Design of novel bis-benzimidazole derivatives as DNA minor groove binding agents

Article abstract of DOI:10.1016/j.cclet.2014.01.019

A new series of bis-benzimidazole derivatives were designed and synthesized. In vitro cytotoxicity evaluation showed that these compounds exhibited high activity against the selected tumor cells. Among them, compound 9 owned the best potential, its IC₅₀ values being 5.95 μmol/L (mononuclear tumor cell line (U937)) and 5.58 μmol/L (cervical cancer cell (HeLa)). Fluorescence and UV-vis studies showed that compound 9 could bind into the minor groove of DNA.

Full text of DOI:10.1016/j.cclet.2014.01.019

Chinese Chemical Letters 25 (2014) 589–592
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design of novel bis-benzimidazole derivatives as DNA minor groove
binding agents
b
Xiu-Jun Wang a,b, Ming-Li Yang b, , Lan-Ping Zhang , Tong Yao a,b, Cheng Chen b,
*
Lian-Gang Mao a,b, Yin Wang b, Jie Wu c,
*
a The 716th Institute of CSIC, Lianyungang 222006, China
b JARI Pharmaceutical Co., Ltd, Lianyungang 222006, China
c Department of Pharmaceutical Engineering, Wuhan Bioengineering Institute, Wuhan 430415, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new series of bis-benzimidazole derivatives were designed and synthesized. In vitro cytotoxicity
Received 5 October 2013
evaluation showed that these compounds exhibited high activity against the selected tumor cells.
Received in revised form 18 December 2013
Accepted 19 December 2013
Available online 14 January 2014
Among them, compound 9 owned the best potential, its IC50 values being 5.95
mmol/L (mononuclear
tumor cell line (U937)) and 5.58 mol/L (cervical cancer cell (HeLa)). Fluorescence and UV–vis studies
m
showed that compound 9 could bind into the minor groove of DNA.
ß 2014 Ming-Li Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
Keywords:
Bis-benzimidazole
Anti-tumor
DNA minor groove
1. Introduction
of compound 9 with CT (calf thymus)-DNA has been investigated
using UV–vis, fluorescence spectroscopy. These results showed that
Benzimidazole derivatives are one of the most extensively
studied classes of heterocyclic compounds. These compounds have
many activities, such as anti-histamine [1], anti-microbial [2], and
anti-tumor [3] properties.
compound 9 interacted with the DNA by binding into the minor
groove. All of these compounds were screened for anti-tumor
activity in vitro. Among them, compound 9 had good activity against
three tumor cell lines (HeLa, HL60 and U937).
Bis-benzimidazole derivatives have been proven to be potent
antitumor agents via DNA minor groove binding, such as Hoechst
33258 (Scheme 1). Hoechst 33258 [3], a fluorescent compound
with a head-to-tail bis-benzimidazole structure, was initially
found to be active against L1210 murine leukemia. During phase I
clinical trials in humans, positive responses were observed in
pancreatic cancer. However, a subsequent phase II clinical trial
failed to show any objective responses [4].
Due to the high DNA minor groove binding ability of Hoechst
33258, several groups have synthesized bis-benzimidazole deriva-
tives derived from Hoechst 33258 [5–8]. To the best of our
knowledge, mostderivativesofHoechst33258areplanarmolecules,
and there is little literature related to adding a linker between two
benzimidazoles. Molecular modeling studies (shown in Fig. 1)
suggest that the novel benzimidazole (9) could effectively bind into
the DNA minor groove. Therefore we combined two benzimidazole
derivatives into one molecule with an oxygen atom. The interaction
2. Experimental
2.1. Molecular modeling
An AutoDock 3.05 was used to perform theoretical docking
calculations [9]. The molecular structure of the A-tract DNA
dodecamer d(CGCAAATTTGCG) (PDB code: 2DND) was as receptor.
The graphical front-end, AutoDock Tools, was used to add polar
hydrogens and partial charges for proteins using the Kollman
United Atom charges. Atomic solvation parameters were assigned
using the add solubility function in the AutoDock package. Affinity
grid fields were generated using the auxiliary program Auto-
Grid3.0. The Lamarckian genetic algorithm (LGA) was used to find
the appropriate binding positions, orientations, and conformations
of the ligands. The optimized AutoDocking parameters are as
follows: The maximum number of energy evaluations was
increased to 25,000,000 per run; the iterations of Solis & Wets
local search was 3000; the number of individuals in the population
was 300. All other parameters were maintained as default. Cluster
analysis with AutoDock results was performed to determine if
different binding sites have been produced from multiple runs.
*
Corresponding authors.
E-mail addresses: Ymlwxj@126.com (M.-L. Yang), tomson-wu@foxmail.com
(J. Wu).
1001-8417/$ – see front matter ß 2014 Ming-Li Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2014.01.019

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X.-J. Wang et al. / Chinese Chemical Letters 25 (2014) 589–592
OH
for 3 h. The reaction was monitored by TLC. To stop the reaction,
triethylamine (25 mL, 179.4 mmol) was added dropwise, neutraliz-
ing the solution and producing a white solid, which was filtrated,
washed with acetone, and dried to obtain the compound 3. Then,
obtained compound 3 (6.2 g, 16.6 mmol) was dissolved with acetic
acid (50 mL) and stirred at 0 8C for 10 min. Fuming nitric acid (8 mL,
171.7 mmol) was added dropwise to the above solution over 2 h.
After that, the ice-bath was removed and the reaction mixture was
further stirred at room temperature for 2 h. The mixture was poured
into ice (100 mL) and the resulting yellow solid (4) was filtered,
washed with water, dried in vacuum, dissolved in a mixed water
(10 mL) and ethanol (30 mL) solution of potassium hydroxide (7.3 g,
106.7 mmol), and then refluxed for 4 h. After stopped the reaction,
the above solution was poured into ice (100 mL). The resulting red
solid was filtrated, washed with water, and dried to give 5.2 g
(17.9 mmol) of compound 5. Then, it was dissolved in methanol
(50 mL) with Pd-C (0.3 g, 10%) in it, enabling hydrogen gas passing
through continuously at a flow rate of 10 mL/min for 3 h. After that, it
was filtrated to collect the filtrate and evaporated under vacuum to
give 3.7 g of compound 6. Then, compound 6 with potassium
ethylxanthate gave 2,20-dithiol-5,50-bis-1H,10H-benzimidazole ether
(7), which reacted with various substituted chloromethylpyridine
analogs in ethanol at reflux to synthesize the desired compounds 8–
15 [11].
H
N
H
N
N
N
N
N
H C
3
1 (Hoechst 33258)
Scheme 1. The structure of Hoechst 33258.
2.2. Chemical and instruments
All commercially available reagents and solvents were used
without further purification unless otherwise specified. Solvents
were dried and re-distilled prior to use according to standard
methods. Melting points were determined on a Bu¨chi Melting Point
B-540 apparatus (Bu¨chi Labortechnik, Flawil, Switzerland) and are
uncorrected. 1H NMR spectra were measured in DMSO-d6 on a
Bruker ARX 300 spectrometer (Bruker, Rheinstetten, Germany).
Chemical shifts are reported in parts per million (ppm) using
tetramethylsilane (TMS) as the internal standard if not specifically
mentioned (J in Hz). Mass spectra were measured on a Waters
Micromass Quattro Micro API mass spectrometer (Waters Corpo-
ration, Milford, United States). The UV–vis spectral measurements
were recorded on a Cary Varian double beam spectrophotometer
(Cary BIO 100, Australia). The sample cuvette used was a pair
quartz cells of 1.00 cm path length. All scanning parameters were
optimized to obtain the best spectra and in general the parameters
were scanned in a wavelength range of 230–300 nm with a step of
0.5 and all measurements were carried out at room temperature.
Fluorescence measurements were performed using a Spectrofluo-
rimeter model FS920 of Edinburgh Instruments, U.K. equipped
with a xenon arc lamp. The temperature of the sample holder was
regulated with a peltier cooled thermostat. Quartz cuvettes of 3 mL
capacity and path length 1 cm were used for all measurements.
2.4. Biological assays
The anti-proliferational effects of the compounds on tumor cells
were tested by the same methods. Tumor cells in RPMI1640
medium with 10% fetal bovine serum were plated in 96-well
microtiter plates (4.0 ꢀ 104 cells per well) and allowed to adhere at
37 8C with 5% CO2 for 4 h. The test compound was then added, and
the cells were incubated at 37 8C with 5% CO2 for 72 h. The cell
viability was assessed using a standard MTT assay [12].
3. Results and discussion
Molecular modeling studies (shown in Fig. 1) suggest that the
novel symmetric head-to-head benzimidazole (9) could effectively
bind into the DNA minor groove. The predicted binding free energy
was ꢁ14.35 kcal/mol. From the modeling, it was shown that the
compound should adopt a concave shape, exactly fitting the
convex minor groove, allowing for deeper penetration into the
DNA minor groove.
The designed compounds were successfully synthesized as
described in Scheme 2. Protection of the amino group of
commercially available 4,40-diaminodiphenyl ether with acetic
anhydride at 0 8C 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), followed by reduction of the nitro group to afford 3,30,4,40-
tetraaminodiphenyl ether (6). Treatment of compound 6 with
Potassium ethylxanthate gave 2,20-dithiol-5,50-bis-1H,10H-benz-
imidazole ether (7), which reacted with sodium hydroxide and
various substituted chloromethylpyridine analogs in ethanol at
reflux to synthesize the desired compounds 8–15.
2.3. General procedure for the synthesis of substituted compounds 8–
15
The key intermediate compounds 3–6 were prepared according
to described procedures using 4,40-diaminodiphenyl ether as
a starting material [10]. To a stirred acetone (50 mL) solution of
4,40-diaminodiphenyl ether (2) (5.0 g, 19.6 mmol) at 0 8C, acetic
anhydride (10.0 mL, 105.8 mmol) was added dropwise and reacted
In vitro antitumor activities of all the above final products were
screened against three selected tumor cell lines. The biological assay
results are summarized in Table 1. It was found that all compounds
showed noticeable antitumor activities. Worthy of noting, com-
pounds8–10withoutsubstituentsonthepyridineringshowed more
potent antitumor activities than compounds 11–15, which contain
electron-withdrawing halogen substituents (Cl, F or Br) or electron-
donating substituents (hydroxyl or methoxyl) on the pyridine ring.
Compounds 8–10 showed low cytotoxicity at a concentration of
Fig. 1. Close-up view of compound 9 binding in the minor groove.
15
mmol/L, while compounds 11–15 were less potent with IC50

X.-J. Wang et al. / Chinese Chemical Letters 25 (2014) 589–592
591
O
O
O N
2
O
NO
2
a
c
b
AcHN
NHAc
H N
NH
NO
NH
AcHN
HS
NHAc
2
2
2
2
2
3
4
H
N
O N
O
2
H N
2
O
NH
2
O
N
e
d
SH
H N
N
H
N
2
H N
NH
2
2
6
5
7
H
N
O
N
f
S
S
N
H
N
R
R
8-15
Scheme 2. Reagents and conditions: (a) (CH3CO)2O/NEt3, acetone, 0 8C, 2 h, 98%; (b) conc. HNO3, acetic acid, 0–50 8C, 5 h, 98%; (c) 42% KOH aq, MeOH, reflux, 2 h, 97%; (d)
NH2NH2 H2O, MeOH, r.t., 6 h, 89%; (e) KOH, CS2, ethanol, reflux, r.t., 6 h, 82%; (f) various hy-CH2Cl (heterocyclic), NaOH, MeOH, reflux, 3 h, 54–93%.
Table 1
Structure and anti-tumor activity of bis-benzimidazoles.a
Compound
Substitute
R
IC50
(mmol/L)
HL60
HeLa
13.83
U937
9.55
8
Pyrid-2-yl
12.17
9.41
9
Pyrid-4-yl
5.95
14.75
30.56
33.64
5.58
10
Pyrid-3-yl
10.36
14.13
11
3,5-Dimethyl-4-methoxypyrid-2-yl
3,4-Methoxypyrid-2-yl
3-Methyl, 4-Trifluoroethoxypyrid-2-yl
3-Methoxyl, 4-chloropyrid-2-yl
6-Chloropyrid-2-yl
>50
>50
41.23
29.58
>50
>50
29.58
>50
>50
22.19
12
13
>50
>50
18.77
14
15
H. 33258
5-FU
Paclitaxel
15.62
18.23
24.56
16.59
0.007
31.36
13.80
0.005
0.004
a
The experiments were performed twice and the average values were obtained from two independent experiments.
values more than 20
potent with IC50 values of 5.95
and 5.58 mol/L for the HeLa tumor cell line.
UV–vis spectroscopy on CT-DNA in the presence of the small
molecules provided useful information related to the nature
of the interaction between the two. Fig. 2a shows the absorbance
of CT-DNA at 257 nm progressively increasing when the
m
mol/L. Among them, compound 9 was most
concentration of compound 9 solution was increased from 0 to
5 mmol/L. Not only was there an increase in the absorbance of
CT-DNA upon the addition of compound 9, the appearance of a
distinct blue shift upon the formation of the DNA-compound 9
complex in the 257 nm region was also observed. These results
indeed indicate that compound 9 may bind in the minor groove
of CT-DNA.
mmol/L for the U937 tumor cell line
m
Fig. 2. (a) The absorption spectra of calf thymus DNA (1, 7.8 ꢀ 10ꢁ5 mol/L) in Tris–HCl buffer upon addition of compound 9 (2–5, 1–4 ꢀ 10ꢁ6 mol/L, respectively); (b)
fluorescence emission spectra (excited at 520 nm) of EB, EB–DNA complexes in the absence (1) and presence (2–5) of increasing concentrations of the compound 9 (2 mmol/L,
1 L per scan).

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X.-J. Wang et al. / Chinese Chemical Letters 25 (2014) 589–592
Table 2
DNA-binding abilities of compounds 9 and Hoechst 33258 determined by ultrafiltration assay using calf thymus DNA.
Compound
Abs. DNA (ꢁ)a
Abs. DNA (+)a
DNA-binding abilityb
Scatchard analyses
Binding constant
(Ka) (ꢀ103 L/mol)
Binding sites
Correlation
(n) (per bp)
coefficient (R)
Hoechst 33258
0.468
0.503
0.141
0.172
69.9
65.8
7.3
6.5
0.4
0.4
0.78
0.79
9
a
The UV-absorption was measured at lmax 341 nm after 5 dilution with 1.0 buffer.
DNA binding ability (%) =(1 ꢁ AbsDNA+/AbsDNAꢁ) ꢀ 100.
b
[9] A.J. Olson, D.S. Goodsell, Automated docking and the search for HIV protease
inhibitors, SAR QSAR Environ. Res. 8 (1998) 273–285.
[10] F. Dawans, C.S. Marvel, Polymers from ortho aromatic tetraamines and aromatic
dianhydrides, J. Polym. Sci. A: Gen. Pap. 3 (1965) 3549–3571.
Fluorescence quenching measurements are effective to monitor
the binding nature of the small molecules to DNA. The molecular
fluorophore EB (ethidium bromide) has conjugate planar
a
[11] Analytical data for compounds 7–15:;
structure, and its fluorescence intensity is very weak, but it emits
intense fluorescence at about 600 nm in the presence of DNA due
to its strong intercalation between the adjacent DNA base pairs.
Many DNA minor groove agents could quench the intense
fluorescence [13]. Similar quenching was observed in compound
9 (Fig. 2b). Therefore, it is concluded that compound 9 could bind
into the minor groove of DNA.
The DNA-binding ability of compound 9 was also evaluated by
the ultrafiltration method (Table 2) [14,15]. It can be seen that
the DNA-binding ability for compound 9 was 65.8 and 69.9 for
Hoechst 33258. The binding constant (Ka) for compound 9 was
6.5 ꢀ 103 L/mol for Hoechst 33258. The similar DNA-binding
ability and similar binding constant (Ka) indicate that compound
9 may bind in the minor groove of CT-DNA.
Compound 7: yield: 54.8%, mp 211.6ꢁ213.8 8C; 1H NMR (300 MHz, DMSO-d6): d
6.84 (d, 1H, J = 8.4, Bz-7-H), 7.30 (d, 1H, J = 1.8), 7.52 (dd, 1H, J = 8.4, 1.8, Bz-6-H),
12.61 (br, s, 2H), 13.56 (br, s, 2H); 13C NMR (100 MHz, DMSO-d6): d 105.6, 112.6,
115.2, 133.8, 138.1, 151.7, 168.0; ESI-HRMS: m/z 314.0288. (Calcd. for
C
26H20N6OS2: 314.0296). Compound 8: yield (last step): 92.6%, mp
120.3ꢁ122.2 8C; 1H NMR (300 MHz, DMSO-d6): d 4.65 (s, 4H), 6.80–7.20 (m,
4H), 7.27 (m, 2H), 7.36 (d, 2H, J = 7.8 Hz), 7.51 (m, 2H), 7.73 (d, 2H, J = 7.8), 8.51
(m, 2H), 12.65 (br s, 2H); 13C NMR (100 MHz, DMSO-d6): d 41.2, 106.7, 114.2,
115.1, 124.5, 125.3, 134.2, 136.7, 139.4, 148.6, 149.3, 151.9, 159.6; ESI-HRMS:
m/z 496.1152. (Calcd. for C26H20N6OS2: 496.1140). Compound 9: yield (last
step): 88.7%, mp 118.9ꢁ122.2 8C; 1H NMR (300 MHz, DMSO-d6): d 4.55(s, 4H),
6.85 (d, 2H, J = 8.6 Hz), 7.00 (s, 2H), 7.33 (s, 2H), 7.44 (d, 2H, J = 8.6 Hz), 7.86 (d,
2H, J = 7.7 Hz), 8.47 (d, 2H, J = 7.7 Hz), 8.54 (s, 2H), 12.59 (br s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 39.6, 106.7, 114.2, 115.1, 125.3, 134.2, 139.4, 146.7,
148.4, 148.6, 151.9; ESI-HRMS: m/z 496.1146. (Calcd. for C26H20N6OS2:
496.1140). Compound 10: yield (last step): 89.3%, mp 134.1ꢁ134.9 8C; 1H
NMR (300 MHz, DMSO-d6): d 4.53 (s, 4H), 6.83 (dd, 2H, J = 8.7, 2.1 Hz), 6.98
(s, 2H), 7.43 (d, 4H, J = 8.7 Hz), 7.44 (s, 2H), 8.48 (d, 4H, J = 2.1 Hz), 12.60 (br s,
2H); 13C NMR (100 MHz, DMSO-d6): d 38.3, 106.7, 114.2, 115.1, 125.9, 133.4,
134.2, 134.8, 139.4, 148.6, 151.9, 152.6; ESI-HRMS: m/z 496.1149. (Calcd. for
4. Conclusion
C
26H20N6OS2: 496.1140). Compound 11: yield (last step): 90.7%, mp
94.9ꢁ96.1 8C; 1H NMR (300 MHz, DMSO-d6): d 3.79 (s, 12H), 3.88 (s, 6H),
4.64 (s, 4H), 6.80–7.20 (m, 4H), 7.07 (d, 2H, J = 5.7 Hz), 8.15 (d, 2H,
J = 5.7 Hz), 12.60 (br s, 2H); 13C NMR (100 MHz, DMSO-d6): d 19.5, 20.6, 42.5,
57.3, 106.7, 114.2, 115.1, 118.4, 132.0, 139.4, 134.2, 143.1, 148.2, 148.6, 150.8,
151.9; ESI-HRMS: m/z 612.1981. (Calcd. for C32H32N6O3S2: 612.1977). Com-
pound 12: yield (last step): 90.7%, mp 94.9ꢁ96.1 8C; 1H NMR (300 MHz, DMSO-
d6): d 3.79 (s, 6H), 3.88 (s, 6H), 4.64 (s, 4H), 6.80–7.20 (m, 4H), 7.07 (d, 2H,
J = 5.7 Hz), 7.42 (m, 2H), 8.15 (d, 2H, J = 5.7 Hz), 12.60 (br s, 2H); 13C NMR
(100 MHz, DMSO-d6): d 35.1, 56.2, 56.9, 106.2, 106.8, 114.2, 115.1, 134.2, 142.7,
139.4, 142.6, 148.2, 148.6, 151.9, 157.9; ESI-HRMS: m/z 616.1567. (Calcd. for
C30H28N6O5S2: 616.1563). Compound 13: yield (last step): 87.2%, mp
123.7ꢁ126.5 8C; 1H NMR (300 MHz, DMSO-d6): d 2.25 (s, 6H), 4.71 (s, 4H),
4.91 (dd, 4H, J = 8.7, 5.4 Hz), 6.80–7.00 (m, 4H), 7.09 (d, 2H, J = 5.4 Hz), 7.45 (s,
2H), 8.31 (d, 2H, J = 8.7 Hz), 12.60 (br s, 2H); 13C NMR (100 MHz, DMSO-d6): d
16.7, 39.0, 83.9, 105.8, 106.7, 112.3, 114.2, 115.1, 124.6, 134.2, 139.4, 148.9,
148.6, 151.9, 161.2, 168.7; ESI-HRMS: m/z 720.1428. (Calcd. for
In summary, we have successfully designed and synthesized a
new series of bis-benzimidazole derivatives based upon molecular
modeling experiments of docking within the minor grooves of
DNA. These compounds exhibited desirable anti-tumor activity
and have better DNA minor groove binding ability. Such
compounds are of interest in the context of the future development
of novel anti-tumor agents.
Acknowledgment
This study was supported by Wuhan City Department of
Education (No. 2009K106).
C
32H26F6N6O3S2: 720.1412). Compound 14: yield (last step): 76.2%, mp
121.8ꢁ124.1 8C; 1H NMR (300 MHz, DMSO-d6): d 3.96 (6H, s), 4.79 (s, 4H),
6.88 (dd, 2H, J = 8.6, 2.1 Hz), 7.04 (s, 2H), 7.47 (d, 2H, J = 8.6 Hz), 7.57 (d, 2H,
J = 2.1 Hz), 8.27 (m, 2H), 12.61 (br s, 2H); 13C NMR (100 MHz, DMSO-d6): d 35.6,
56.3, 106.7, 114.2, 115.1, 122.8, 124.0, 134.2, 139.4, 143.9, 148.6, 149.2, 151.9,
156.2; ESI-HRMS: m/z 624.0560. (Calcd. for C28H22Cl2N6O3S2: 624.0572). Com-
pound 15: yield (last step): 53.7%, mp 111.2ꢁ113.1 8C; 1H NMR (300 MHz,
DMSO-d6): d 4.54 (s, 2H), 6.80–7.30 (m, 2H), 7.43 (m, 1H), 7.46 (d, 1H,
J = 8.1 Hz), 7.93 (dd, 1H, J = 8.1, 2.4 Hz), 8.47 (d, 1H, J = 2.4 Hz), 12.61 (br s,
1H); 13C NMR (100 MHz, DMSO-d6): d 39.0, 106.7, 114.2, 115.1, 122.4, 124.1,
134.2, 139.4, 140.6, 148.2, 148.6, 151.9, 160.8; ESI-HRMS: m/z 496.1152. (Calcd.
for C26H18Cl2N6OS2: 496.1140).
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