Synthesis and biological evaluation of novel benzimidazole derivatives and their binding behavior with bovine serum albumin

Article abstract of DOI:10.1016/j.ejmech.2012.07.015

A series of novel benzimidazole derivatives were synthesized and characterized by ¹H NMR, ¹³C NMR, MS, IR and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by two-fold serial dilution t

Full text of DOI:10.1016/j.ejmech.2012.07.015

European Journal of Medicinal Chemistry 55 (2012) 164e175
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel benzimidazole derivatives
and their binding behavior with bovine serum albumin
1
*
*
Shao-Lin Zhang, Guri L.V. Damu , Ling Zhang, Rong-Xia Geng , Cheng-He Zhou
Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Beibei, Tiansheng Road, Chongqing 400715, China
a r t i c l e i n f o
a b s t r a c t
A series of novel benzimidazole derivatives were synthesized and characterized by 1H NMR, 13C NMR,
MS, IR and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro
by two-fold serial dilution technique. Bioactive assay manifested that the bis-benzimidazole derivative
11d and its hydrochloride 13b exhibited remarkable antimicrobial activities, which were comparable or
even better than the reference drugs Norfloxacin, Chloromycin and Fluconazole. The interaction evalu-
ation of compound 11d with bovine serum albumin (BSA) by Fluorescence and UVevis absorption
spectroscopic method showed that BSA could generate fluorescent quenching under approximately
human physiological conditions by the prepared benzimidazole compound 11d as result of the formation
of ground-state compound 11deBSA complex. The thermodynamic parameters indicated that the
hydrogen bonds and van der Waals forces played major roles in the strong association of benzimidazole
11d and BSA.
Article history:
Received 7 April 2012
Received in revised form
21 June 2012
Accepted 7 July 2012
Available online 22 July 2012
Keywords:
Benzimidazole
Antibacterial
Antifungal
Fluorescence spectrum
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
synthesized and found to exhibit good antimicrobial efficacy
[16,17], especially benzimidazole compound 1a displayed signifi-
Benzimidazoles have been frequently found to display a variety
of biological activities such as antihelmintic [1], antihistaminic [2],
anticancer [3], antiviral [4], antiinflammatory [5], antiproliferative
[6], antioxidant [7], anticoagulant properties [8]. Many benzimid-
azole drugs like antiparasitic thiabendazole, mebendazole and
albendazole, antihistaminic norastemizole and mizolastine, as well
as antihypertensive telmisartan etc. have been successfully devel-
oped and extensively used in clinic. This has attracted increasing
interest to investigate the possible applications of benzimidazole
derivatives in other medicinal aspects. Recently, a lot of literature
has revealed that benzimidazole derivatives could effectively
prevent the synthesis of microbial nucleic acids and proteins [9],
thus inhibiting the growth of various microorganisms [10e13],
which suggests that benzimidazole compounds should have large
potential as a new type of antibacterial and antifungal agents [14].
Much effort has been directed toward the development of struc-
turally novel benzimidazole compounds as antimicrobial agents
[15]. So far several active benzimidazole derivatives have been
cant activity against Methicillin-resistant Staphylococcus aureus
(MRSA) [18], while compound 1b gave 2- to 4-fold more efficient
antifungal activities against Candida krusei and Candida glabrata
than Fluconazole [19]. Moreover, benzimidazole hydrazone deriv-
ative 2 also showed remarkable antifungal activity against Candida
species, which was superior to antifungal Ketoconazole [20]. All
these indicated that the benzimidazole moiety played quite an
important role in exerting antimicrobial efficacy (Fig. 1).
It is well known that the combination of two or more types of
heterocycles into one molecule could afford a novel entity with
increased bioactivities and extended antimicrobial spectrum
[21,22]. Many researches suggested that the incorporation of
heterocyclic groups like furan [23], imidazole [24], thiadizole [25]
etc. into benzimidazole scaffold produced new benzimidazole
derivatives which exhibited significant antimicrobial activities.
Among these heterocyclic groups, thiophene moiety was widely
used in drug design [26] because its rich electronic cloud density
endowed thiophene derivatives with a property of ready binding
with a variety of enzymes and receptors [27] in biological system
via diverse non-covalent interactions, thereby effectively
improving the pharmacokinetic properties [28].
* Corresponding authors. Tel./fax: þ86 23 68254967.
E-mail addresses: geng0712@swu.edu.cn (R.-X. Geng), zhouch@swu.edu.cn
(C.-H. Zhou).
Considering the importance of benzimidazole and thiophene
nucleus, it is worthwhile to design and synthesize a new skeleton of
both benzimidazole and thiophene moieties, which was expected
to possess good antimicrobial activities. Herein, a series of novel
1
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT),
Hyderabad 500 607, India.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.015

S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
165
hybrid compounds were designed and synthesized. The antibac-
terial and antifungal activities of all newly synthesized compounds
were evaluated in vitro against four Gram-positive bacteria, five
Gram-negative bacteria and two fungi. The target compounds were
designed by the following considerations:
R1
Cl
N
N
R4
1. Hydrazide moiety was reported to easily bind with transport
proteins and target enzymes via coordination bonds [29],
hydrogen bonds etc., thus modulating the physicochemical and
pharmacokinetics properties of compounds. Therefore, hydra-
zide group was introduced into the designed compounds.
2. Many literature evidenced that alkyl groups could improve
biological potency by regulating the lipid-water partition
coefficients [30]. So compounds 11aef with different lengths of
alkyl chains were synthesized to explore the influence of chain
lengths on biological activities.
3. Halobenzyl groups were reported to significantly affect anti-
microbial efficacy [31]. Reasonably, diverse halobenzyl moie-
ties including chlorobenzyl and fluorobenzyl groups were
introduced into the target compounds 12aef to investigate
their contributions to antimicrobial efficiency.
1a−b
R2
R3
1a: R1 = Cl, R2 = Cl, R3 = SO2CH2CH2N(Et)2, R4 = H
1b: R1 = F, R2 = H, R3 = H, R4 =
N
N CH3
Br
O
O
H
N
NH
HN N
N
2
Fig. 1. Structures of some antimicrobial benzimidazole derivatives.
4. The transformation of azoles into their hydrochlorides was
proved to be potent in reinforcing water solubility, thus
enhancing antibacterial and antifungal efficacy and broadening
H3COOC
NC
CH3
CH3
NC
S
Cl
CH3
CH3
CH3
S
b
d
a
c
S
NC
S
3
CN
CH3
H3COOC
N
O
O
6
NC
5
4
NH2
S
e
H3C
CH3
H3C
H3C
CH3
CH3
O
O
O
O
O
O
O
H2NHN
H2NHN
S
S
HN
NH
CH3
HN
HN
NH
NH
h
g
NH
N
HN
N
NH2
NH
HN
HN
HN
S
CH3
CH3
CH3
n
N
n
N
N
NH
N
HN
N
7
10
11a−f
9
j
11: a, n = 2; b, n = 5
c, n = 7; d, n = 9
i
H3C
CH3
H3C
CH3
e, n = 11; f, n = 17
H3C
CH3
O
O
O
HN
O
O
O
S
F
S
F
R2
R3
R3
R2
S
HN
NH
NH
HN
R1
NH
R1
NH
4HCl
HN
NH
4HCl
j
HN
NH
N
HN
N
CH3
n
CH3
N
N
N
N
n
N
N
N
N
N
N
13: a, n = 2
b, n = 9
14
12a−f
13a−b
NH2
12: a, R1 = H, R2 = Cl, R3 = Cl; b, R1 = F, R2 = H, R3 = F
N
c, R1 = H, R2 = H, R3 = F ; d, R1 = H, R2 = H, R3 = Cl
f
Cl
COOH
e, R1 = H, R2 = Cl, R3 = H ; f, R1 = Cl, R2 = H, R3 = H
Cl
N
H
NH2
8
Scheme 1. Synthetic route of target compounds.

166
S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
antimicrobial spectrum [32,33]. For this reason, the benz-
imidazole derivatives 11a, 11d and 12c were converted into the
corresponding hydrochlorides.
sodium hydroxide and sodium ethoxide were investigated to form
a linkage of benzyl groups or alkyl chains onto the eNH of hydra-
zide, however, the results were unsatisfactory. The synthetic route
of target compounds 11e14 was outlined in Scheme 1.
In view of the above considerations, further studies on binding
behaviors between serum albumin and the target compounds have
been performed. Serum albumins are the most important and
abundant macromolecule proteins in the circulatory system. When
drugs or other bioactive small molecules bind reversibly [34,35] to
them, serum albumins could deliver drugs or other bioactive small
molecules to the binding sites. Given these critical roles of serum
albumins, thorough studies involving the interaction of drugs or
bioactive small molecules with serum albumins are not only
beneficial to provide a proper understanding of the absorption,
transportation, distribution, metabolism and excretion properties
of drugs [36], but also significant to the design, modification and
screening of drug molecules. As the homologous proteins of human
serum albumins (HSA) [37e39], BSA was selected as protein model
because of its medical importance, low cost, ready availability,
unusual ligand-binding and intrinsic fluorescence properties
[40,41]. Therefore, it is important to study the interactions of our
newly synthesized bioactive small molecules with this protein. In
this work, interaction information including fluorescence quench-
ing mechanisms, binding parameters, thermodynamic parameters
and binding mode between BSA and target compound was ob-
tained by fluorescence and UVevis absorption spectroscopy on
molecular level.
2.2. Spectral analysis
All new compounds were confirmed by 1H NMR, 13C NMR, MS,
IR and HRMS spectra. The spectral analyses were in accordance
with the assigned structures, and listed in the experimental section.
2.2.1. IR spectra
In IR spectra, compounds 7 and 10 gave moderate absorption
bands in 3289e3238 cmꢀ1 and 3280e3207 cmꢀ1, which were
attributed to the stretching vibration of eNH2 group, while the
disappearance of the characteristic primary amino group stretching
frequencies in compounds 11e14 and 9 suggested that the primary
amino group underwent N-alkylation. Moreover, it was found that
the stretching frequencies of C]O moiety in compounds 9e14 and
7 shifted to lower wave number at 1659e1630 cmꢀ1 due to the
presence of electron-donating effect of thiophene and eNHe
groups which increased the electronic cloud density of the
carbonyl moiety. However, the eNeHe group in compounds 11e14
and 9 gave higher wave numbers at 3438e3418 cmꢀ1 (Table 1) in
comparison with primary amino group, which was mainly
responsible for the inductive effects of the electron-withdrawing
character of carbonyl group.
2.2.2. 1H NMR spectra
2. Results and discussion
As seen in Table 1, the singlet peak at 4.81 ppm was assigned to
the methylene protons Hb of compound 9, and the bis-
2.1. Chemistry
The preparation of target benzimidazole derivatives started
from commercially available chloroacetonitrile. The reaction of
chloroacetonitrile with sodium sulfide yielded bis(cyanomethyl)
sulfide 3, which was cyclizated with butane-2,3-dione to produce
thiophene derivative 4, and further treated with thionyl chloride to
afford 3,4-dimethylthiophene-2,5-dicarbonitrile 5. The 2,5-
dicarboxylate thiophene 6 was efficiently prepared by the esteri-
fication of compound 5 in methanol catalyzed by concentrated
sulfuric acid, then condensed with hydrazine hydrate to give 3,4-
dimethylthiophene-2,5-dicerbohydrazide 7 with moderate yield
of 68.3%. Bis-benzimidazole 9 and mono-benzimidazole 10 were
conveniently synthesized from compound 7 by N-alkylation with
2-substituted benzimidazole 8. Bis-benzimidazole 9 then further
was treated with alkyl or aryl halides respectively in the presence of
potassium carbonate in ethanol to produce target compounds
11aef and 12aef with yields ranging from 64.7% to 87.2%, then
compounds 11a, 11d and 12c were selected to react with hydro-
chloric acid (4 M) affording their corresponding hydrochlorides
13aeb and 14.
Table 1
Some 1H NMR data (
d/ppm) of target compounds 11e14 and intermediate 9.
H2C Ha
H2C Ha
H3C
H3C
O
O
O
O
S
Hd
S
Hd
N He
Hb
N He
Hb
HN
HN
NH
N
NH
N
N
Hc
Hc
4HCl
R
N
N
N
N
N
R
N
N
R
R
13a−b, 14
9, 11a−f, 12a−f
Compds
IR (
n
/cmꢀ1
)
1H NMR (
Ha
d
/ppm)
Hb
NeHe
NeHd
Hc
In the experiment, excessive anhydrous potassium carbonate
was employed which could not only serve as base to neutralize the
reaction system, but also effectively absorb moisture to prevent
amide from hydrolyzation. Moreover, the effects of bases on this
reaction were explored, if potassium carbonate was absent during
the synthesis of compounds 11aef and 12aef, no target
compounds were obtained after the reaction system refluxed in
ethanol for 24 h, however, target compounds formed immediately
once the base was added (monitored by TLC, petroleum/ethyl
acetate, 1/1, V/V). Interestingly, the introduced groups including
halobenzyl or alkyl chains with different lengths were all connected
to the N-1 position of benzimidazole instead of eNH of hydrazide,
which were verified by the remarkable downfield shifts of meth-
ylene protons in 1H NMR spectra. Furthermore, strong bases such as
9
3431
3421
3423
3425
3428
3426
3429
3421
3419
3432
3420
3431
3418
3436
3431
3438
3292
3299
3298
3296
3305
3294
3296
3297
3289
3303
3299
3302
3291
3309
3306
3319
3.48
3.39
3.38
3.39
3.40
3.38
3.39
3.38
3.36
3.37
3.37
3.38
3.36
3.49
3.49
3.46
4.81
4.76
4.75
4.76
4.76
4.76
4.75
4.72
4.76
4.70
4.70
4.72
4.70
5.48
5.46
5.52
e
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
13a
13b
14
4.20
4.21
4.22
4.22
4.22
4.22
5.43
5.49
5.43
5.44
5.45
5.57
4.73
4.72
6.07

S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
167
benzimidazole derivatives 11e12 with alkyl or halobenzyl substi-
tution displayed upfield shifts for protons Hb, but the hydrochlo-
to the National Committee for Clinical Laboratory Standards
(NCCLS) [42], the biological tests were carried out in triplicate.
rides
13e14
exhibited
remarkable
downfield
shifts
Minimal inhibitory concentration (MIC, mg/mL) was defined as the
(0.78e0.70 ppm) in contrast to compounds 11e12. This fact man-
ifested that alkyl or halobenzyl groups caused shielding effect on
Hb, while the positive charge in nitrogen atoms obviously affected
the chemical shifts of bis-benzimidazole compounds. In addition,
the protons Hc in phenyl substituted compounds displayed large
downfield shifts at the region of 5.49e5.43 ppm in comparison
lowest concentration of new compounds that completely inhibited
the growth of bacteria (Supplementary information 1). The values
of clog p (a partition coefficient) and MR (molar refractivity) were
calculated using ChemDraw Ultra 10.0 software integrated with
Cambridge soft Software (Cambridge Soft Corporation), log p data
were measured by traditional saturation shake flask approach and
UVevis spectrophotometric method (Supplementary information
2). The antimicrobial, clog p, log p data and MR values were
depicted in Tables 2 and 3, respectively.
with alkyl substituted ones (
d
¼ 4.22e4.20 ppm), which was
probably due to the electron-withdrawing character of halophenyl
moieties. Moreover, large downfield chemical shifts at
3.48e3.38 ppm were observed for protons Ha because of the exis-
tence of a conjugation system between thiophene ring and amide
group, as well as the strong electron-withdrawing of the amide
linkage.
2.3.1. Antibacterial activity
The antibacterial results showed that compounds 11aef and
12aef exhibited moderate to excellent efficacy against all tested
bacterial strains, especially compound 11d displayed remarkable
antibacterial activities, which indicated that compound 11d could
be employed to further study for potent novel board-spectrum
antimicrobial agents.
2.2.3. 13C NMR spectra
The 13C NMR spectral analyses were consistent with the
assigned structures. In compound 12b, the split signals observed at
d
163.58e161.30 and 161.21e158.93 ppm were assigned to the C-2
Results in Table 2 demonstrated the significant effects of alkyl
chain with different lengths on biological activities. Most of alkyl
benzimidazole derivatives showed moderate to excellent efficacy
against tested bacteria. Particularly, compound 11d with decyl
pendant displayed the best antibacterial activities, especially for
MRSA, Escherichia Coli, Pseudomonas aeruginosa, which was more
potent than Norfloxacin and Chloromycin respectively. When the
alkyl chain was extended to dodecyl group, benzimidazole deriv-
ative 11e gave more efficient inhibition against Shigella dysenteriae
and MRSA than the standard drug Chloromycin at the concentra-
and C-4 position of 2,4-difluorobenzyl group respectively due to
the existence of F atom with strong electronegativity. The formation
of hydrochlorides of compounds 11a, 11d and 12c resulted in
downfield 13C chemical shifts with 16 ppm for methylene carbon of
eCH2eR and 10 ppm for the eNHNHCH2. Additionally, all the other
aromatic and aliphatic carbons also appeared at the appropriate
chemical regions.
2.3. Pharmacology
tions of 8 and 16
mg/mL. Regretfully, propyl- and octadecyl-con-
taining compounds 11a and 11f were unfavorable for the
antibacterial efficiency even at high concentration, this might be
explained by the presence of their short alkyl chain with poor lip-
ophilicity and long alkyl chain with good lipophilicity, which make
All newly synthesized compounds were evaluated for their
in vitro antimicrobial activities against four Gram-positive bacteria,
five Gram-negative bacteria and two fungi using the standard two
folds serial dilution method in 96-well microtest plates according
Table 2
Antimicrobial data as MIC (
m
g/mL)a,b,c for compounds 7e14.
Compds
Gram-positive bacteria
Gram-negtive bacteria
Fungi
MRSA
S. aureus
B. subtilis
M. luteus
E. coli
S. dysenteriae
P. aeruginosa
B. proteus
E. typhosa
C. albicans
C. mycoderma
7
8
9
10
>512
>512
>512
256
32
4
8
2
8
512
>512
128
128
32
16
16
32
2
>512
>512
>512
256
512
32
32
2
128
256
32
64
128
32
128
32
256
2
>512
>512
128
64
256
32
512
512
256
128
256
64
512
256
512
512
256
8
512
512
512
512
256
64
16
8
16
512
>512
256
128
32
128
32
128
8
>512
>512
>512
256
128
32
>512
>512
256
128
256
64
512
512
512
64
256
64
512
512
>512
256
512
32
16
4
256
>512
>512
64
64
16
128
32
32
512
>512
256
128
256
32
4
2
64
256
>512
256
32
32
64
128
128
4
8
e
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
13a
13b
14
32
4
16
16
32
4
16
4
>512
8
64
4
512
>512
512
256
32
32
128
32
>512
>512
>512
256
128
16
32
16
256
8
64
128
512
512
128
32
8
32
32
256
4
128
512
>512
128
128
32
128
64
32
>512
>512
>512
128
128
32
128
16
16
256
64
>512
128
128
16
256
128
4
256
2
16
16
64
32
2
128
32
4
256
32
8
64
32
16
256
16
16
32
8
32
A
32
8
e
B
C
8
e
0.5
e
1
e
2
e
16
e
4
e
16
e
8
e
4
e
e
e
1
4
a
Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
b
c
A ¼ Chloromycin, B ¼ Norfloxacin, C ¼ Fluconazole.
MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus, Staphylococcus aureus ATCC25923; B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus ATCC 4698;
E. Coli, Escherichia coli DH52; S. dysenteriae, Shigella dysenteriae; P. aeruginosa, Pseudomonas aeruginosa; E. typhosa, Eberthella typhosa; B. proteus, Bacillus proteus; C. albicans,
Candida albicans; C. mycoderma, Candida mycoderma.

168
S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
Table 3
2.0
clog p, log p and molar refractivity (MR) of compounds 7e12.
T = 310 K
T = 304 K
T = 298 K
Compounds
clog p
log p
MR (cm3/mol)
1.6
1.2
0.8
0.4
0.0
7
8
9
10
ꢀ1.27
0.69
1.08
ꢀ1.41 ꢁ 0.13
1.44 ꢁ 0.14
1.25 ꢁ 0.14
0.84 ꢁ 0.10
3.70 ꢁ 0.24
6.62 ꢁ 0.19
8.19 ꢁ 0.22
NE
61.38
43.47
135.98
98.68
164.78
192.38
211.29
230.49
249.87
278.93
213.4
ꢀ0.10
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
3.23
6.40
8.38
10.33
12.36
18.22
7.24
5.22
4.93
6.07
6.07
NE
NE
7.16 ꢁ 0.33
4.96 ꢁ 0.06
5.25 ꢁ 0.11
6.19 ꢁ 0.18
6.34 ꢁ 0.11
6.25 ꢁ 0.10
196.6
195.8
204.2
204.2
6.07
204.2
0
1
2
3
4
5
6
105 Q mol/L
NE ¼ No experimental data. It is difficult to obtain the log p data of compounds
11def due to their quite low water solubility.
Fig. 3. SterneVolmer plot at three different temperatures.
both of them unfavorable for being delivered to the binding sites.
Therefore, suitable lipophilicity is necessary for better antibacterial
activities in drug design.
strains, which displayed much better efficacy than standard drug
Chloromycin. The enhanced activities for all these bis-
benzimidazole hydrochlorides might be attributed to the
improvement of the water solubility in comparison with their
corresponding precursors.
Among the benzimidazole derivatives 12aef bearing a variety
of halobenzyl groups, some compounds gave comparable inhibi-
tory potency to Chloromycin, but were relatively weaker than the
alkyl ones. Dichlorobenzyl compound 12a and difluorobenzyl
derivative 12b showed weak activities against both Gram-
negative and Gram-positive bacteria, whereas mono-substituted
ones 12cef gave better efficacy against all these strains than the
bis-substituted ones. This manifested that electron-withdrawing
group was a unfavorable for the antibacterial efficiency in
compounds 12aef. Notably, compound 12d showed low inhibi-
2.3.2. Antifungal activity
The antifungal evaluation in vitro revealed that the activities of
most compounds were relatively weaker compared to their anti-
bacterial activities (Table 2). However, alkyl substituted compounds
11ced and halobenzyl substituted compound 12d exhibited
significant antifungal activities against tested strains. Especially for
Candida mycoderma, compound 11d showed quite low inhibitory
tory concentration (MIC ¼ 8
a potential anti-E. Coli agent in comparison with clinical drug
g/mL).
mg/mL) against E. Coli, which was
concentration (MIC ¼ 2
mg/mL) which was 2-fold more effective
than the first-line antifungal drug Fluconazole. Hydrochlorides 13a
and 14 showed better antifungal capabilities than their corre-
sponding precursors bis-benzimidazole derivatives, but the trans-
formation of compound 11d into hydrochloride decreased its
antifungal efficiency. To our surprise, all newly synthesized
compounds displayed better antibacterial efficiency and antifungal
activity than their precursors 7 and 8, some even gave better
antimicrobial activities compared to the reference drugs.
Chloromycin (MIC ¼ 32
m
Compounds 11a, 11d and 12c were transformed into water-
soluble hydrochlorides 13aeb and 14, this modification resulted
in improvement of their antibacterial efficacy. Among these salts,
compound 13b showed the best antibacterial activity with MIC
values ranging from 2 to 16
mg/mL against all tested bacterial
1.0
BSA
2.3.3. Analysis of clog p, log p and MR (molar refractivity)
a
l
Lipophilicity/hydrophilicity plays a significant role in deter-
mining where drugs are distributed and how rapidly they are
metabolized and excreted in the body, for example, hydrophobic
drugs are preferentially distributed to hydrophobic compartments
such as lipid bilayers of cells while hydrophilic drugs are prefer-
entially found in hydrophilic compartments like blood serum.
Therefore, the lipophilicity/hydrophilicity expressed as log p was
calculated theoretically using ChemDraw Ultra 10.0 software and
experimentally by traditional saturation shake flask approach
combining with UVevis spectrophotometric method. The obtained
results were given in Table 3, the clog p of compounds 11aef
increased with the length of the alkyl groups increasing, and an
enhancement of the antimicrobial activities was observed in
compounds 11aed, but decreased in that of compounds 11eef,
these might be explained by the possibility that higher lipophilic
compounds were unfavorable for being delivered to the binding
sites. So suitable lipophilicity is necessary for good antimicrobial
activities in drug design.
0.8
0.6
0.4
0.2
Compound 11d only
0.0
330
360
390
420
450
Wavelength (nm)
Fig. 2. Emission spectra of BSA in the presence of various concentrations of compound
11d. c(BSA) 1.0 ꢂ 10ꢀ5 M; c(compound 11d)/(10ꢀ5 M), ael: from 0.0 to 5.5 at incre-
ments of 0.50; blue line shows the emission spectrum of compound 11d only;
The molar refractivity (MR), describing steric effects, represents
size and polarizability of a molecule and has been calculated to
T ¼ 298 K,
l
ex ¼ 295 nm. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
169
Table 4
10
SterneVolmer quenching constants for the interaction of compound 11d with BSA at
T = 310 K
T = 304 K
T = 298 K
various temperatures.
pH
7.4
T (K)
10ꢀ4 KSV (L/mol)
Ra
S.D.b
8
6
4
2
295
304
310
3.325
3.160
3.080
0.999
0.999
0.998
0.024
0.030
0.031
Ra is the correlation coefficient. S.D.b is standard deviation.
explain the activity behavior of the synthesized compounds
(Table 3). The para-chlorobenzyl substituted benzimidazole 12d
was more active against most of the tested strains than the ortho-
chlorobenzyl substituted 12f because the ortho-substitution on
benzene ring could easily generate steric hindrance. For this reason,
ortho-substituted compound is less active than the corresponding
para-substituted one on same nucleus.
0.0
0.4
0.8
1.2
-1
1.6
2.0
10-5 [Q] (L/mol)
2.4. Binding discussion
Fig. 5. Modified SterneVolmer plots of compound 11deBSA system at different
temperatures.
2.4.1. Quenching mechanism
In this experiment, the concentration of BSA solution was
1.0 ꢂ 10ꢀ5 M, and the concentrations of compound 11d solution
increased progressively from 0 to 5.5 ꢂ 10ꢀ5 M at an increment of
0.5 ꢂ 10ꢀ5 M. The effect of compound 11d on fluorescence of BSA
intensity at 298 K was shown in Fig. 2. It was observed that
a gradual decrease of the fluorescence intensity was caused by the
increment of compound 11d, accompanied with the red shift of
wavelength (from 345 to 352 nm) in the albumin spectrum. In
Fig. 2, the blue line showed the only emission spectrum of
compound 11d, which indicated that compound 11d did not posses
significant fluorescence features, thus at the excitation wavelength
(295 nm), the effect of compound 11d on fluorescence of BSA would
be negligible.
In this equation the F0 and F represent steady-state fluorescence
intensities in the absence and presence of compound 11d, respec-
tively. KSV is the SterneVolmer quenching constant, and [Q] is the
concentration of compound 11d. Hence, KSV was calculated by
linear regression of plot of F0/F versus [Q] (Fig. 3).
Quenching mechanisms are often classified as dynamic
quenching, static quenching etc. depending on temperature and
viscosity. Because higher temperatures result in larger diffusion
coefficients, the quenching constants are expected to increase with
a
gradually increasing temperature in dynamic quenching.
However, the increasing of temperature is likely to result in
a smaller static quenching constant due to the dissociation of
weakly bound complexes [44].
Therefore, the KSV was calculated from SterneVolmer plots at
each temperature. The result (Table 4) demonstrated that the
quenching constant KSV was inversely correlated with temperature,
which indicated that the probable quenching mechanism of
compound 11deBSA binding reaction was initiated by ground-state
complex formation (static quenching).
The decreased fluorescence intensity is usually described by the
SterneVolmer [43] equation:
F0
F
¼ 1 þ KSV½Qꢃ
(1)
0.42
0.39
0.36
0.33
0.30
0.27
0.24
0.21
In order to reconfirm that the probable quenching mechanism
of fluorescence of BSA by compound 11d was mainly initiated by
complex formation, the difference absorption spectroscopy was
employed. The UVevis absorption spectrum of BSA and the
difference absorption spectrum between BSAecompound 11d
complex and compound 11d at the same concentration could not
be superposed (Fig. 4), this result suggested that the probable
quenching mechanism of fluorescence of BSA by compound 11d
was mainly a static quenching procedure [34].
B
A
3
C
A
2
250 260 270 280 290 300
1
D
C
2.4.2. Binding constant and site
For a static quenching procedure, the data was analyzed
according to the Modified SterneVolmer [45] equation:
0
Table 5
200
250
300
350
Binding constant and sites of compound 11deBSA interaction at pH ¼ 7.4
Wavelength (nm)
T (K)
Modified SterneVolmer
Scatchard method
method
Fig. 4. UVevis spectra of BSA in the presence of compound 11d: A, absorption spec-
trum of BSA only; B, absorption spectrum of compound 11d/BSA 1:1 complex; C,
difference between absorption spectrum of compound 11d/BSA 1:1 complex and
compound 11d; D, absorption spectrum of compound 11d only. c(BSA) ¼ c(compound
11d) ¼ 1.0 ꢂ 10ꢀ5 M. The curve A and C for the wavelength ranging from 250 to 300 nm
are depicted in the inset.
10ꢀ4 Ka/(L/mol)
Ra
10ꢀ4 Kb (L/mol)
Ra
n
298
304
310
3.584
2.564
1.803
0.998
0.999
0.999
3.335
2.641
1.953
0.993
0.997
0.991
0.997
1.08
1.26

170
S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
Table 6
Thermodynamic parameters of compound 11deBSA interaction at different
temperatures.
0.28
0.24
0.20
0.16
0.12
T = 310 K
T = 304 K
T = 298 K
T (K)
DH (kJ/mol)
DG (kJ/mol)
D
S (J/mol K)
298
304
310
ꢀ43.96
ꢀ25.99
ꢀ25.63
ꢀ25.27
ꢀ60.30
The decreasing trend of Ka and Kb with increasing temperatures
was in accordance with KSV’s dependence on temperatures. The
value of the binding sites n was approximately 1, which showed the
interaction of compound 11d with BSA seemed to be the presence
of one high affinity binding site. Moreover, the interaction between
BSA and compound 11d accelerated with the temperature
increasing, hence the high affinity binding sites of the interaction
were slightly strengthened, which were accordance with the
results shown in Table 5. The results also showed that the binding
constant was moderate and the effect of temperature was not
significant, thus compound 11d might be stored and carried by this
protein [34].
0.1
0.2
0.3
0.4
r
0.5
0.6
0.7
Fig. 6. Scatchard plots of compound 11deBSA system at different temperatures.
F0
1
1
1
¼
þ
(2)
D
F
faKa ½Qꢃ fa
2.4.3. Binding mode and thermodynamic parameters
The interaction forces between bioactive small molecules or
drugs and biomolecules include electrostatic interactions, multiple
hydrogen bonds, van der Waals interactions, hydrophobic and
steric contacts within the antibody-binding site and so on. If the
enthalpy change (DH) does not vary significantly over the studied
temperature range, then its value and that of entropy change (DS)
D
F is the difference of fluorescence in the absence and presence
of compound 11d at concentration [Q], fa is the fraction of accessible
fluorescence, and Ka is the effective quenching constant for the
accessible fluorophores. The dependence of F0/DF on the reciprocal
value of concentration [Q]ꢀ1 is linear with the slope equaling to the
value of (faKa)ꢀ1. The Modified Stern-Volmer plots were Fig. 5 and
the calculated results were depicted in Table 5.
can be evaluated from the van’t Hoff equation:
When small molecules bind to a set of equivalent sites on
macromolecule, the equilibrium binding constant and the
numbers of binding sites can also be calculated according to the
Scatchard equation [46]:
D
RT
H
D
S
R
a
lnK ¼ ꢀ
þ
(4)
K is analogous to the associative binding constants at the cor-
responding temperature and R is the gas constant. In order to
explain the interaction between compound 11d and BSA, the
thermodynamic parameters were calculated from the van’t Hoff
r
Df
¼ nKb ꢀ rKb
(3)
plots. The enthalpy change (DH) was estimated from the slope of
Df is the molar concentration of free small molecules, r is the
the van’t Hoff relationship (Fig. 7). The free energy change (
DG) was
moles of small molecules bound per mole of protein, n is binding
sites multiplicity per class of binding sites, and Kb is the equilibrium
binding constant. The Scatchard plots were Fig. 6 and the Kb and n
were listed in Table 5.
then calculated from the following equation:
D
G ¼
D
H ꢀ T
D
S
(5)
Figs. 5 and 6 showed the Modified SterneVolmer and Scatchard
plots for the compound 11deBSA system at different temperatures.
Table 6 summarized the values of
D
H, DG and DS. The negative
values of free energy G suggested the binding process was spon-
D
taneous, the negative entropy S and enthalpy H values of the
D
D
interaction of compound 11d with BSA indicated that the binding
was mainly enthalpy-driven and the entropy was unfavorable for it,
moreover, hydrogen bonds and van der Waals forces played a major
role in the reaction [47].
InKa = -7.2523 + 5288/T
R = 0.999
10.4
10.2
10.0
9.8
3. Conclusion
In conclusion, a series of novel benzimidazole derivatives were
designed and synthesized in good yields via an easy, convenient and
efficient synthetic route starting from commercially available chlor-
oacetonitrile, and all the new compounds were characterized by 1H
NMR, 13C NMR, MS, IR and HRMS spectra. The in vitro antimicrobial
activities of these benzimidazole derivatives and their intermediates
were evaluated against nine bacterial strains and two fungal strains.
The biological results revealed that the intermediates 7 and 8
exhibited poor or no biological activities, while most of newly
synthesized compounds displayed moderate to excellent antibacte-
rial and antifungal activities against all the tested strains compared
to the reference drugs Chloromycin, Norfloxacin and Fluconazole.
3.24
3.27
3.30
3.33
3.36
1000/T K-1
Fig. 7. Van’t Hoff plots of compound 11deBSA system.

S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
171
Compounds 11aef with alkyl chains of different lengths gave better
antimicrobial efficiency than compounds 12aef incorporated hal-
obenzyl groups. Especially bis-benzimidazole compound 11d
exhibited the best bacterial growth inhibitory activities against
3289, 3238 (NH2), 2961, 2926 (CH3), 1631 (C]O), 1607, 1493, 1463
(aromatic frame), 1298, 1088, 863, 671; 1H NMR (300 MHz, DMSO-
d6):
d 7.82 (s, 2H, CONH), 4.52 (s, 4H, NH2), 2.54 (s, 6H, CH3) ppm;
MS (ESI): m/z 229 [M þ H]þ.
MRSA (MIC ¼ 2
m
g/mL), E. coli (MIC ¼ 4
mg/mL), P. aeruginosa
(MIC ¼ 4 g/mL), 8 or 16-fold to Chloromycin. The transformation of
m
4.1.3. 2-(Chloromethyl)-1H-benzoimidazole (8)
Compound 8 was prepared according to the literature procedure
[51]. Yield: 89.3%; m.p.: 218e219 ꢄC.
compounds 11a, 11d and 12c into water-soluble hydrochlorides
improved their antimicrobial activities. The specific interaction of
compound 11d with BSA was studied by fluorescence and UVevis
absorption spectroscopy. The experimental results displayed that
the quenching mechanism of fluorescence of BSA by compound 11d
was a static quenching procedure, binding constant and binding sites
were obtained according to the classical equations. The thermody-
namic parameters indicated the binding process was spontaneous,
2
5
4.1.4. N0 ,N0 -Bis((1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (9)
A mixture of compound 7 (114 mg, 0.5 mmol), potassium
carbonate (1.4 g, 10 mmol) and compound 8 (216 mg, 1.3 mmol) in
ethanol (25 mL) was stirred under reflux. After the reaction was
completed (monitored by TLC, eluent, petroleum/ethyl acetate, 1/1,
V/V), the mixture was cooled to room temperature, then the solvent
was evaporated under vacuum, and water (30 mL) was added. The
resulting mixture was extracted with dichloromethane (3 ꢂ 20 mL),
then the combined organic phase was dried over anhydrous
sodium sulfate and subsequently the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluent, petroleum/ethyl acetate, 2/1, V/V) to give
the compound 9 (129 mg) as yellow solid. Yield: 53.3%; m.p.:
the negative entropy DS and enthalpy DH values of the interaction of
compound 11d and BSA suggested that the binding should be mainly
enthalpy-driven and the entropy was unfavorable for it, moreover,
hydrogen bonds and van der Waals forces played major roles in the
interaction.
4. Experimental protocols
4.1. General methods
143e144 ꢄC; IR (KBr, cmꢀ1
2925 (CH3), 1653 (C]O), 1614 (C]N), 1515, 1457 (aromatic frame),
1301, 1163, 969, 846, 670; 1H NMR (300 MHz, CDCl3):
7.62 (d,
) n: 3431, 3292 (NH), 3026 (AreH), 2967,
Melting points were uncorrected and recorded on X-6 melting
point apparatus. TLC analysis was done using pre-coated silica gel
plates. FT-IR spectra were carried out on Bruker RFS100/S spec-
trophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in
the 400e4000 cmꢀ1 range. NMR spectra were recorded on a Bruker
AV 300 spectrometer using TMS as an internal standard. The
chemical shifts were reported in parts per million (ppm), the
coupling constants (J) were expressed in hertz (Hz) and signals
were described as singlet (s), doublet (d), triplet (t), as well as
multiplet (m). The following abbreviations were used to designate
aryl groups: Thio, thiophenyl; Benim, benzimidazolyl; Ph, phenyl.
The mass spectra were recorded on LCMS-2010A and the high-
resolution mass spectra (HRMS) were recorded on an IonSpec FT-
ICR mass spectrometer with ESI resource. All fluorescence spectra
were recorded on F-2700 Spectrofluorimeter (Hitachi, Tokyo,
Japan) equipped with 1.0 cm quartz cells, the widths of both the
excitation and emission slit were set as 2.5 nm, and the excitation
wavelength was 295 nm. Fluorescence spectra were recorded at
298, 304, 310 K in the range of 300e450 nm. The UV spectrum was
recorded at room temperature on a TU-2450 spectrophotometer
(Puxi Analytic Instrument Ltd. of Beijing, China) equipped with
1.0 cm quartz cells. BSA was obtained from SigmaeAldrich (St.
Louis, MO, USA). Tris, NaCl, HCl, ethanol were analytical purity. BSA
was dissolved in TriseHCl buffer solution (0.05 M Tris, 0.15 M NaCl,
pH ¼ 7.4), compound 11d was dissolved in ethanol [48]. Sample
masses were weighed on a microbalance with a resolution of
0.1 mg. All other chemicals and solvents were commercially avail-
able, and were used without further purification.
d
J ¼ 3.0 Hz, 4H, benim 4,7-H), 7.28 (d, J ¼ 4.7 Hz, 4H, benim 5,6-H),
6.98 (bs, 4H, benim 1-H, CONH), 4.81 (s, 4H, CH2), 3.48 (s, 6H, CH3),
2.17 (NH) ppm; 13C NMR (75 MHz, CDCl3):
d 150.04 (C]O), 142.12
(thio 2,5-C), 135.21 (benim 2-C), 122.71 (benim 8,9-C), 121.90
(benim 4,7-C),119.82 (benim 5,6-C),109.76 (thio 3,4-C), 67.74 (CH2),
45.56 (CH3); MS (ESI): m/z 489 [M þ H]þ; HRMS (ESI) calcd. for
C24H24N8O2S [M þ H]þ, 489.1821; found, 489.1825.
2
4.1.5. N0 -((1H-Benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (10)
Compound 10 was prepared according to the experimental
procedure described for compound 9, starting from thiophene
derivative 7 (114 mg, 0.5 mmol), potassium carbonate (1.4 g,
10 mmol) and compound 8 (216 mg, 1.3 mmol). The desired
compound 10 (32 mg) was obtained as slight yellow solid. Yield:
19.2%; m.p.: 203e204 ꢄC; IR (KBr, cmꢀ1
) n: 3407 (CONH), 3280,
3207 (NH2), 3053 (AreH), 2973, 2926 (CH3), 1654 (C]O), 1613 (C]
N), 1504, 1451 (aromatic frame), 1294, 1163, 973, 867, 664; 1H NMR
(300 MHz, CDCl3):
d
7.89 (d, J ¼ 7.0 Hz, 1H, CONHNH2), 7.42 (d,
J ¼ 8.1 Hz, 2H, benim 4,7-H), 7.31e7.26 (m, 2H, benim 5,6-H), 6.98 (s,
1H, CONHNHCH2), 5.53 (s, 2H, NH2), 4.89 (s, 2H, CH2), 3.44 (s, 3H,
thio 3-CH3), 2.19 (s, 3H, thio 4-CH3), 2.17 (s, 1H, NH) ppm; 13C NMR
(75 MHz, CDCl3):
d 152.29 (CONHNH2), 150.14 (CONHNH), 143.56
(thio 2-C), 142.33 (thio 5-C), 135.26 (benim 2-C), 122.77 (benim 8,9-
C), 121.85 (benim 4,7-C), 119.86 (benim 5,6-C), 111.52 (thio 3-C),
109.76 (thio 4-C), 67.72 (CH2), 45.51 (thio 4-CH3), 44.89 (thio 3-
CH3); MS (ESI): m/z 359 [M þ H]þ; HRMS (ESI) calcd. for
C16H18N6O2S [M þ H]þ, 359.1290; found, 359.1293.
4.1.1. Synthesis of compounds 3e6
Compounds 3e6 were prepared according to the literature
procedures [49,50] (Supplementary Information 3).
2
5
4.1.6. N0 ,N0 -Bis((1-propyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11a)
4.1.2. 3,4-Dimethylthiophene-2,5-dicarbohydrazide (7)
A mixture of compound 8 (200 mg, 0.4 mmol), potassium
carbonate (1.4 g, 10 mmol) and 1-bromopropane (154 mg,
1.2 mmol) in ethanol was reflux for 5 h. After the reaction was
completed (eluent, petroleum/ethyl acetate, 4/1, V/V), the mixture
was cooled to room temperature. The reaction solvent was evap-
orated, and then water (30 mL) was added. The resulting mixture
was extracted with dichloromethane (3 ꢂ 20 mL), the combined
organic phase was dried over anhydrous sodium sulfate and
A mixture of compound 6 (2.3 g, 10 mmol) and excessive
hydrazine hydrate (6 mL) in methanol (50 mL) was stirred under
reflux for 5 h. Upon completion of the reaction, the mixture was
cooled to room temperature, white solid was collected by filtration
and washed with cold methanol. The crude product was recrys-
tallized from methanol to afford compound 7 (1.5 g) as white
crystal. Yield: 68.3%; m.p.: 212e215 ꢄC; IR (KBr, cmꢀ1): 3419 (NH),

172
S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
subsequently the solvent was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
(eluent, petroleum/ethyl acetate, 8/1, V/V) to give the desired
(223 mg) was obtained as yellow oil. Yield: 72.5%; IR (KBr, cmꢀ1
) n:
3428, 3305 (NH), 3037 (AreH), 2925, 2823 (CH3), 1659 (C]O), 1615
(C]N), 1510, 1466 (aromatic frame), 1370, 1094, 1008, 959, 739; 1H
compound 11a (193 mg) as yellow oil. Yield: 84.4%; IR (KBr, cmꢀ1
)
n
:
NMR (300 MHz, CDCl3):
d
7.76 (d, J ¼ 6.8 Hz, 2H, benim 4-H), 7.37 (d,
3421, 3299 (NH), 3054 (AreH), 2921, 2877 (CH3), 1664 (C]O), 1615
(C]N),1508,1466 (aromatic frame),1295,1091,1006, 917, 858, 732;
J ¼ 7.2 Hz, 2H, benim 7-H), 7.32e7.22 (m, 4H, benim 5,6-H), 4.76 (s,
4H, benim 2-CH2), 4.22 (t, J ¼ 7.5 Hz, 4H, benim 1-CH2), 3.40 (s, 6H,
thio-CH3), 2.07 (CONHNH), 1.85e1.83 (m, 4H, benim 1-CH2CH2),
1.34e1.26 (m, 28H, benim 1-CH2CH2(CH2)7), 0.89e0.87 (m, 6H,
1H NMR (300 MHz, CDCl3):
d
7.77 (d, J ¼ 6.9 Hz, 2H, benim 4-H), 7.37
(d, J ¼ 7.1 Hz, 2H, benim 7-H), 7.32e7.20 (m, 4H, benim 5,6-H), 4.76
(s, 4H, benim 2-CH2), 4.20 (t, J ¼ 7.3 Hz, 4H, benim 1-CH2), 3.39 (s,
6H, thio-CH3), 2.32 (CONHNH), 1.85e1.92 (m, 4H, CH2), 0.99 (t,
CH3) ppm; 13C NMR (75 MHz, CDCl3):
d 150.10 (C]O), 142.19 (thio
2,5-C),135.47 (benim 2-C),122.85 (benim 8,9-C), 121.99 (benim 4,7-
C), 119.95 (benim 5,6-C), 109.74 (thio 3,4-C), 67.17 (NHCH2), 58.17
(benim 1-CH2), 44.12 (thio-CH3), 31.79 (CH2), 29.81 (CH2), 29.43
(CH2), 29.40 (CH2), 29.21 (CH2), 29.17 (CH2), 26.92 (CH2), 22.60
(CH2), 14.05 (CH3); MS (ESI): m/z 769 [M þ H]þ; HRMS (ESI) calcd.
for C44H64N8O2S [M þ H]þ, 769.4951; found, 769.4956.
J ¼ 7.3 Hz, 6H, CH3) ppm; 13C NMR (75 MHz, CDCl3):
d 150.12 (C]O),
142.14 (thio 2,5-C),135.48 (benim 2-C),122.84 (benim 8,9-C),121.98
(benim 4,7-C), 119.89 (benim 5,6-C), 109.78 (thio 3,4-C), 67.08
(NHCH2), 58.14 (benim 1-CH2), 45.56 (thio-CH3), 23.08 (CH2),
11.34(CH3); MS (ESI): m/z 573 [M þ H]þ; HRMS (ESI) calcd. for
C30H36N8O2S [M þ H]þ, 573.2760; found, 573.2766.
2
5
4.1.10. N0 ,N0 -Bis((1-dodcyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11e)
2
5
4.1.7. N0 ,N0 -Bis((1-hexyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11b)
Compound 11e was prepared according to the procedure depic-
Compound 11b was prepared according to the experimental
procedure for compound 11a, starting from compound 8 (75 mg,
0.15 mmol), 1-bromohexane (66 mg, 0.4 mmol) and potassium
carbonate (1.4 g, 10 mmol). The desired compound 11b (72 mg) was
ted for compound 11a, starting from compound 8 (75 mg,
0.15 mmol), 1-bromododecane (100 mg, 0.4 mmol) and potassium
carbonate (1.4 g, 10 mmol). The product 11e (96 mg) was obtained as
yellow oil. Yield: 78.3%; IR (KBr, cmꢀ1
) n: 3426, 3294 (NH), 3051
obtained as yellow oil. Yield: 73.2%; IR (KBr, cmꢀ1
)
n
: 3423, 3298
(AreH), 2924, 2853 (CH3), 1652 (C]O), 1610 (C]N), 1508, 1463
(NH), 3053 (AreH), 2925, 2854 (CH3), 1659 (C]O), 1615 (C]N),
(aromatic frame), 1374, 1091, 1007, 960, 738; 1H NMR (300 MHz,
1510, 1464 (aromatic frame), 1374, 1091, 1008, 959, 739; 1H NMR
CDCl3):
d
7.76 (d, J ¼ 6.8 Hz, 2H, benim 4-H), 7.37 (d, J ¼ 7.2 Hz, 2H,
(300 MHz, CDCl3):
d
7.76 (d, J ¼ 7 Hz, 2H, benim 4-H), 7.34 (d,
benim 7-H), 7.31e7.24 (m, 4H, benim 5,6-H), 4.76 (s, 4H, benim 2-
CH2), 4.22 (t, J ¼ 7.4 Hz, 4H, benim 1-CH2), 3.40 (s, 6H, thio-CH3),
2.28 (CONHNH), 1.83e1.85 (m, 4H, benim 1-CH2CH2), 1.28e1.10 (m,
36H, benim 1-CH2CH2(CH2)9), 0.90e0.88 (m, 6H, CH3) ppm; 13C NMR
J ¼ 7.6 Hz, 2H, benim 7-H), 7.32e7.22 (m, 4H, benim 5,6-H), 4.75 (s,
4H, benim 2-CH2), 4.21 (t, J ¼ 7.1 Hz, 4H, benim 1-CH2), 3.39 (s, 6H,
thio-CH3), 2.28 (CONHNH), 1.85e1.78 (m, 4H, benim 1-CH2CH2),
1.33e1.26 (m, 12H, benim 1-CH2CH2(CH2)3), 0.88e0.86 (m, 6H, CH3)
(75 MHz, CDCl3): d 150.10 (C]O), 142.29 (thio 2,5-C), 136.34 (benim
ppm; 13C NMR (75 MHz, CDCl3):
d
150.10 (C]O), 142.03 (thio 2-C),
2-C), 122.87 (benim 8,9-C), 122.03 (benim 4,7-C), 119.99 (benim 5,6-
C), 109.73 (thio 3,4-C), 67.19 (NHCH2), 58.17 (benim 1-CH2), 44.14
(thio-CH3), 31.90 (CH2), 31.87 (CH2), 29.84 (CH2), 29.68 (CH2), 29.57
(CH2), 29.50 (CH2), 29.43 (CH2), 29.30 (CH2), 26.94 (CH2), 22.65 (CH2),
14.08 (CH3); MS (ESI): m/z 826 [M þ H]þ; HRMS (ESI) calcd. for
C48H72N8O2S [M þ H]þ, 825.5577; found, 825.5579.
135.55 (benim 2-C), 122.81 (benim 8,9-C), 122.03 (benim 4,7-C),
119.93 (benim 5,6-C), 109.82 (thio 3,4-C), 67.14 (NHCH2), 58.11
(benim 1-CH2), 45.50 (thio-CH3), 31.88 (CH2), 29.77 (CH2), 29.42
(CH2) 23.08 (CH2), 11.35 (CH3); MS (ESI): m/z 657 [M þ H]þ; HRMS
(ESI) calcd. for C36H48N8O2S [M þ H]þ, 657.3699; found, 657.3694.
2
5
2
5
4.1.8. N0 ,N0 -Bis((1-octyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11c)
4.1.11. N0 ,N0 -Bis((1-octadeyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11f)
Compound 11c was prepared according to the experimental
procedure described for compound 11a, starting from compound 8
(50 mg, 0.1 mmol),1-bromooctane (58 mg, 0.3 mmol) and potassium
carbonate (1.4 g, 10 mmol). The desired compound 11c (54 mg) was
Compound 11f was prepared according to the experimental
procedure described for compound 11a, starting from compound 8
(75 mg, 0.15 mmol), 1-bromooctadecane (132 mg, 0.4 mmol) and
potassium carbonate (1.4 g, 10 mmol). The desired compound 11f
obtained as yellow oil. Yield: 76.3%; IR (KBr, cmꢀ1
)
n: 3425, 3296 (NH),
(102 mg) was obtained as yellow oil. Yield: 69.2%; IR (KBr, cmꢀ1
) n:
3062 (AreH), 2923, 2850 (CH3), 1657 (C]O), 1613 (C]N), 1510, 1462
3429, 3292 (NH), 3053 (AreH), 2923, 2852 (CH3), 1651 (C]O), 1612
(aromatic frame), 1365, 1088, 1010, 950, 734; 1H NMR (300 MHz,
(C]N), 1509, 1464 (aromatic frame), 1373, 1092, 1008, 960, 739; 1H
CDCl3):
d
7.72 (d, J ¼ 6.8 Hz, 2H, benim 4-H), 7.37 (d, J ¼ 7.4 Hz, 2H,
NMR (300 MHz, CDCl3):
d
7.75 (d, J ¼ 7.2 Hz, 2H, benim 4-H), 7.37 (d,
benim 7-H), 7.33e7.24 (m, 4H, benim 5,6-H), 4.76 (s, 4H, benim 2-
CH2), 4.22 (t, J ¼ 7.5 Hz, 4H, benim 1-CH2), 3.37 (s, 6H, thio-CH3),
2.19 (CONHNH), 1.84e1.77 (m, 4H, benim 1-CH2CH2), 1.42e1.18 (m,
20H, benim 1-CH2CH2(CH2)5), 0.88e0.86 (m, 6H, CH3) ppm; 13C NMR
J ¼ 7.6 Hz, 2H, benim 7-H), 7.30e7.23 (m, 4H, benim 5,6-H), 4.75 (s,
4H, benim 2-CH2), 4.22 (t, J ¼ 7.4 Hz, 4H, benim 1-CH2), 3.39 (s, 6H,
thio-CH3), 2.27 (CONHNH), 1.84e1.82 (m, 4H, benim 1-CH2CH2),
1.26 (m, 40H, benim 1-CH2CH2 (CH2)10), 0.86e0.85 (d, 26H,
(75 MHz, CDCl3):
d
150.12 (C]O),141.98 (thio 2,5-C),135.49 (benim 2-
(CH2)5CH3) ppm; 13C NMR (75 MHz, CDCl3):
d 150.10 (C]O), 141.91
C), 122.79 (benim 8,9-C), 122.10 (benim 4,7-C), 119.89 (benim 5,6-C),
109.77 (thio 3,4-C), 67.10 (NHCH2), 58.09 (benim 1-CH2), 45.53 (thio-
CH3), 31.77 (CH2), 29.70 (CH2), 29.67 (CH2), 29.60 (CH2), 29.56 (CH2),
23.08 (CH2), 11.36 (CH3); MS (ESI): m/z 713 [M þ H]þ; HRMS (ESI)
calcd. for C40H56N8O2S [M þ H]þ, 713.4325; found, 713.4326.
(thio 2,5-C), 135.45 (benim 2-C), 122.81 (benim 8,9-C), 122.14
(benim 4,7-C), 119.91 (benim 5,6-C), 109.74 (thio 3,4-C), 67.13
(NHCH2), 58.11 (benim 1-CH2), 45.55 (thio-CH3), 31.81 (CH2), 29.74
(CH2),
29.60
(CH2),
29.56
(CH2),
29.52e29.11
(CH2CH2CH2CH2CH2CH2CH2), 23.22 (CH2), 23.18 (CH2), 22.88
(CH2), 22.72 (CH2), 14.16 (CH3); MS (ESI): m/z 994 [M þ H]þ; HRMS
(ESI) calcd. for C60H96N8O2S [M þ H]þ, 993.7455; found, 993.7458.
2
5
4.1.9. N0 ,N0 -Bis((1-decyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide (11d)
2
5
Compound 11d was prepared according to the experimental
procedure described for compound 11a, starting from compound 8
(200 mg, 0.4 mmol), 1-bromodecane (266 mg, 1.2 mmol) and
potassium carbonate (1.4 g, 10 mmol). The desired compound 11d
4.1.12. N0 ,N0 -Bis((1-(3,4-dichlorobenzyl)-1H-benzoimidazole-2-
yl)methyl)-3,4-dimethylthiophene-2,5-dicarbohydrazide (12a)
A mixture of compound 8 (50 mg, 0.1 mmol), potassium
carbonate (1.4 g, 10 mmol) and 3,4-dichlorobenzyl chloride (78 mg,

S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
173
0.4 mmol) in ethanol was stirred under reflux for 12 h. After the
reaction was completed, the mixture was cooled to room temper-
ature, and the solvent was evaporated, then water (30 mL) was
added. The resulting mixture was extracted with dichloromethane
(3 ꢂ 20 mL), the combined organic phase was dried over anhydrous
sodium sulfate and subsequently the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluent, petroleum/ethyl acetate, 4/1, V/V) to give
compound 12a (52 mg) as light yellow solid. Yield: 64.7%; m.p.:
FPh 2,6-C), 123.34 (benim 8,9-C), 122.41 (benim 4,7-C), 120.11
(benim 5,6-C), 115.62, 115.91 (4-FPh 3,5-C), 109.92 (thio 3,4-C),
67.31 (NHCH2), 58.35 (benim 1-CH2), 46.71 (thio-CH3); MS (ESI): m/
z 705 [M þ H]þ; HRMS (ESI) calcd. for C38H34F2N8O2S [M þ H]þ,
705.2572; found, 705.2577.
2
5
4.1.15. N0 ,N0 -Bis((1-(4-chlorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarbohydrazide (12d)
Compound 12d was prepared according to the procedure
depicted for compound 12a, starting from compound 8 (100 mg,
0.2 mmol), potassium carbonate (1.4 g, 10 mmol) and
4-chlorobenzyl chloride (81 mg, 0.5 mmol). The product 12d
(105 mg) was obtaineꢀd1as slight yellow solid. Yield: 71.2%; m.p.:
103e104 ꢄC; IR (KBr, cmꢀ1
) n: 3421, 3297 (NH), 3055, 3005 (AreH),
2923, 2823 (CH3), 1631 (C]O), 1611 (C]N), 1518, 1462 (aromatic
frame), 1366, 1221, 1094, 1031, 961, 832, 762, 742; 1H NMR
(300 MHz, CDCl3):
d
7.80 (d, J ¼ 7.1 Hz, 2H, benim 4-H), 7.37 (d,
J ¼ 8.2 Hz, 2H, benim 7-H), 7.30e7.26 (m, 4H, benim 5,6-H), 7.36 (d,
J ¼ 7.5 Hz, 2H, 3,4-Cl2Ph 5-H), 7.21 (d, J ¼ 3 Hz, 2H, 3,4-Cl2Ph 2-H),
6.93 (d, J ¼ 8.2 Hz, 2H, 3,4-Cl2Ph 6-H), 5.43 (s, 4H, benim 1-CH2),
4.72 (s, 4H, benim 2-CH2), 3.38 (s, 6H, thio-CH3), 2.17 (CONHNH)
84e85 ꢄC; IR (KBr, cm
) n: 3420, 3299 (NH), 3048, (AreH), 2929,
2820 (CH3), 1636 (C]O), 1614 (C]N), 1492, 1446 (aromatic frame),
1221, 1085, 962, 852, 749; 1H NMR (300 MHz, CDCl3):
d
7.80 (d,
J ¼ 6.2 Hz, 2H, benim 7-H), 7.28 (s, 2H, benim 4-H), 7.25 (m, 4H,
benim 5,6-H), 7.23 (m, 4H, 4-ClPh 3,5-H), 7.04 (d, J ¼ 7.5 Hz, 4H, 4-
ClPh 2,6-H), 5.44 (s, 4H, benim 1-CH2), 4.70 (s, 4H, benim 2-CH2),
3.37 (s, 6H, thio-CH3), 2.26 (CONHNH) ppm; 13C NMR (75 MHz,
ppm; 13C NMR (75 MHz, CDCl3):
d 150.65 (C]O), 142.29 (thio 2,5-
C), 136.34 (3,4-Cl2Ph 3-C), 135.48 (benim 2-C), 133.07 (3,4-Cl2Ph 4-
C), 132.02 (3,4-Cl2Ph 2-C), 130.82 (3,4-Cl2Ph 5-C), 128.55 (3,4-Cl2Ph
1-C), 125.83 (3,4-Cl2Ph 6-C), 123.54 (benim 8,9-C), 122.61 (benim
4,7-C), 120.29 (benim 5,6-C), 109.69 (thio 3,4-C), 67.39 (NHCH2),
58.41 (benim 1-CH2), 46.35 (thio-CH3); MS (ESI): m/z 805 [M þ H]þ;
HRMS (ESI) calcd. for C38H32Cl4N8O2S [M þ H]þ, 805.1201; found,
805.1205.
CDCl3):
d 150.40 (C]O), 142.18 (thio 2,5-C), 135.59 (benim 2-C),
134.55 (4-ClPh 4-C), 133.67 (4-ClPh 3,5-C), 129.01 (4-ClPh 1-C),
127.87 (4-ClPh 2,6-C), 123.40 (benim 8,9-C), 122.48 (benim 4,7-C),
120.14 (benim 5,6-C), 109.87 (thio 3,4-C), 67.30 (NHCH2), 58.38
(benim 1-CH2), 46.75 (thio-CH3); MS (ESI): m/z 737 [M þ H]þ;
HRMS (ESI) calcd. for C38H34Cl2N8O2S [M þ H]þ, 737.1981; found,
737.1983.
2
5
4.1.13. N0 ,N0 -Bis((1-(2,4-difluorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarbohydrazide (12b)
2
5
Compound 12b was prepared according to the procedure
depicted for compound 12a, starting from compound 8 (100 mg,
0.2 mmol), potassium carbonate (1.4 g, 10 mmol) and 2,4-
difluorobenzyl bromide (103 mg, 0.5 mmol). The product (98 mg)
was obtained as slight yellow solid. Yield: 66.2%; m.p.: 80e81 ꢄC; IR
4.1.16. N0 ,N0 -Bis((1-(3-chlorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarbohydrazide (12e)
Compound 12e was prepared according to the procedure
depicted for compound 12a, starting from compound 8 (150 mg,
0.3 mmol), potassium carbonate (1.4 g, 15 mmol) and
3-chlorobenzyl chloride (122 mg, 0.76 mmol). The product 12e
(153 mg) was obtained as slight yellow solid. Yield: 68.6%; m.p.:
(KBr, cmꢀ1
)
n
: 3419, 3289 (NH), 3079, 3010 (AreH), 2924, 2821
(CH3), 1630 (C]O), 1610 (C]N), 1504, 1465 (aromatic frame), 1227,
1090, 963, 860, 745; 1H NMR (300 MHz, CDCl3):
d
7.79 (d, J ¼ 5.8 Hz,
93e94 ꢄC; IR (KBr, cmꢀ1
)
n
: 3431, 3302 (NH), 3073, 3036 (AreH),
2937, 2826 (CH3), 1632 (C]O), 1610 (C]N), 1508, 1451 (aromatic
frame), 1220, 1014, 960, 861, 743; 1H NMR (300 MHz, CDCl3):
7.80
2H, benim 4-H), 7.27 (m, 6H, benim 5,6,7-H), 6.88 (d, J ¼ 4.5 Hz, 2H,
2,4-F2Ph 6-H), 6.82 (m, 2H, 2,4-F2Ph 5-H), 6.75 (m, 2H, 2,4-F2Ph 3-
H), 5.49 (s, 4H, benim 1-CH2), 4.76 (s, 4H, benim 2-CH2), 3.36 (s, 6H,
thio-CH3), 2.17 (CONHNH) ppm; 13C NMR (75 MHz, CDCl3):
d
(d, J ¼ 6.8 Hz, 2H, benim 4-H), 7.29 (d, J ¼ 7.4 Hz, 2H, benim 7-H),
7.29e7.24 (m, 4H, benim 5,6-H), 7.24e7.19 (m, 4H, 3-ClPh 4,5-H),
7.13 (s, 2H, 3-ClPh 2-H), 6.96 (d, J ¼ 6.2 Hz, 2H, 3-ClPh 6-H), 5.45 (s,
4H, benim 1-CH2), 4.72 (s, 4H, benim 2-CH2), 3.38 (s, 6H, thio-CH3),
d
163.58, 163.44, 161.51, 161.30 (2,4-F2Ph 2-C), 161.21, 161.12, 159.11,
158.93 (2,4-F2Ph 4-C), 150.67 (C]O), 142.33 (thio 2-C), 135.52
(benim 2-C), 132.11, 132.03 (2,4-F2Ph 6-C), 123.55 (benim 8,9-C),
122.64 (benim 4,7-C),120.33 (benim 5,6-C),119.38,119.22 (2,4-F2Ph
1-C), 111.95, 111.65 (2,4-F2Ph 5-C), 109.66 (thio 3-C), 104.51, 104.33,
103.78 (2,4-F2Ph 3-C), 58.45 (NHCH2), 46.32 (thio-CH3), 67.50
(benim 1-CH2); MS (ESI): m/z 741 [M þ H]þ; HRMS (ESI) calcd. for
C38H32F4N8O2S [M þ H]þ, 741.2383; found, 741.2386.
2.04 (CONHNH) ppm; 13C NMR (75 MHz, CDCl3):
d 150.40 (C]O),
142.23 (thio 2,5-C), 138.13 (3-ClPh 3-C), 135.63 (benim 2-C), 134.80
(3-ClPh 1-C), 130.14 (3-ClPh 2-C), 126.68 (3-ClPh 6-C), 124.61 (3-
ClPh 5-C), 123.43 (benim 8,9-C), 122.49 (benim 4,7-C), 120.17
(benim 5,6-C), 1128.07 (3-ClPh 4-C), 109.83 (thio 3,4-C), 67.34
(NHCH2), 58.37 (benim 1-CH2), 46.75 (thio-CH3); MS (ESI): m/z 737
[M þ H]þ; HRMS (ESI) calcd. for C38H34Cl2N8O2S [M þ H]þ,
737.1981; found, 737.1986.
2
5
4.1.14. N0 ,N0 -Bis((1-(4-fluorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarb hydrazide (12c)
2
5
Compound 12c was prepared according to the experimental
procedure described for compound 12a, starting from compound 8
(100 mg, 0.2 mmol), potassium carbonate (1.4 g, 10 mmol) and 4-
fluorobenzyl chloride (73 mg, 0.5 mmol). The desired compound
12c (123 mg) was obtained as slight yellow solid. Yield: 87.3%; m.p.:
4.1.17. N0 ,N0 -Bis((1-(2-chlorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarbohydrazide (12f)
Compound 12f was prepared according to the procedure
depicted for compound 12a, starting from compound 8 (150 mg,
0.3 mmol), potassium carbonate (1.4 g, 10 mmol) and
2-chlorobenzyl chloride (122 mg, 0.76 mmol). The product 12f
(165 mg) was obtained as slight yellow solid. Yield: 75.2%; m.p.:
72e73 ꢄC; IR (KBr, cmꢀ1
)
n
: 3432, 3303 (NH), 3088, 3054 (AreH),
2930, 2824 (CH3), 1634 (C]O), 1613 (C]N), 1498, 1448 (aromatic
frame), 1223, 1092, 960, 825, 743; 1H NMR (300 MHz, CDCl3):
7.79
d
95e96 ꢄC; IR (KBr, cmꢀ1
)
n
: 3418, 3291 (NH), 3075, 3037 (AreH),
2930, 2831 (CH3), 1636 (C]O), 1613 (C]N), 1509, 1453 (aromatic
frame), 1220, 1003, 960, 854, 737; 1H NMR (300 MHz, CDCl3):
7.82
(d, J ¼ 7.3 Hz, 2H, benim 4-H), 7.26 (d, J ¼ 8.5 Hz, 2H, benim 7-H),
7.24 (m, 4H, benim 5,6-H), 7.09 (m, 4H, 4-FPh 3,5-H), 6.98 (m, 4H, 4-
FPh 2,6-H), 5.43 (s, 4H, benim 1-CH2), 4.70 (s, 4H, benim 2-CH2),
3.37 (s, 6H, thio-CH3), 2.27 (CONHNH) ppm; 13C NMR (75 MHz,
d
(d, J ¼ 7.6 Hz, 2H, benim 4-H), 7.44 (d, J ¼ 7.8 Hz, 2H, 2-ClPh 3-H),
7.30 (m, 2H, benim 7-H), 7.26e7.23 (m, 4H, benim 5,6-H), 7.21e7.19
(m, 2H, 2-ClPh 4-H), 7.06 (d, J ¼ 7.4 Hz, 2H, 2-ClPh 6-H), 6.52e6.49
(m, 2H, 2-ClPh 5-H), 5.57 (s, 4H, benim 1-CH2), 4.70 (s, 4H, benim
CDCl3):
d 163.87,160.60 (4-FPh 4-C),150.40 (C]O),142.20 (thio 2,5-
C), 135.63 (benim 2-C), 131.82, 131.78 (4-FPh 1-C), 128.32, 128.21 (4-

174
S.-L. Zhang et al. / European Journal of Medicinal Chemistry 55 (2012) 164e175
2-CH2), 3.36 (s, 6H, thio-CH3), 2.27 (CONHNH) ppm; 13C NMR
(75 MHz, CDCl3): 150.41 (C]O),142.26 (thio 2,5-C),135.64 (benim
4H, benim 1-CH2), 5.52 (s, 4H, benim 2-CH2), 3.46 (s, 6H, thio-CH3);
13C NMR (75 MHz, DMSO-d6):
163.90, 160.66 (4-FPh 4-C), 150.40
d
d
2-C), 133.60 (2-ClPh 2-C), 132.21 (2-ClPh 1-C), 129.60 (2-ClPh 3-C),
128.88 (2-ClPh 6-C), 127.21 (2-ClPh 4-C), 126.96 (2-ClPh 5-C),
123.43 (benim 8,9-C), 122.52 (benim 4,7-C), 120.18 (benim 5,6-C),
109.89 (thio 3,4-C), 67.24 (NHCH2), 58.44 (benim 1-CH2), 46.53
(thio-CH3); MS (ESI): m/z 737 [M þ H]þ; HRMS (ESI) calcd. for
C38H34Cl2N8O2S [M þ H]þ, 737.1981; found, 737.1985.
(C]O), 142.41 (benim 2-C), 142.35 (thio 2,5-C), 132.48 (4-FPh 1-C),
131.05, 131.01 (4-FPh 2,6-C), 126.58 (benim 8,9-C), 125.39 (benim
4,7-C), 117.25 (benim 5,6-C), 115.96, 115.39 (4-FPh 3,5-C), 113.67
(thio 3,4-C), 83.97 (NHCH2), 69.57 (benim 1-CH2), 46.85 (thio-CH3);
MS (ESI): m/z 705 [M þ H ꢀ 4HCl]þ; HRMS (ESI) calcd. for
C38H38Cl4F2N8O2S [M þ H ꢀ 4HCl]þ, 705.2572; found, 705.2574.
2
5
4.1.18. N0 ,N0 -Bis((1-propyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide hydrochloride (13a)
To a stirring solution of compound 11a (114 mg, 0.2 mmol) in
ethyl ether/chloroform (4/1, V/V) was added hydrochloric acid (4 M,
2 mL) dropwise at room temperature. The reactants were stirred for
12 h at 40 ꢄC. After the reaction was completed (monitored by TLC,
eluent, ethyl acetate/petroleum ether, 1/1, V/V), the formed
precipitate was filtered, and washed with chloroform, dried by
freeze drying oven to afford hydrochloride 13a (90 mg) as slight
Acknowledgments
This work was partially supported by National Natural Science
Foundation of China [No. 21172181, 81250110089 (The Research
Fellowship for International Young Scientists from International
(Regional) Cooperation and Exchange Program)], the key program
from
Natural
Science
Foundation
of
Chongqing
(CSTC2012jjB10026), the Specialized Research Fund for the
Doctoral Program of Higher Education of China (SRFDP
20110182110007) and the Research Funds for the Central Univer-
sities (XDJK2011D007, the key program XDJK2012B026).
yellow solid. Yield: 63.3%; m.p.: 105e106 ꢄC; IR (KBr, cmꢀ1
) n: 3436,
3309 (NH), 3059 (AreH), 2921, 2859 (CH3), 1672 (C]O), 1618 (C]
N), 1502, 1461 (aromatic frame), 1291, 1094, 1003, 905, 852, 731; 1H
NMR (300 MHz, DMSO-d6):
d
8.06 (d, J ¼ 5.6 Hz, 2H, benim 4-H),
Appendix A. Supplementary information
7.90e7.83 (m, 2H, benim 7-H), 7.65e7.57 (m, 4H, benim 5,6-H), 5.48
(s, 4H, benim 2-CH2), 4.73 (t, J ¼ 7.4 Hz, 4H, benim 1-CH2), 3.49 (s,
6H, thio-CH3), 1.89e1.82 (m, 4H, benim 1-CH2CH2), 0.95 (t,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.07.
015.
J ¼ 7.3 Hz, 6H, CH3) ppm; 13C NMR (75 MHz, DMSO-d6):
d 150.07
(C]O), 142.76 (benim 2-C), 142.55 (thio 2,5-C), 126.84 (benim 8,9-
C), 125.72 (benim 4,7-C), 118.04 (benim 5,6-C), 113.59 (thio 3,4-C),
83.78 (NHCH2), 69.56 (benim 1-CH2), 46.70 (thio-CH3), 22.59 (CH2),
11.24 (CH3); MS (ESI): m/z 573 [M þ H ꢀ 4HCl]þ; HRMS (ESI) calcd.
for C30H40Cl4N8O2S [M þ H ꢀ 4HCl]þ, 573.2760; found, 573.2765.
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4790e4794.
2
5
4.1.19. N0 ,N0 -Bis((1-decyl-1H-benzoimidazol-2-yl)methyl)-3,4-
dimethylthiophene-2,5-dicarbohydrazide hydrochloride (13b)
Compound 13b was prepared according to the procedure
depicted for compound 13a, starting from compound 11d (77 mg,
0.1 mmol). The product 13b (65 mg) was obtainedꢀa1s a slight yellow
solid. Yield: 71.4%; m.p.: 99e100 ꢄC; IR (KBr, cm
) n: 3431, 3306
(NH), 3052 (AreH), 2933, 2852 (CH3), 1670 (C]O), 1622 (C]N),
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1513, 1452 (aromatic frame), 1291, 1083, 1010, 911, 853, 731; 1H
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NMR (300 MHz, DMSO-d6):
d
8.02 (d, J ¼ 6.4 Hz, 2H, benim 4-H),
7.84 (d, J ¼ 5.5 Hz, 2H, benim 7-H), 7.61e7.59 (m, 4H, benim 5,6-H),
5.46 (s, 4H, benim 2-CH2), 4.72 (t, J ¼ 6.8 Hz, 4H, benim 1-CH2), 3.49
(s, 6H, thio-CH3), 1.82e1.80 (m, 4H, benim 1-CH2CH2), 1.31e1.23 (m,
28H, benim 1-CH2CH2(CH2)7), 0.97e0.95 (m, 6H, CH3) ppm; 13C
}
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NMR (75 MHz, DMSO-d6):
d 149.90 (C]O), 142.49 (benim 2-C),
142.34 (thio 2,5-C), 126.67 (benim 8,9-C), 125.38 (benim 4,7-C),
117.94 (benim 5,6-C), 113.55 (thio 3,4-C), 83.69 (NHCH2), 69.57
(benim 1-CH2), 46.43 (thio-CH3), 31.67 (CH2), 29.42e28.74
(CH2CH2CH2CH2CH2), 26.32 (CH2), 22.49 (CH2), 14.34 (CH3); MS
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(ESI): m/z 769 [M
þ
H
ꢀ
4HCl]þ; HRMS (ESI) calcd. for
C44H68Cl4N8O2S [M þ H ꢀ 4HCl]þ, 769.4951; found, 769.4948.
2
5
4.1.20. N0 ,N0 -Bis((1-(4-fluorobenzyl)-1H-benzoimidazole-2-yl)
methyl)-3,4-dimethylthiophene-2,5-dicarbo hydrazide
hydrochloride (14)
Compound 14 was prepared according to the procedure depic-
ted for compound 13a, starting from compound 12c (105 mg,
0.15 mmol). The product 14 (83 mg) was obtained ꢀas1 a slight yellow
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54e60.
ꢀ
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1427e1435.
solid. Yield: 60.3%; m.p.: 160e161 ꢄC; IR (KBr, cm
) n: 3438, 3319
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(aromatic frame), 1291, 1081, 1002, 919, 854, 731; 1H NMR
(300 MHz, DMSO-d6):
d
7.87 (d, J ¼ 6.8 Hz, 2H, benim 4-H), 7.81 (d,
_
ꢀ
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