Design, synthesis, and anti-tumor evaluation of novel symmetrical bis-benzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2008.07.021

A novel symmetrical bis-benzimidazole was designed as DNA minor groove binder. Molecular modeling study showed that it could dock into the minor groove of DNA. Several derivatives were synthesized and confirmed by IR, MS, and ¹H NMR. All these novel compounds were screened for cytotoxic activity on SKOV-3, HeLa, and BGC-823 cell lines in vitro. Some compounds showed IC₅₀s in the single-digit micromolar range for cytotoxicity in several tumor cell lines.

Full text of DOI:10.1016/j.ejmech.2008.07.021

European Journal of Medicinal Chemistry 44 (2009) 1808–1812
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Design, synthesis, and anti-tumor evaluation of novel symmetrical
bis-benzimidazoles
,
a
a
a
a
b
Yan-Hui Yang a, Mao-Sheng Cheng a , Qing-He Wang , Han Nie , Na Liao , Jian Wang , Hong Chen
*
a Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, PR China
b Staff Room of Pharmacognosy, Medical College of Chinese People’s Armed Police Force, Tianjin 300162, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel symmetrical bis-benzimidazole was designed as DNA minor groove binder. Molecular modeling
study showed that it could dock into the minor groove of DNA. Several derivatives were synthesized and
confirmed by IR, MS, and 1H NMR. All these novel compounds were screened for cytotoxic activity on
SKOV-3, HeLa, and BGC-823 cell lines in vitro. Some compounds showed IC50s in the single-digit
micromolar range for cytotoxicity in several tumor cell lines.
Received 14 December 2007
Received in revised form 8 July 2008
Accepted 11 July 2008
Available online 24 July 2008
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Minor groove binder
Symmetrical bis-benzimidazole
Molecular modeling
Anti-tumor
1. Introduction
DNA minor groove binders (MGBs) is a novel family of anti-
tumor agents and some of them have entered clinical trials [1].
During the last decade, many synthetic minor groove binders have
been reported, including analogues and conjugates of naturally
occurring minor groove-binding agents, such as distamycin (Dst),
netropsin (Net), CC-1065, anthramycin (Atm), and Hoechst 33258
[2]. Hoechst 33258 (1), a fluorescent reagent with a head-to-tail
bis-benzimidazole structure, was initially found to be active against
L1210 murine leukemia. During phase I trials in human, some
responses were seen in pancreatic cancer. However, a subsequent
phase II trial did not show any objective responses [3]. X-ray crys-
tallographic and NMR studies on complexes of Hoechst 33258 with
AT-containing oligonucleotides have shown that this drug fits the
minor groove snugly, with the planar benzimidazole groups
oriented parallel to the direction of the groove. Each inner-facing
nitrogen atom hydrogen could bond in a bifurcated manner to
a pair of adjacent hydrogen-bond donors on the edge of the AT base
pairs [3]. 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 [4–7]. Among them, a series of bis-benzimidazoles
were reported. Neidle et al. reported upon a series of head-to-head
linked bis-benzimidazoles, such as compound 2. This compound
not only selectively recognized AT bases sequence with four base
pairs but also possessed remarkably selective cytotoxicity at the
micromolar level, with activity significantly greater than that
* Corresponding author. Tel.: þ86 24 23986413; fax: þ86 24 23995043.
E-mail address: mscheng@syphu.edu.cn (M.-S. Cheng).
shown by Hoechst 33258 in a group of ovarian carcinoma cell lines
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.021

Y.-H. Yang et al. / European Journal of Medicinal Chemistry 44 (2009) 1808–1812
1809
[8]. These symmetrical bis-benzimidazoles had favorable cellular
uptake properties [9,10]. They also designed a dimeric bis-benz-
imidazole molecule with a high degree of sequence selectivity for
AT-rich regions of DNA, which could recognize a run of almost 10
consecutive base pairs [8].
are van der Waals contacts between compound 8 and the narrow
minor groove. Compound 8 adopts a concave shape, which exactly
fits into the convex minor groove in the model. These two factors
indicate that compound 8 is able to penetrate deep into the minor
groove of DNA. In addition, two hydrogen bonds between
compound 8 and DNA are formed between one benzimidazole NH
group and T20-O2 (2.20 Å), and the other benzimidazole NH group
and A18-N3 (1.87 Å). Therefore, compound 8 can effectively bind at
the central AT region in the minor groove.
Most derivatives of Hoechst 33258 contained a phenyl group in
2-position of benzimidazole, which kept whole molecules to planar
structures. To our knowledge, no literature related the alkyl chain
substituents in 2-position of benzimidazole as the derivatives of
Hoechst 33258 were reported so far. Occasionally, the thioether
derivative of omeprazole, a compound with a substituted pyridyl-
methylene thioyl group at 2-position of benzimidazole, attracted
our attention. On the basis of structural similarities between
symmetrical bis-benzimidazole and omeprazole thioether, we
combined two omeprazole thioethers into one molecule with
a single bond. Therefore, a novel symmetrical bis-benzimidazole,
2,20-di-[[(3,5-dimethyl-4-methoxy)pyrid-2-yl]methylenethio]-5,
50-bis-1H,10H-benzimidazole (8) was synthesized. We hypothesized
that this symmetrical bis-benzimidazole could selectively bind to
the DNA minor groove. In the present paper, modeling study was
performed with compound 8 and a series of derivatives were
synthesized on the basis of modeling results. All of these
compounds were screened for anti-tumor activity in vitro.
3. Chemistry
The synthesis of these bis-benzimidazoles is outlined in Scheme 1.
The common intermediate was 2,20-dithiol-5,50-bis-1H,10H-benz-
imidazole (7), which reacted with sodium hydroxide and various
substituted chloromethylpyridine analogues in ethanol at reflux to
synthesize the desired compounds 8–17. The intermediate 7 was
prepared from the condensation of tetraminobiphenyl (TAB) with
disulfide carbon in ethanol. The synthesis of TAB was carried out
according to Vogel’s method [15], with minor improvements. The
overall yield of 7 was 49%.
4. Biological investigation
Preliminary cytotoxic activity of these bis-benzimidazoles on
SKOV-3, HeLa, BGC-823 cell lines was investigated in vitro. Briefly,
tumor cell lines in RPMI1640 medium with 10% fetal bovine serum
were plated in 96-well microtiter plates (4.0 ꢀ 104 cells/well), and
allowed to adhere at 37 ꢁC with 5% CO2 for 4 h. The test compound
was then added, andthecells were incubated at 37 ꢁC with 5% CO2 for
72 h. The cell viability was assessed using standard MTT assay [16].
2. Molecular modeling
A molecular modeling study was carried out by the Sybyl/FlexX
program to determine the binding ability of compound 8 to bind to
the minor groove of DNA. Usually, electrostatic, van der Waals,
hydrophobic, and hydrogen bonding forces dominate binding
ability. Sequence specificity is often attributed to key hydrogen
bonds between a base pair and the small molecule. Another crucial
structural requirement is that the ligand has a crescent shape and
can adopt an ‘‘isohelical’’ conformation to fit the minor groove [11].
In our model, computational calculations were performed by the
Sybyl 6.9.1 package (Tripos Ltd.) [12] on a Fuel Workstation (SGI
Ltd.) with the Irix 6.5 platform. The X-ray crystallographic structure
of the DNA dodecamer d(CGCAAATTTGCG) with a bifurcated
hydrogen-bonded conformation of the AT base pairs and its
complex with distamycin A was selected from the Protein Data
Bank (PDB code: 2DND) for the docking study [13]. During the
docking process, the binding site atoms and the ligand atoms were
set to be flexible. An incremental construction algorithm was
applied to generate and minimize the possible pose. A represen-
tative model of the dodecamer complexed to compound 8 is
depicted in Fig. 1 with DS Visualizer v1.5 (Accelrys Ltd.) [14]. There
5. Results and discussion
As shown in Table 1, for SKOV-3 cell line, compounds 8, 15, and
16 showed cytotoxicity below
Compounds 10 and 14 were less cytotoxic with concentrations
below 50 M. The other five compounds showed smaller effects
a concentration of 10 mM.
m
under the same conditions. For HeLa cell line, compound 14 was as
effective as cisplatin. Compounds 10, 11, and 16 were less potent
with concentrations below 50
were much less effective. For BGC-823, only compounds 14 and 16
showed cytotoxicity with concentrations below 50 M.
mM and the other six compounds
m
For pyrid-2-yl compounds, bulky substituents at the 3- and 4-
positions in pyridine reduce activity, but a methyl at the 5-position
can evidently improve activity. For compounds 14, 15, and 16, they
Fig. 1. Two views of compound 8 binding to the sequence d(CGCAAATTTGCG) (PDB code 2DND). (a) Close-up view of binding in the minor groove, highlighting the electrostatic
potential surface of the oligonucleotide. (b) Close-up view of hydrogen bonds between compound 8 and the DNA minor groove. The hydrogen bond distances were measured to be
2.20 Å between T20-O2 and one benzimidazole NH group of compound 8, 1.87 Å between A18-N3 and the other benzimidazole NH. Additionally, the corresponding hydrogen bond
angles are 159ꢁ and 165ꢁ, respectively.

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Y.-H. Yang et al. / European Journal of Medicinal Chemistry 44 (2009) 1808–1812
Scheme 1. Reagents and conditions: (a) (CH3CO)2O/NEt3, acetone, 0 ꢁC; (b) Conc. HNO3, acetic acid, 0–50 ꢁC; (c) 42% KOH aq, ethanol, reflux; (d) H2/Pd-C, methanol, r.t.; (e) KOH, CS2,
ethanol, reflux; (f) various substituted pyridines, NaOH, ethanol, reflux.
almost shared same structures, except the position of nitrogen in
pyridyl group. Pharmacological results in SKOV-3 cell line demon-
strated that pyrid-3-yl and pyrid-4-yl compounds showed higher
activity than pyrid-2-yl compound. However, bioevaluation in HeLa
cell line, pyrid-2-yl compound was more effective than pyrid-4-yl
compound, and pyrid-3-yl compound was least effective. In BGC-
823 cell line, pyrid-2-yl compound was equally effective as pyrid-
4-yl compound, and pyrid-3-yl compound was least effective.
Nevertheless, further research is still needed in order to investigate
further aspects of these effective compounds.
7.1. Synthesis of N,N0-diacetyl-4,40-diamino-p-biphenyl (4)
To a stirred solution of benzidine (3) (5.0 g, 27.2 mmol) in acetone
(50 ml) with an ice-bath, acetic anhydride (10.0 ml,105.8 mmol) was
slowly added dropwise and reaction was carried out for 3 h. Then,
triethylamine (25 ml, 179.4 mmol) was added dropwise. The reac-
tion was monitored by TLC. A white solid was obtained after filtra-
tion, and washed with acetone. After drying, the yield of compound
4 was 6.8 g (93%), m.p. 339–342 ꢁC. GC–MS (m/z): [M]þ 268.
7.2. Synthesis of 3,30-dinitro-4,40-diamino-p-biphenyl (5)
6. Conclusion
Compound 4 (6.8 g, 25.4 mmol) in solution with acetic acid
(50 ml) was stirred in an ice-bath. Fuming nitric acid (8 ml,
171.7 mmol) was slowly added dropwise for 2 h. Then, the ice-bath
was removed and the reaction mixture was incubated at r.t. for 2 h.
The mixture was poured onto ice (100 ml) and stirred. The resultant
yellow solid was filtered and washed with water. After drying as
much as possible, the yellow compound was mixed with a solution
of potassium hydroxide (7.3 g, 106.7 mmol) in water (10 ml) and
ethanol (30 ml), and then heated to reflux for 4 h. Then the mixture
was poured onto ice (100 ml) and stirred. The resultant red solid
was filterated and washed with water. After drying, the yield of the
compound 5 was 5.3 g (76%, two steps), m.p. 280–282 ꢁC. GC–MS
(m/z): [M]þ 274. 1H NMR (300 MHz, DMSO-d6, ppm): 8.15 (d, J ¼ 2.0,
1H, 2-H), 7.76 (dd, J ¼ 8.8, J ¼ 2.1, 1H, 6-H), 7.51 (s, 2H, NH2), 7.11 (d,
J ¼ 8.8, 1H, 5-H).
A series of novel bis-benzimidazoles were synthesized as novel
DNA minor groove binders and evaluated for anti-tumor activity.
Molecular modeling was used to predict the binding of compound 8
with the DNA minor groove, and the results showed that compound
8 could effectively fit into the minor groove. Preliminary anti-
tumorogenic activities indicated that compounds 8 and 16 were
most effective on SKOV-3 cell line. And compounds 14 and 16
showed broad activities on these tumor cell lines. This is the first
paper focused on a series of alkyl chain substituted bis-benzimid-
azoles as novel DNA minor groove binders. This work enriched the
structural types of bis-benzimidazoles and provided references for
the further development of minor groove binders. Further studies
in this area are in progress and will be reported upon in the future.
7. Experimental protocols
7.3. Synthesis of 3,30,4,40-tetraamino-p-biphenyl (6)
All commercially available reagents and solvents were
employed without further purification unless specified. Solvents
were dried and re-distilled prior to use using standard methods.
Melting points are uncorrected and were determined in a Bu¨chi
Melting Point B-540 apparatus. Infrared spectra were recorded in
a Bruker IFS-55 spectrophotometer. 1H NMR spectra were recorded
in Bruker ARX 300 MHz, using TMS as the internal standard if not
specifically mentioned (chemical shifts in d values, J in Hz). Mass
spectra were obtained on Shimazu GCMS-QP5050A and Waters
MicromassÒ Quattro microÔ API mass spectrometer. Column
chromatography was performed on silica gel H and analytical TLC
data on silica gel HF254. Hydrogen gas was produced by a QL-300
hydrogen generator.
Hydrogen gas was passed through
a stirred mixture of
compound 5 (5.3 g, 19.3 mmol), Pd-C (0.3 g, 10%) and methanol
(50 ml) at a flow rate of 10 ml/min for 3 h. After filtration, the
filtrate was evaporated under reduced pressure to give 3.7 g of
compound 6 with a yield of 89%, m.p. 174–176 ꢁC. GC–MS (m/z):
[M]þ 214.
7.4. Synthesis of 2,20-dithiol-5,50-bis-1H,10H-benzimidazole (7)
Compound 6 (3.7 g, 17.3 mmol) was dissolved in ethanol (10 ml)
and the resulting solution was added to a mixture of potassium
hydroxide (8.4 g, 122.8 mmol), water (10 ml), carbon disulfide

Y.-H. Yang et al. / European Journal of Medicinal Chemistry 44 (2009) 1808–1812
1811
7.5.3. Data for 2,20-di-[[[3-methyl-4-(2,2,2-trifluoroethyl)]prid-2-
Table 1
Structure and anti-tumor activity of bis-benzimidazoles against SKOV-3, HeLa, and
BGC-823 tumor cell lines
yl]methylenethio]-5,50-bis-1H,10H-benzimidazole (10)
Yield: 80%; m.p.: 177–180 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.68 (1H, br s, Bz-NH), 8.31 (1H, d, J ¼ 5.7, Py-6-H), 7.40–7.80
(3H, m, Bz-4-H, Bz-6-H, Bz-7-H), 7.09 (1H, d, J ¼ 5.7, Py-5-H), 4.90
(2H, q, J ¼ 8.7, OCH2CF3), 4.75 (2H, s, SCH2), 2.26 (3H, s, Py-3-CH3);
ESI-MS (m/z): 705.6 [M þ H]þ; IR (KBr, cmꢂ1): 3430, 2936, 1626,
1580,1474,1431,1384,1282,1256,1170,1110, 979, 857, 803, 663, 578.
7.5.4. Data for 2,20-di-[[(3-methoxy-4-chloro)prid-2-
Compound
Substituents
Pyridyl
IC50
SKOV-3
0.00134
(
m
M)a
yl]methylenethio]-5,50-bis-1H,10H-benzimidazole (11)
Yield: 69%; m.p.: 124–127 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.66 (1H, br s, Bz-NH), 8.25 (1H, d, J ¼ 5.2, Py-6-H), 7.66 (1H,
s, Bz-4-H), 7.55 (1H, d, J ¼ 5.2, Py-5-H), 7.49 (1H, d, J ¼ 8.3, Bz-7-H),
7.41 (1H, d, J ¼ 8.5, Bz-6-H), 4.80 (2H, s, SCH2), 3.92 (3H, s, Py-3-
OCH3); ESI-MS (m/z): 609.3 [M þ H]þ; IR (KBr, cmꢂ1): 3438, 1626,
1564, 1461, 1401, 1384, 1281, 1233, 1082, 897, 818, 664.
R
HeLa
–
1.6
>50
>50
18.3
45.4
>50
>50
7.1
>50
32.4
>50
BGC-823
Taxol
Cisplatin
8
9
10
11
12
13
14
15
16
17
–
–
1.3
Pyrid-2-yl
Pyrid-2-yl
Pyrid-2-yl
Pyrid-2-yl
Pyrid-2-yl
Pyrid-2-yl
Pyrid-2-yl
Pyrid-3-yl
Pyrid-4-yl
Pyrid-3-yl
3,5-CH3, 4-OCH3
3,4-OCH3
3-CH3, 4-OCH2CF3
3-OCH3, 4-Cl
3-CH3, 4-OCH3
3-CH3, 4-OCH2CH3
H
H
2.95
>50
22.96
>50
>50
>50
38.60
8.73
2.81
>50
>50
>50
>50
>50
>50
16.4
>50
11.0
>50
7.5.5. Data for 2,20-di-[[(3-methyl-4-methoxy)prid-2-
yl]methylenethio]-5,50-bis-1H,10H-benzimidazole (12)
Yield: 79%; m.p.: 159–162 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.67 (1H, br s, Bz-NH), 8.26 (1H, d, J ¼ 5.6, Py-6-H), 7.67 (1H,
s, Bz-4-H), 7.50 (1H, d, J ¼ 8.3, Bz-7-H), 7.42 (1H, dd, J ¼ 8.3, J ¼ 1.0,
Bz-6-H), 6.96 (1H, d, J ¼ 5.7, Py-5-H), 4.71 (2H, s, SCH2), 3.85 (3H, s,
OCH3), 2.21 (3H, s, Py-3-CH3); ESI-MS (m/z): 569.5 [M þ H]þ; IR
(KBr, cmꢂ1): 3439, 1625, 1584, 1467, 1431, 1384, 1297, 1097, 807, 600.
H
6-Cl
>50
a
The drug concentrations that inhibited cell growth by 50% (IC50) were deter-
mined from semilogarithmic dose-response plots.
(5.0 ml, 82.7 mmol), and ethanol (30 ml) dropwise. The solution
was heated to reflux for 4 h and monitored by TLC. The mixture was
evaporated under reduced pressure. Water (50 ml) was added to
the residue, and conc. HCl was added to adjust the pH to about 3.
The solution was filterated to yield the solid and dried. The yield of
compound 7 was 4.0 g (78%), with an m.p. >400 ꢁC. GC–MS (m/z):
[M]þ 298; 1H NMR (300 MHz, DMSO-d6, ppm): 12.61 (d, 2H, 1-NH
and 2-SH), 7.40 (dd, J ¼ 8.3, J ¼ 1.6, 1H, Bz-6-H), 7.29 (s, 1H, Bz-4-H),
7.21 (d, J ¼ 8.3, 1H, Bz-7-H).
7.5.6. Data for 2,20-di-[[(3-methyl-4-ethoxy)prid-2-
yl]methylenethio]-5,50-bis-1H,10H-benzimidazole (13)
Yield: 75%; m.p.: 125–128 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.68 (1H, br s, Bz-NH), 8.24 (1H, d, J ¼ 5.6, Py-6-H), 7.69 (1H,
s, Bz-4-H), 7.52 (1H, d, J ¼ 8.3, Bz-7-H), 7.44 (1H, dd, J ¼ 8.3, J ¼ 1.2,
Bz-6-H), 6.94 (1H, d, J ¼ 5.7, Py-5-H), 4.71 (2H, s, SCH2), 4.11 (2H, q,
J ¼ 6.9, OCH2CH3), 2.21 (3H, s, Py-3-CH3), 1.35 (3H, t, J ¼ 6.9,
OCH2CH3); ESI-MS (m/z): 597.4 [M þ H]þ; IR (KBr, cmꢂ1): 3439,
1625, 1583, 1467, 1384, 1299, 1279, 1092, 809.
7.5. General process for the synthesis of compounds 8–17
7.5.7. Data for 2,20-di-[(prid-2-yl)methylenethio]-5,50-bis-1H,10H-
benzimidazole (14)
After a mixture of compound 7 (1.0 g, 3.36 mmol), ethanol
(30 ml), sodium hydroxide (1.0 g, 24.0 mmol), and water (10 ml)
was stirred for 30 min, substituted chloromethylpyridine hydro-
chloride (6.90 mmol) was added and heated to reflux for 8 h. The
mixture was evaporated under reduced pressure. Water (50 ml)
was added to the residue, and glacial acetic acid was added to
adjust the pH to about 5. The solution was filterated to yield the
solid and dried. Compounds 8–17 were purified by gel column
chromatography (fluent CHCl3:CH3OH ¼ 90:1).
Yield: 81%; m.p.: 91–94 ꢁC; 1H NMR (300 MHz, DMSO-d6, ppm):
12.68 (1H, br s, Bz-NH), 8.52 (1H, d, J ¼ 4.7, Py-6-H), 7.75 (1H, t,
J ¼ 7.5, Py-4-H), 7.60–7.72 (1H, m, Bz-4-H), 7.54 (1H, d, J ¼ 7.7, Py-3-
H), 7.40–7.60 (2H, m, Bz-6-H, Bz-7-H), 7.29 (1H, dd, J ¼ 7.2, J ¼ 5.1,
Py-5-H), 4.69 (2H, s, SCH2); ESI-MS (m/z): 481.4 [M þ H]þ; IR (KBr,
cmꢂ1): 3427, 1623, 1593, 1475, 1435, 1384, 1341, 1276, 1004, 804,
748, 585.
Compounds 8–17 were characterized as follows.
7.5.8. Data for 2,20-di-[(prid-3-yl)methylenethio]-5,50-bis-1H,10H-
benzimidazole (15)
7.5.1. Data for 2,20-di-[[(3,5-dimethyl-4-methoxy)prid-2-
yl]methylenethio]-5,50-bis-1H,10H-benzimidazole (8)
Yield: 74%; m.p.: 103–106 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.70 (1H, br s, Bz-NH), 8.66 (1H, d, J ¼ 1.6, Py-2-H), 8.43 (1H,
dd, J ¼ 4.8, J ¼ 1.1, Py-6-H), 7.87 (1H, dt, J ¼ 7.8, J ¼ 1.6, Py-4-H), 7.67
(1H, s, Bz-4-H), 7.51 (1H, d, J ¼ 8.3, Bz-7-H), 7.43 (1H, dd, J ¼ 8.4,
J ¼ 0.8, Bz-6-H), 7.33 (1H, dd, J ¼ 7.8, J ¼ 4.8, Py-5-H), 4.59 (2H, s,
SCH2); ESI-MS (m/z): 481.4 [M þ H]þ; IR (KBr, cmꢂ1): 3423, 1623,
1479, 1430, 1384, 1343, 1278, 1126, 1030, 975, 803, 709, 668.
Yield: 80%; m.p.: 109–112 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.68 (1H, br s, Bz-NH), 8.19 (1H, s, Py-6-H), 7.67 (1H, s, Bz-4-
H), 7.50 (1H, d, J ¼ 8.3, Bz-7-H), 7.42 (1H, dd, J ¼ 8.3, J ¼ 1.5, Bz-6-H),
4.71 (2H, s, SCH2), 3.72 (3H, s, OCH3), 2.30 (3H, s, Py-3-CH3), 2.21
(3H, s, Py-5-CH3); ESI-MS (m/z): 597.2 [M þ H]þ; IR (KBr, cmꢂ1):
3439, 1624, 1569, 1475, 1433, 1384, 1273, 1078, 1000, 805.
7.5.9. Data for 2,20-di-[(prid-4-yl)methylenethio]-5,50-bis-1H,10H-
benzimidazole (16)
7.5.2. Data for 2,20-di-[[(3,4-dimethoxy)prid-2-yl]methylenethio]-
5,50-bis-1H,10H-benzimidazole (9)
Yield: 87%; m.p.: 126–129 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.69 (1H, br s, Bz-NH), 8.45 (2H, m, J ¼ 4.5, Py-2-H and Py-6-
H), 7.54 (1H, s, Bz-4-H), 7.45 (2H, m, Py-3-H and Py-5-H), 7.36 (1H,
d, J ¼ 8.3, Bz-7-H), 7.22 (1H, dd, J ¼ 8.3, J ¼ 1.4, Bz-6-H), 4.52 (2H, s,
SCH2); ESI-MS (m/z): 481.4 [M þ H]þ; IR (KBr, cmꢂ1): 3424, 3077,
1605, 1561, 1498, 1417, 1385, 1338, 1279, 1227, 1068, 1004, 977, 803,
754, 714, 665.
Yield: 89%; m.p.: 202–205 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.68 (1H, br s, Bz-NH), 8.17 (1H, d, J ¼ 5.7, Py-6-H), 7.40–7.70
(3H, m, Bz-4-H, Bz-7-H, Bz-6-H), 7.09 (1H, d, J ¼ 5.7, Py-5-H), 4.69
(2H, s, SCH2), 3.89 (3H, s, OCH3), 3.82 (3H, s, OCH3); ESI-MS (m/z):
601.1 [M þ H]þ; IR (KBr, cmꢂ1): 3441, 1626, 1587, 1489, 1428, 1384,
1302, 1281, 1227, 1071, 995, 788.

1812
Y.-H. Yang et al. / European Journal of Medicinal Chemistry 44 (2009) 1808–1812
7.5.10. Data for 2,20-di-[(6-chloro-prid-3-yl)methylenethio]-5,50-
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Yield: 71%; m.p.: 214–217 ꢁC; 1H NMR (300 MHz, DMSO-d6,
ppm): 12.66 (1H, s, Bz-NH), 8.50 (1H, s, Py-2-H), 7.40–8.10 (5H, m,
Bz-4-H, Bz-7-H, Bz-6-H, Py-4-H, and Py-5-H), 4.58 (2H, s, SCH2);
ESI-MS (m/z): 549.2 [M þ H]þ; IR (KBr, cmꢂ1): 3446, 1626, 1587,
1461, 1435, 1384, 1280, 1106, 1027, 806, 668.
¨
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