order to investigate the effect of the compound 2c on
the intracellular signaling of the SMCs, we examined
whether the compound 2c activates the SAPK/JNK
protein in the RAoSMCs by immunoblot assay.18 As
shown in Figure 2, the compound 2c had little effect on
the expression of the SAPK/JNK protein but dramati-
cally increased the phosphorylation of the protein
Tables 1 and 2, the most active potential among the
benzimidazole-4,7-dione series 2–5 was found for 6-
arylamino-5-chloro-2-methyl-benzimidazole-4,7-diones
2a–l, which showed generally good activity. Actually,
many compounds of benzimidazole-4,7-diones 4a–i and
5a–f exhibited good activity. The activity of these com-
pounds is superior or comparable to that of MPA. In
contrast, compounds 3a–f did not show significant
activity.
The most active potential among the series was found
for the compounds 2b, 2c, 2f, 2g, and 2k, which showed
half maximal inhibition of the PDGF-stimulated pro-
liferation of the RAoSMCs tested at the level of 0.8 lM.
The results indicate that the 6-arylamino-5-chloro-2-
methyl-benzimidazole-4,7-diones 2 are a promising lead
Figure 2. The compound 2c activated SAPK/JNK protein in rat aortic
SMC. The SMCs treated with the compound 2c for 0.5, 1, 2, 4, and 8 h
were immunoblotted with anti-SAPK/JNK polyclonal antibody and
phospho-SAPK/JNK 6 monoclonal antibody, respectively.

3566
S.-Y. Hong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3563–3566
11. Robinson, J. R.; Cobb, M. H. Curr. Opin. Cell Biol. 1997,
9, 180.
12. Mayr, M.; Xu, Q. Exp. Gerontol. 2001, 36, 969.
13. Middleton, R. W.; Monney, H.; Parrick, J. Synthesis 1984,
740.
(phospho-SAPK/JNK), which means that the com-
pound 2c activated SAPK/JNK pathway. SAPK/JNK,
one of various MAPKs is potentially activated by a
variety of environmental stress, including UV and
gamma radiation, ceramide, inflammatory cytokine and,
some instance, by growth factors. The result suggests
that the compound 2c could induce cell cycle arrest or
apoptosis by activating SAPK/JNK pathway, which
resulted in the inhibition of the SMC proliferation. On
the base of the result, we propose that the antiprolifer-
ative benzimidazole-4,7-dione series, as derivatives of
the compound 2c, could activate SAPK/JNK pathway
in the SMCs. Further pharmacological investigations of
these compounds and the structural optimization are in
progress.
ꢀ
14. March, L. C.; Joullie, M. M. J. Heterocyclic Chem. 1970,
395.
15. SMC proliferation assay: The RAoSMCs were isolated
from rat thoracic aorta by method described previously.16
The cells were maintained in Dulbeccos’s modified eagle
medium (DMEM, Gibco BRL, Grand Island, NY, USA)
supplemented with 10% fetal bovine serum, 100 units/mL,
and 100 lg/mL streptomycin. The SMCs were seeded in
triplicate at a concentration of 1Â103 cells/well in 200 lL
of DMEM containing 10% (v/v) fetal bovine serum in 96-
well flat-bottom plates (Costar, Corning, NY, USA). After
24 h incubation, the complete medium was replaced with
DMEM containing 0.2% FBS, and incubated for an
additional 72 h. And then the cells were treated with test
compounds in 100 lL of DMEM containing PDGF (5 ng/
mL) and 5% (v/v) fetal bovine serum for 48 h. Proliferation
of the cells was determined using a colorimetric assay kit
based on the uptake of WST by viable cells (Premix WST-
1 cell proliferation assay system, Takara Bio Inc, Otsu,
Japan). The assay kit is dependent on the reduction of
tetrazolium salt WST-1, which results in formation of a
dark red formazan product, by various mitochondrial
dehydrogenase of viable cells.
Acknowledgements
This study was supported by a grant of the Korea Sci-
ence and Engineering Foundation (KOSEF R06-2002-
011-01004-0) and a grant of the National Research Lab
from the Ministry of the Science and Technology
(M10203000098-02J0000-04610).
16. Sohn, Y. D.; Lim, H. J.; Hwang, K. C.; Kwon, J. H.; Park,
H. Y.; Chung, K.-H.; Cho, S. Y.; Jang, Y. Biochem.
Biophys. Res. Commun. 2001, 284, 931.
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