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Table 2
Cytotoxic activities of compounds 6a, 6f, 6o and 6q against a panel of cancer cells
a
Compounds
IC50 (lM)
K562
A431
HepG2
Hela
MDA-MB-435S
6a
6f
6o
6q
Taxol
Colchicine
0.012 0.006
0.045 0.036
0.006 0.001
0.021 0.001
0.004 0.003
0.001 0.000
0.036 0.012
0.075 0.014
0.026 0.003
0.020 0.008
0.007 0.002
0.008 0.006
2.336 1.160
6.691 1.283
1.774 0.374
8.309 2.915
0.990 0.178
1.710 0.294
1.121 0.1624
0.187 0.052
0.452 0.129
1.613 0.356
0.410 0.106
0.704 0.125
0.055 0.036
0.087 0.058
0.052 0.025
0.052 0.024
0.009 0.001
0.007 0.004
a
The data are means of three independent experiments performed in duplicate.
We further investigated the effects of R2 substitution on the
anti-proliferative activities by fixing R1 as an aminomethyl group.
It was found that R2 also showed great impact on the cytotoxic
activities of the resulting compounds. When R2-phenyl group in
6a was replaced with a cyclohexyl (6b) or 1-naphthyl (6c), or
benzyl (6d) moiety, the potencies were decreased about 4–75
folds. Further investigation suggested that the meta-position of
the phenyl group in 6a could be an optimal position to introduce
different substituent to maintain or improve its antiproliferative
activity. However, a substituted group at the ortho- or para-posi-
tion is detrimental to the potency. For instance, the 3-methoxyl
compound 6i displayed 3–6 times greater potency than that of
the 2-methoxyl (6h) and 4-methoxyl (6j) analogues. The study also
revealed that the 3,5-dimethoxyl compound 6l is significantly
more potent than the 3,4-dimethoxyl (6k) and 3,4,5-trimethoxyl
(6m) compounds.
Arg-2
Asp-251
Glu-71
Other meta-substituted derivatives were also designed and
synthesized, and almost all of them exhibited promising anti-
proliferative activities against NCI-H460 and Colo205 cancer cells
Figure 4. The predicted binding mode of 6o with
a and b tubulin.
with IC50 values ranged from 0.04 to 1.72 lM. Encouragingly, the
3-nitrile (6o), 3-trifluoromethyl (6p), 3-isppropyl (6q) and 3-ethyl
(6u) compounds displayed comparable potencies to that of well
known anticancer agents taxol and colchicines. It was noteworthy
that compounds 6o and 6p contained strong electron withdrawing
groups (i.e., nitrile, 3-trifluoromethyl) and might display greater
metabolic stability than compound 6a.
Further investigation also revealed that these compounds
indeed displayed strong inhibition against the growth of a panel
of various organs sourced human cancer cells (Table 2). For
example, compound 6o potently inhibited the growth of K562,
A431, Hela and MDA-MB-435S cancer cells with IC50 values of
These results support the potential inhibitory function of 6o
against tubulin. A further investigation demonstrated that the
compound dose-dependently induced G2/M phase arrest in NCI-
H460 non-small cell lung cancer cells (Supporting information).
A computational study was also performed to investigate the
potential binding mode of compound 6o with b tubulin. Based on
the previous study by Elias Georges et al.,27 we first fixed the
Ala63-Lys103 in the N-terminal of b tubulin as a centroid of the grid
and used SiteMap to identify the potential binding site of compound
6o. The possible binding poses of the compounds were then gener-
ated by Glide XP (the crystal structure of b tubulin was taken from
PDB (ID: 1SA1)). It was predicted that 6o locates in the interface
0.006, 0.026, 0.452 and 0.052 lM, respectively. However, it was
interestingly found that almost all of the compounds displayed
relatively lower potencies to the HepG2 cancer cells.
of
a
and b tubulin (Fig. 4). The benzimidazole and urea groups in
tubulin)
6o form three critical hydrogen bonds with Arg2, Asp251 (
a
In order to validate the tubulin inhibition of the new benzimid-
azole compounds, we further investigated the 6o’s effect on micro-
tubule skeleton by utilizing an immunofluorescence technique, in
which DMSO was used as blank control. As shown in Figure 3,
exposuring of NCI-H460 cells to different concentrations (25 nM,
50 nM, 100 nM) of 6o for 8 h induced a dose-dependent depoly-
merization of the microtubule cytoskeleton and 6o profoundly
inhibited the spindles formation at the concentration of 100 nM.
and Glu71 (b tubulin) residues, while the phenyl group protrude
toward the solvent-exposed section of the protein and the cyano-
group form another hydrogen bond with Arg2 (a tubulin).
In summary, a series of benzimidazole-2-urea derivatives were
identified as new tubulin inhibitors to potently suppress the prolif-
eration of a panel of human cancer cells with low nM IC50 values.
One of the most promising compounds 6o obviously inhibited
NCI-H460 cancer cells spindles formation and dose-dependent
induced cell cycle arrest at G2/M phase. A computational study
suggested the compound might bind with b tubulin in a new
binding site different from the three well-known ones. The strong
cell growth inhibitory effects support the compounds as new
promising leads for further investigation.
Acknowledgments
We thank the National Natural Science Foundation
(#21302186), and the Key Project on Innovative Drug of Guangdong
Province (#2012A080201014) for their financial support.
Figure 3. Compound 6o disturbs the microtubule skeleton in NCI-H460 cells. Cells
were exposed to various concentrations of 6o for 8 h and stained with anti-
tubulin antibody (green) and DAPI (blue) (scale bar = 20 ms).
a-
l