Design, synthesis and evaluation of 2-phenyl-1H-benzo[d]imidazole-4,7-diones as vascular smooth muscle cell proliferation inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.03.066

A series of 2-phenyl-1H-benzo[d]imidazole-4,7-diones were synthesized and tested for their inhibitory activity on the PDGF-stimulated proliferation of rat aortic vascular smooth muscle cells. Among the tested compounds, 6-arylthio-5-chloro-2-phenyl-1H-benzo[d]imidazole-4,7-diones exhibited an potent antiproliferative activity.

Full text of DOI:10.1016/j.bmcl.2008.03.066

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2948–2951
Design, synthesis and evaluation of 2-phenyl-1H-benzo[d]-
imidazole-4,7-diones as vascular smooth muscle
cell proliferation inhibitors
Chung-Kyu Ryu,a,* Ra-Young Lee,a Seung-Yon Lee,a Hwa-Jin Chung,a
Sang Kook Leea and Kwang-Hoe Chungb
aCollege of Pharmacy, Ewha Womans University, Seodaemun-ku, Seoul 120-750, Republic of Korea
bThrombosis & Vascular Biochemistry Laboratory, Department of Biochemistry, College of Medicine, Pochon CHA University,
Sungnam 463-836, Republic of Korea
Received 17 December 2007; revised 5 March 2008; accepted 24 March 2008
Available online 27 March 2008
Abstract—A series of 2-phenyl-1H-benzo[d]imidazole-4,7-diones were synthesized and tested for their inhibitory activity on the
PDGF-stimulated proliferation of rat aortic vascular smooth muscle cells. Among the tested compounds, 6-arylthio-5-chloro-2-phe-
nyl-1H-benzo[d]imidazole-4,7-diones exhibited an potent antiproliferative activity.
Ó 2008 Elsevier Ltd. All rights reserved.
The proliferation and migration of vascular smooth
muscle cells (SMCs) play an important role in the pro-
gression of atherosclerosis and restenosis.1 Abnormal
arterial injury results in the migration of SMCs into
the intimal layer of the arterial wall, where they prolifer-
ate and synthesize extracellular matrix components.
Many growth factors induced the proliferation and
migration of arterial SMCs.2 Among them, platelet-de-
rived growth factor (PDGF) is one of the most potent
promoters of the proliferation and migration of SMCs.3
SMCs (Fig. 1). We describe herein our results on the
synthesis of 2-phenyl-1H-benzo[d]imidazole-4,7-dione
series 2–3 and their antiproliferative activity on the rat
aortic SMCs. Additional data for the antiproliferative
activity of other 2-pyridyl-1H-benzo[d]imidazole-4,7-
diones 4 are also provided.
Additional data for the mechanism of SMCs antiprolif-
erative activity of one representative 1H-benzo[d]
imidazole-4,7-dione 3a was also performed. The mito-
gen-activated protein kinase (MAPK) cascade known
as the extracellular signal-regulated kinase (ERK) path-
way mediates mitogenic responses induced by a wide
variety of growth factor receptors in many cell types,
Quinonoid compounds represent an important class of
biologically active molecules.4 Therefore, we designed,
synthesized and evaluated the antiproliferative effects
of various quinone derivatives on PDGF-stimulated
SMC proliferation. Previously, we reported that benz-
imidazole-4,7-dione derivatives 1 exhibited a potent
inhibition for the smooth muscle cells (SMCs) prolifera-
tion as preliminary results. The arylamino, arylthio- or
phenyl-substituents of quinones have been improved
several biological activities.5 On this line, we further ex-
tended to synthesize 1H-benzo[d]imidazole-4,7-diones
2–4, which would be analogues of quinones 1, and eval-
uated their antiproliferative activity on the rat aortic
O
O
O
O
H
N
H
N
W
Cl
R1
R2
R1
R2
Y
X
Z
R3
1
N
N
2
R , R = H, X, amino ..
1
2
R , R = H, F, Cl, ..
3
R = H, Al or Ar
2: W = NH; X,Y, Z = CH
3: W = S; X,Y, Z = CH
1
Keywords: 2-Phenyl-1H-benzo[d]imidazole-4,7-dione; Smooth muscle
cell; Antiproliferative activity; Substitution effects.
4: W = NH or S; X,Y, Z = N or CH
*
Figure 1. 1H-Benzo[d]imidazole-4,7-dione derivatives.
Corresponding author. E-mail: ckryu@mm.ewha.ac.kr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.066

C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2948–2951
2949
OCH3
The method used to synthesize 6-arylamino-5-chloro-2-
phenyl-1H-benzo[d]imidazole-4,7-diones 2 is shown in
Scheme 1. 2,3-Diamino-1,4-dimethoxybenzene (5) was
prepared according to the known method.10 Cycliza-
tions of compound 5 with benzaldehyde gave 4,7-dime-
thoxy-2-phenyl-benzimidazole (6) resulting in 72%
OCH3
OCH3
H
N
H2N
H2N
a
b
N
OCH3
5
7
6
yields.
5,6-Dichloro-2-phenyl-1H-benzo[d]imidazole-
O
O
O
O
H
N
H
4,7-dione (7) was synthesized by oxidizing compound 6
with HNO3/HCl variation resulting in 46% yields. 2-
Phenyl-1H-benzo[d]imidazole-4,7-diones 2a–i (Table 1)
were prepared by nucleophilic substitution on com-
pound 7 with appropriate arylamines. Most of these
substitutions went as expected and had overall high
yields of 75–92%.
Cl
Cl
X
R1
R2
N
N
c or d
N
Cl
R1, R2 = H, F, Cl ...
2a-i: X=NH
3a-f: X=S
Scheme 1. Synthesis of 2-phenyl-1H-benzo[d]imidazole-4,7-diones.
Reagents and conditions: (a) benzaldehyde (1 equiv)/toluene/reflux/
4 h/72%; (b) concd HCl/concd HNO3/reflux/0.5 h/46%; (c) arylamine
(1 equiv)/EtOH/reflux/5 h/75–92%/; (d) arylthiol (1 equiv)/EtOH/
reflux/24 h/65–90%.
In a similar manner, 6-arylthio-5-chloro-2-phenyl-1H-
benzo[d]imidazole-4,7-diones 3a–f (Table 1) were syn-
thesized by nucleophilic substitution on the compound
7 with appropriate arylthiols in good yields. 2-Pyridyl-
1H-benzo[d]imidazole-4,7-diones 4a–t (Table 2) were
prepared according to a method previously reported.5
including SMCs.6 MAPKs play an important role in
regulating cell growth and survival, and are also in-
volved in both mitogenic and stress responses of cells.7
MAPK is activated through a specific phosphorylation
cascade. In general, the ERK pathway plays a major
role in regulating cell growth and differentiation, being
highly induced in response to growth factors and cyto-
kines.8 The ERK pathway is required for cell cycle ar-
rest, apoptosis and growth of the SMCs.9 In order to
investigate the effect of the compound 3a on the prolif-
eration of SMCs and its mechanism of action, we exam-
ined the effect of the compound on ERK activation and
cell cycle regulation.
The 1H-benzo[d]imidazole-4,7-diones 2–4 were tested
in vitro for their antiproliferative activity on the rat aor-
tic SMC proliferation. Inhibition of PDGF-stimulated
proliferation was determined by colorimetric assay.11
The IC50 values were determined by comparison to
mycophenolic acid (MPA)12 as a standard agent. As
indicated in Tables 1 and 2, 6-arylthio-2-phenyl-1H-
Table 2. Structures and IC50 values of 2-pyridyl-1H-benzo[d]imida-
zole-4,7-diones for inhibition of SMC proliferation
O
H
W
Cl
Y
Z
N
N
Table 1. Structures and IC50 values of 2-phenyl-1H-benzo[d]imida-
zole-4,7-diones for inhibition of SMC proliferation
X
R
O
O
H
b
Compound
X
Y
Z
W
R
SMCa IC50 (lM)
X
R1
R2
N
N
4a
4b
4c
4d
4e
4f
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
CH CH
N
N
N
N
N
N
N
NH
NH Cl
F
9.4
12.1
6.5
Cl
NH Br
O
NH OCH3
NH CH3
NH CF3
NH OCF3
20.0
50.0
1.0
b
Compound
X
R1
R2
SMCa IC50 (lM)
2a
2b
2c
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
H
H
H
H
OCH3
CH3
H
2.0
3.0
4.0
1.5
3.0
9.4
50.0
5.3
10.7
1.0
1.0
1.5
1.5
2.0
2.0
1.0
4g
4h
4i
20.0
21.0
20.0
50.0
42.0
50.0
25.0
100.0
50.0
4.0
CH
CH
CH
CH
N
N
N
N
N
CH NH
F
2d
2e
OCF3
CH3
CF3
CH NH Cl
CH NH Br
CH NH OCH3
CH3
H
4j
2f
4k
4l
2g
2h
2i
H
OCH2CH3
Cl
CH CH NH
F
H
4m
4n
4o
4p
4q
4r
N
CH CH NH Cl
CH CH NH Br
CH CH NH OCH3
H
CH2CH3
OCH3
CH3
H
N
3a
3b
3c
H
N
S
H
CH CH
CH CH
CH CH
CH CH
CH CH
N
N
N
N
N
S
S
S
S
S
Cl
Br
S
H
12.0
4.2
3d
3e
S
H
Br
OH
OCH3
CH3
CH2CH3
S
H
CH3
4s
2.5
7.0
3f
MPA
S
H
4t
MPA
1.0
a SMCs were isolated from rat thoracic aorta.
b The inhibitory activity against the PDGF-induced proliferation of
SMCs.
a SMCs were isolated from rat thoracic aorta.
b The inhibitory activity against the PDGF-induced proliferation of
SMCs.

2950
C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2948–2951
benzo[d]imidazole-4,7-diones 3a–f showed generally
good activity. Actually, many compounds of 6-arylami-
not greatly influence the inhibitory activity. This sug-
gests that 1H-benzo[d]imidazole-4,7-dione structure is
mainly responsible for the activities.
no-2-phenyl-1H-benzo[d]imidazole-4,7-diones
2a–i
exhibited the potent activity. In contrast, 2-pyridyl-1H-
benzo[d]imidazole-4,7-diones 4a–t did not show signifi-
cant their antiproliferative activity, although some com-
pounds of them exhibited good antiproliferative activity.
1H-Benzo[d]imidazole-4,7-diones 3a, 3b and 4f inhibited
the PDGF-stimulated proliferation of the SMC tested at
the IC50 of 1.0 lM. The activity of these compounds is
comparable to that of MPA.
Further mechanistic study on the antiproliferative activ-
ity was performed using one representative compound
3a in cultured SMCs. As illustrated in Figure 2, when
SMCs were exposed to 1 lg/mL of the compound 3a
for 48 h morphological changes also revealed that the
cell density was decreased by observation under the
phase-contrast microscope.
In terms of structure–activity relationship, 2-phenyl-1H-
benzo[d]imidazole-4,7-diones
To explore whether the antiproliferative effects on the
compound 3a were mediated by the modulation of the
cell cycle in SMCs, DNA contents were analyzed by flow
cytometry. As shown in Figure 3, when SMCs were trea-
ted with the compound 3a for 48 h, the DNA contents
were accumulated in the S phase of the cell cycle com-
pared to vehicle-treated control group.
2 and 3 showed, in
general, more potent activity than 2-pyridyl-1H-benz-
imidazole-4,7-dione series 4. The 2-phenyl-substituted
compounds 2 and 3 exhibited the greatest activity, indi-
cating a correlation that may offer insight into the mode
of action of these compounds. The 2-pyridyl-moiety of
compounds 4 did not appeared to contribute partially
toward biological potency.
To investigate whether cell cycle arrest mediated by test
compounds was related to the expression of regulatory
proteins, Western blot analysis was performed. As
shown in Figure 4, when SMCs were treated with
In addition, the quinone moiety in 1H-benzo[d]imidaz-
ole-4,7-diones 2–4 might be essential for the antiprolifer-
ative activity. For example, non-quinonoid compounds
6 lost the antiproliferative activity. The results of their
QSAR study would imply that alteration of R, R1,
and R2 on 1H-benzo[d]imidazole-4,7-diones 2–4 did
Figure 4. Effect of compound 3a on protein expression in cultured
SMCs. (A) Total cell lysates from SMCs treated with the compound 3a
(1 lg/mL) for 48 h were analyzed for pRb, cyclin D1, PCNA, cdk 2,
and cyclin A. (B) The compound 3a was exposed to SMCs for 1 h, and
the expression of phosphorylation of ERK and phosphorylation of
Akt was examined.
Figure 2. Morphological change in cultured SMCs treated with
compound 3a. SMCs treated with DMSO alone (control) or the
compound 3a (1 lg/mL) for 48 h were observed under the phase-
contrast microscope and photographed.
Figure 3. Effect of compound 3a on cell cycle progression in cultured SMCs. Cells were treated with the compound 3a (1 lg/mL) for 48 h and then
the cell cycle was analyzed by flow cytometry analysis.

C.-K. Ryu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2948–2951
2951
1 lg/mL of the compound 3a for 48 h, the level of cdk 2
was markedly reduced. In addition, the down-regulation
of cyclin A, which binds to cdk 2 and promotes progres-
sion through the S phase of cell cycle, was observed in
compound 3a-treated cells. However, the expression lev-
els of pRb and cyclin D1, which promotes progression
through the G1 into S phase of cell cycle, was not ob-
served in compound 3a-treated cells. The cell-prolifera-
tion biomarker PCNA was down-regulated which is
well correlated with the antiproliferative effect of the
compound 3a. The expression of protein was not af-
fected by the compound 3a. These results indicate that
the compound 3a might affect the exit of S phase cell cy-
cle and thus accumulate the DNA contents of S phase in
the cells.
part to suppression of ERK signaling activation. Fur-
ther pharmacological investigations of these compounds
and the structural optimization are in progress.
Acknowledgments
This study was supported by the Korea Science and
Engineering Foundation (KOSEF) grant funded by
the Korea government (MOST: R01-2006-000-10020-0).
References and notes
1. Ross, R. Nature 1993, 362, 801.
2. Indolfi, C.; Mongiardo, A.; Curcio, A.; Torella, D. Trends
Cardiovas. Med. 2003, 13, 142.
3. Newby, A. C.; George, S. J. Cardiovasc. Res. 1993, 27,
1173.
4. Middleton, R. W.; Parrick, J.. In The Chemistry of the
Quinonoid Compounds; Patai, S., Rappoport, Z., Eds.;
John Wiley & Sons: London, 1988; Part 2, pp 1019–1066.
5. Ryu, C.-K.; Song, E. H.; Shim, J.-Y.; You, H.-J.; Choi, K.
U.; Choi, I. H.; Lee, E. Y.; Chae, M. J. Bioorg. Med.
Chem. Lett. 2003, 13, 17.
6. Bornfeldt, K. E.; Campbell, J. S.; Koyama, H.; Argast, G.
M.; Leslie, C. C.; Raines, E. W.; Krebs, E. G.; Ross, R.
J. Clin. Invest. 1997, 100, 875.
7. Petrache, I.; Choi, M. E.; Otterbein, L. E.; Chin, B. Y.;
Mantell, L. L.; Horowitz, S.; Choi, A. M. Am. J. Physiol.
1999, 277, 589.
8. Roulston, A.; Reinhard, C.; Amiri, P.; Williams, L. T.
J. Biol. Chem. 1998, 273, 10232.
9. Mayr, M.; Xu, Q. Exp. Gerontol. 2001, 36, 969.
10. Weinberger, L.; Day, A. R. J. Org. Chem. 1959, 24, 1451.
11. 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.
To better understand the molecular mechanisms in-
volved in the compound 3a on the proliferation of
SMCs, we investigated the possible involvement of
ERK and Akt cell signaling pathways. As shown in Fig-
ure 4, a remarkable decrease of ERK phosphorylation,
but not Akt, was detected with the treatment of the com-
pound 3a (1 lg/mL) for 1 h (Fig. 4B), indicating that the
ERK signaling pathway might be involved in the inhibi-
tion of SMC proliferation.
The ERK and Akt are major signal transduction mole-
cules regulating cell proliferation, differentiation, and
apoptosis. In particular, ERK pathway has been known
to play pivotal roles in controlling SMC proliferation.
Several studies have suggested that the inhibition of
SMC proliferation is ERK-dependent. In the present
study, the regulation of ERK by test compound 3a was
manifested, but not much related to the regulation of Akt.
In conclusion, the antiproliferative effect of the com-
pound 3a in SMCs is associated with its blockade of cell
cycle progression which appears to be attributable in
12. Mohacsi, P. J.; Tuller, D.; Hulliger, B.; Wijngaard, P. L.
¨
J. Heart Lung Transplant. 1997, 16, 484.