Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through induction of apoptosis and autophagy

Article abstract of DOI:10.1016/j.bmcl.2012.06.102

In this study, a new series of bis-benzimidazole derivatives were designed and synthesized. Most of these new compounds showed significant anti-tumor activity in vitro compared to Hoechst 33258. Among them, the most potent compound 8 had the IC₅₀

Full text of DOI:10.1016/j.bmcl.2012.06.102

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6297–6300
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Newly synthesized bis-benzimidazole derivatives exerting anti-tumor
activity through induction of apoptosis and autophagy
Xiu-Jun Wang a, Na-Ying Chu b, Qin-He Wang a, Chao Liu a, Chun-guo Jiang a, Xiao-Yu Wang a,
a,
Takashi Ikejima b, , Mao-Sheng Cheng
⇑
⇑
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
PR China
b China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a new series of bis-benzimidazole derivatives were designed and synthesized. Most of these
new compounds showed significant anti-tumor activity in vitro compared to Hoechst 33258. Among
Received 30 April 2012
Revised 21 June 2012
Accepted 29 June 2012
Available online 7 July 2012
them, the most potent compound 8 had the IC50 values of 0.56
lM for HL60 (Human promyelocytic leu-
kemia cells) tumor cell line and 0.58 M for U937 (Human leukemic monocyte lymphoma cells) tumor
l
cell line. Subsequent toxicity study on human peripheral blood mononuclear cells (PBMC) showed that
compound 8 exhibited less toxicity than 5-FU. We also found that apoptosis and autophagy were simul-
taneously induced by compound 8 in HL60 cells, and inhibition of autophagy by 3-MA decreased com-
pound 8-induced apoptosis, indicating that they acted in synergy to exert tumor cell death.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Bis-benzimidazole
Anti-tumor
Apoptosis
Autophagy
PBMC
Apoptosis (type I cell death) and autophagy (type II cell death)
are maior types of programmed cell death.1 Apoptosis (type I cell
death) and autophagy (type II cell death) are maior types of pro-
grammed cell death. Apoptosis is induced either through mito-
chondrial (intrinsic) pathway or death receptor (extrinsic)
pathway in multicellular organisms.2 It is characterized by a series
of morphological changes including membrane blebbing, loss of
cell membrane asymmetry and attachment, cell shrinkage, chro-
matin condensation and chromosomal DNA fragmentation and
others.3 Deregulation of apoptosis is a hallmark of all cancer cells,
and the agents that activate apoptosis in cancer cells could be used
as valuable anti-cancer therapeutics.4 As type II programmed cell
death, autophagy is an intracellular degradation system that deliv-
ers cytoplasmic constituents to the lysosome. It is involved in cell
growth, development and homeostasis, helping to maintain a bal-
ance between synthesis and degradation. Autophagy is not only a
survival response to either growth factor or nutrient deprivation
but also a mechanism for tumor cell suicide induced by chemo-
therapy or radiation.5 Recent studies have demonstrated that the
co-regulation of both apoptosis and autophagy may participate in
mammalian cell death.6
Bis-benzimidazole derivatives are one of the most extensively
studied classes of heterocyclic compounds. They have been proven
to be potent anti-tumor agents via DNA minor groove binding. A
representative compounds, Hoechst 33258, is listed in Scheme 1
Hoechst 332587, a fluorescent compound with a head-to-tail bis-
benzimidazole structure, was initially found to be cytotoxic against
L1210 murine leukemia. Phase I clinical trials of this compound
showed positive pancreatic cancer activity. However, a subsequent
phase II clinical trial failed to yield any objective results.8 Due to
the synthetic accessibility and high binding affinity of Hoechst
33258, several groups have focused on a strategy to utilize the
pharmacophore-like benzimidazole motif derived from Hoechst
33258.9–12 In 2011, Singh et al. synthesized several 2-aryl-5-
substituted-2,5-bisbenzimidazole derivatives and evaluated their
antitumor activity in vitro.13
OH
H
N
N
N
N
N
H
N
H3C
⇑
Corresponding authors. Fax: +86 24 23844463 (T.I.); fax: +86 24 23995043
(M.-S.C.).
1 (Hoechst 33258)
E-mail addresses: ikejimat@vip.sina.com (T. Ikejima), mscheng@syphu.edu.cn
(M.-S. Cheng).
Scheme 1. Chemical structures of Hoechst 33258.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.102

6298
X.-J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6297–6300
O
O
O2N
O
NO2
b
a
c
AcHN
NHAc
H2N
NH2
AcHN
NHAc
3
2
4
H
N
O2N
H2N
O
NO2
NH2
O
H2N
H2N
O
NH2
NH2
N
e
d
RCHO
R
R
N
H
N
7
6
5
8 15
12 R = phenyl
8
9
R = 4-OCH3, phenyl
13 R = 4-Br, phenyl
14 R = 2, 4-Cl, phenyl
15 R = 4-F, phenyl
R = 3, 5-OCH3, phenyl
10 R = 3, 4, 5-OCH3, phenyl
11 R = 3-OCH3, 4-OH, phenyl
Scheme 2. Reagents and conditions: (a) (CH3CO)2O/NEt3, acetone, 0 °C, 2 h, 98%; (b) Conc. HNO3, acetic acid, 0–50 °C, 5 h, 98%; (c) 42% KOH aq, MeOH, reflux, 2 h, 97%; (d) Pd-
C, H2, MeOH, rt, 6 h, 89%; (e) NaHSO3, methanol, rt, 8 h, 54–93%.
against MCF7 and A431 cells.15 No literature related adding an oxy-
Table 1
The anti-tumor activities of the compounds (8–15)
gen atom between two benzimidazoles has been reported so far. In
this study, we combined two benzimidazole derivatives into one
molecule with an oxygen atom. All of these compounds were
screened for anti-tumor activity in vitro. Among them, the most
compound 8 had the good activity for three tumor cell lines (HeLa,
HL60 and U937). We also compared the toxic effects of compound
8 and 5-FU on human PBMC. The result indicated that compound 8
had lower toxicity than 5-FU.
In the subsequent study, inhibition of autophagy by 3-MA pro-
moted compound 8-induced apoptosis, indicating that compound
8 could induce autophagy and apoptosis simultaneously and these
PCD both exerted HL60 cells death.
Compound 8–15 were achieved with an efficient synthetic
route (Scheme 2). Protection of the amino group of commercially
available 4,40-diaminodiphenyl ether with acetic anhydride at
0 °C gave 4,40-diacetamido-diphenyl (3). Nitration of compound 3
with nitric acid in acetic acid formed compound 4. Treatment of
compound 4 with a solution of potassium hydroxide in water
and ethanol gave 3,30-dinitro-4,40-diaminodiphenyl ether (5), fol-
lowed by reduction of the nitro group to afford 3,30, 4,40-tetraam-
inodiphenyl ether (6). Treatment of compound 6 with various
substituted benzaldehyde in methanol at reflux gave the desired
compounds.
Compound
IC50
HL60
(lM)
HeLa
4.96 0.53
8.59 0.88
9.37 1.67
U937
8
9
10
0.56 0.04
6.35 1.03
7.56 0.94
0.58 0.06
7.09 1.20
6.92 0.65
11
12
13
14
10.61 1.13
18.35 2.16
20.39 3.78
17.24 2.19
22.76 2.72
51.31 4.56
0.28 0.03
8.17 0.71
5.60 0.53
16.47 2.01
17.22 1.37
15.78 1.96
23.19 2.47
32.43 3.27
0.06 0.01
17.11 3.64
21.67 2.15
20.52 2.68
22.63 3.50
15.42 2.16
1.07 0.08
15
H. 33258
5-FU
Paclitaxel
2.93 0.61 (nM)
2.82 0.41 (nM)
3.37 0.89 (nM)
The data were the mean SD obtained from three independent experiments.
5-Fluorouracil and Paclitaxel were used as the positive control.
To our knowledge, most derivatives of Hoechst 33258 kept
whole molecules to planar. In 2009, Hu et al. synthesized and eval-
uated the antibacterial activities of a new series of bisbenzimidaz-
ole derivatives with various two atom or one-atom as a linker.14
Alpan et al. synthesized a new series of bis-benzimidazole deriva-
tives with alkyl chain as a linker which were found to be cytotoxic
compound 8
5-FU
12 h
compound 8
5-FU
24 h
A
B
D
concentration (μΜ)
concentration (μΜ)
compound 8 5 FU
compound 8
5 FU
72 h
48 h
C
concentration (μΜ)
concentration (μΜ)
Figure 1. Effects of compound 8 and 5-FU on human PBMC cells. The PBMC were obtained from three healthy adult volunteers and were seeded on 96-well culture plates at a
density of 5 ꢀ 105 cells/well. The cells were cultured for 12, 24, 48 and 72 h with various doses of compound 8, 5-FU. n=3, mean SD. ⁄p < 0.05, comparison with 5-FU.

X.-J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6297–6300
6299
Figure 2. (A) The morphologic changes of compound 8-treated cells were observed. (B) The apoptosis changes were observed under a fluorescence microscope by AO
staining. (C) The autophagic changes were observed under a fluorescence microscope by MDC staining. (D) Flow cytometric analysis of compound 8-induced apoptosis
(SubG1 fraction). (E) The cells were treated with 0.5 l
mol Lꢁ1 compound 8 for 0, 24, 48 or 72 h, respectively. The MDC fluorescent intensity was analyzed by flow cytometry.
(F) The cells were treated with 0.5
l
mol Lꢁ1 compound 8 for indicated time period, followed by Western blot analysis for detection of Beclin-1 and LC3 levels. b-actin was
used as an equal loading control.
All the newly synthesized compounds were investigated for
their anti-tumor activities in three cancer cell lines using MTT as-
say.16 In respect to the in vitro cytotoxic activities listed in Table 1,
almost all of the synthesized compounds exhibited high anti-tu-
mor activity when compared with known DNA minor groove agent
Hoechst 33258 in Table 1. As shown in Table 1, compounds 8–11
with electronic-donating substituents (methoxy group) on the
benzene ring showed more potent anti-tumor activities than com-
pounds 13–15, which only contains electronic-withdrawing halo-
gen substituents (Cl, F or Br) on benzene ring. Compounds 8–11
B
A
∗∗
∗∗
showed low cytotoxicity at concentration of 10
pounds 13–15 were less potent with IC50 values more than
10 M. Among them, compound 8 was most potent with IC50
values of 0.56 M for HL60 tumor cell line and 0.58 M for U937
lM while com-
Figure 3. Effects of 3-MA on compound 8-induced autophagy and apoptosis in
HL60 cells. The cells were treated with 0.5 M compound 8 for 24 h in the presence
l
l
or absence of 1 mM 3-MA, the autophagic ratio (A) and the apoptotic ratio (B) were
measured by flowcytometric analysis. n = 3, mean SD. ⁄P < 0.05, ⁄⁄P < 0.01.
l
l
tumor cell line. The standard drugs used for comparison were
5-FU and paclitaxel. In terms of U937 tumor cell line, compound
8 showed similar anticancer activity with 5-FU. On the other hand,
5-FU is widely used as a clinical anti-tumor drugs.17 Its low PBMC
toxicity was an important indicator for its use. By now, bis-benz-
imidazole compounds have no yet went to the clinicals. So we
compared compound 8’s PBMC toxicity with that of 5-FU (Fig. 1).18
When human peripheral blood mononuclear cells (PBMC) were
ities of PBMC were about 98%, 96%, 88% and 79%, respectively. In the
case of 5-FU (5 lM) were about 98%, 94%, 87% and 76%, respectively.
In other concentrations, the viabilities of compound 8 were also
higher than 5-FU. Especially, when human peripheral blood mono-
nuclear cells (PBMC) were cultured with 160 lM compound 8 at 24
and 72 h, compound 8 has lower toxicity to normal PBMC from
cultured with 5 lM compound 8 at 12, 24, 48 and 72 h, the viabil-
healthy volunteers than 5-FU with a significant level (p <0.05).

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X.-J. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6297–6300
To determine the characteristics of HL60 cell death, we exam-
ined the morphological changes and DNA fragmentation of the
compound 8 treated cells. When the cells were cultured with
derivatives as far as we know. We also found that inhibition of
autophagy by 3-MA decreased compound 8-induced apoptosis,
indicating that they act together to exert cell death.
0.5
l
M compound 8 for 24, 48 and 72 h, marked morphological
In conclusion, this is the first report focused on adding an oxy-
gen atom between two benzimidazoles as novel anti-tumor agents.
This work enriched the structural types of bis-benzimidazole. On
the other hand, compound 8 exerted a strong anti-tumor effect
and had a lower toxity on PBMC in comparison to paclitaxel and
5-FU. Thus, compound 8 can be considered to become an appropri-
ate lead compound to develop more potent agents with anti-tumor
promoting activity for clinical use.
changes were observed as compared with the untreated control.
Compound 8-treated HL60 cells underwent retraction of cellular
processes and became round in shape at 24 h. By 48 h, the majority
of HL60 cells had become round, with shrunken nuclei. Some of
these cells showed membrane blebbing and the nuclei were
fragmented into apoptotic bodies. Eventually, dead cells became
non-adherent. Untreated cells did not show these apoptotic char-
acteristics (Fig. 2A). The quantitative analysis of apoptosis by
flow-cytometry also showed that compound 8 induced a signifi-
cant time-dependent increase in the percentage of subG1 cells
(Fig. 2D). When we stained the HL60 cells with acridine orange,
the cells in control medium were stained homogeneously with
AO, whereas treatment with compound 8 resulted in marked chro-
matin condensation and nuclear fragmentation in HL60 cells, a
hallmark of apoptosis (Fig. 2B).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.102. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Autophagy has been reported to co-exist with apoptosis in
many experimental systems,19–21 therefore we tried to examine
the effect of compound 8 on autophagy induction. The formation
of autophagic vacuoles was assessed by staining cells with auto-
phagosomal fluorescent dye MDC (monodansylcadaverine). As
shown in Figure 2C, treatment with compound 8 induced marked
increase in the number of MDC-labeled fluorescent particles in
HL60 cells compared to control cells. For quantitative analysis of
autophagy, the MDC fluorescent intensity of compound 8 treated
cells for the indicated time periods was analyzed by flow-cytome-
try. As shown in Figure 2E, compound 8 induced HL60 cell autoph-
agy in a time-dependent manner. Accordingly, the expression level
of Beclin 1 and the conversion from LC3-I to LC3-II, as autophagic
markers, were increased with time after administration of com-
pound 8 (Figure 2F), suggesting that compound 8 induces autoph-
agy as well as apoptosis in HL60 cells.
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For the first time, we found that bis-benzimidazole derivatives
could induced both autophagy and apoptosis in cancer cells. Such
dual properties have never been reported for bis-benzimidazole