Design, synthesis, and biological evaluation of novel benzimidazole derivatives and their interaction with calf thymus DNA and synergistic effects with clinical drugs

Article abstract of DOI:10.1007/s11426-014-5087-x

A series of new benzimidazole derivatives was synthesized and characterized by IR, ¹H NMR, ¹³C NMR, MS, and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by a twofold serial dilution technique. The bioactive evaluation showed that 3,5-bis(trifluoromethyl)phenyl benzimidazoles were comparably or even more strongly antibacterial and antifungal than the reference drugs Chloromycin, Norfloxacin, and Fluconazole. The combination of 2,4-difluorobenzyl benzimidazole derivative 5l and its hydrochloride 7 respectively with the antibacterials Chloromycin, Norfloxacin, and the antifungal Fluconazole was more sensitive to methicillin-resistant MRSA and Fluconazole-insensitive A. flavus. In addition, the interaction of compound 5l with calf thymus DNA demonstrated that this compound could effectively intercalate into DNA to form a compound 5l-DNA complex that might block DNA replication and thereby exert good antimicrobial activity.

Full text of DOI:10.1007/s11426-014-5087-x

SCIENCE CHINA
Chemistry
May 20 4 Vol.57 No.5: 1–16
doi: 10.1007/s11426-014-5087-x
• ARTICLES •
doi: 10.1007/s11426-014-5087-x
Design, synthesis, and biological evaluation of novel benzimidazole
derivatives and their interaction with calf thymus DNA and
synergistic effects with clinical drugs
ZHANG HuiZhen1†, LIN JianMei2†, RASHEED Syed1# & ZHOU ChengHe1*
1School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
2Sichuan Provincial People's Hospital, Chengdu 610072, China
Received October 31, 2013; accepted November 27, 2013
1
A series of new benzimidazole derivatives was synthesized and characterized by IR, H NMR, 13C NMR, MS, and HRMS
spectra. All the new compounds were screened for their antimicrobial activities in vitro by a twofold serial dilution technique.
The bioactive evaluation showed that 3,5-bis(trifluoromethyl)phenyl benzimidazoles were comparably or even more strongly
antibacterial and antifungal than the reference drugs Chloromycin, Norfloxacin, and Fluconazole. The combination of
2,4-difluorobenzyl benzimidazole derivative 5l and its hydrochloride 7 respectively with the antibacterials Chloromycin, Nor-
floxacin, and the antifungal Fluconazole was more sensitive to methicillin-resistant MRSA and Fluconazole-insensitive A. fla-
vus. In addition, the interaction of compound 5l with calf thymus DNA demonstrated that this compound could effectively in-
tercalate into DNA to form a compound 5l-DNA complex that might block DNA replication and thereby exert good antimicro-
bial activity.
benzimidazole, antibacterial, antifungal, calf thymus DNA, synergistic effect
Benzimidazole is a fused heterocycle of benzene with
1 Introduction
imidazole that possesses a larger conjugated system and
electron-rich properties higher than those of imidazole [3],
thiazole [4], oxazole [5], triazole [68], and tetrazole [9].
These special structures make benzimidazole-based deriva-
tives easily interact with various active targets and exhibit a
broad range of properties in medicinal chemistry, including
antihelmintic, antihistaminic, anticancer, antiviral, anti-
inflammatory, antiproliferative, antioxidant, and anticoagu-
lant bioactivities [1013]. Recently, many benzimidazole
derivatives have been successfully developed in clinics,
such as the antihistaminic Astemizole, anti-anabrotic Ome-
prazole, antihypertensive Candesartan, and antiparasitic
Albendazole. Many studies have demonstrated that ben-
zimidazoles as analogues of purines can compete with pu-
rines to efficiently prevent the synthesis of nucleic acids and
proteins, thereby inhibiting the growth of various microor-
ganisms and killing them. This suggests that, according to
The combination of the rising incidence of bacterial re-
sistance to antibiotic therapy and newly emerging pathogens
has become a serious problem in the past few decades.
Methicillin-resistant Staphylococcus aureus (MRSA) has
been a particularly unusual challenge for public health.
Currently, combination therapy in clinic with two or more
agents, which generally overcomes multi-drug resistance, is
an important approach to improving treatment efficiency
and bioavailability as well as to treating mixed diseases [1,
2]. However, more efforts are being made to develop new
antimicrobial agents with novel structures that possibly use
different mechanisms than traditional clinical drugs [38].
†These two authors contributed equally to this work
# Postdoctoral fellow from Department of Chemistry, Hyderabad University, India
*Corresponding author (email: zhouch@swu.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2014
chem.scichina.com link.springer.com

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Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
their different mechanisms, benzimidazoles should possess
great potential as a new type of antimicrobial agent. This
possibility has encouraged many studies to explore novel
benzimidazole derivatives to new molecular scaffolds with
high efficiency, broad spectra, and low toxicity [10]. Re-
cently, several active benzimidazoles have been developed
that display good or even superior bioactivity [1416]. In
particular, N-1 and C-2 alkylations of the benzimidazole
ring have been actively investigated to explore new com-
pounds that may have different mechanisms. Of these,
C-alkylated benzimidazole amine CABA has displayed ef-
fective inhibition of all the tested bacteria, especially
Klebsiella pneumoniae with a comparable MIC value of 0.5
g/mL to the reference drug Cefoperazone [17, 18]. Nota-
bly, N-alkylated benzimidazole derivative NMBI also ex-
hibited significant antifungal activity against strains of
Candida tropicalis and Candida globrata [19] (Figure 1).
These results suggest that benzimidazole derivatives might
be very effective as new antibacterial and antifungal agents
[10, 20].
Amino groups are important structural fragments that are
frequently present in natural products and synthetic bioac-
tive molecules such as clinical analgesic, antiallergic, and
antitumor drugs that improve affinity and water solubility
by the formation of hydrogen bonds, strong coordination,
and/or quaternary salts. Therefore, the amino moiety is
widely employed in the design and development of new
bioactive molecules [2123]. Recently, we demonstrated
that the benzimidazolylmethene amine ZHZ is an essential
moiety for antimicrobial activity and also displays intri-
guing antibacterial and antifungal activities [16]. This multi-
nitrogen nucleus exerts diverse non-covalent interactions
with active sites by use of hydrogen bonds, coordination,
ion-dipole, cation-, - stacking, and hydrophobic effect,
as well as van der Waals force, to exhibit desirable bioactiv-
ity. In continuation of our studies on the development of
novel antimicrobial agents, we designed a series of new
benzimidazoles. Fluoro-, chloro-, trifluoromethyl- and methyl-
substituted phenyl groups were introduced into target mol-
ecules with the aim of improving the molecules’ pharmaco-
logical properties by enhancing their rate of absorption and
transportation of drugs in vivo [2426]. A variety of ali-
phatic chains were also incorporated to investigate favora-
ble chain-length in modulating molecular flexibility to reg-
ulate the lipid-water partition coefficients [2729]. Because
coumarin compounds containing 1,2-benzopyrone skeletons
structurally similar to clinical anti-infective quinolone drugs
with benzopyridone backbones, have attracted considerable
attention due to their extensive biological activities [3032],
a coumarin ring was introduced into target molecules to
increase their antimicrobial efficiencies and broaden their
antimicrobial spectra. The carbazole fragment of interest
possesses desirable electronic and charge-transport proper-
ties as well as a large -conjugated system [33, 34]; in addi-
tion, its derivatives have been found to easily interact with
microbial targets via non-covalent interactions. Therefore,
carbazoles were also employed to construct antimicrobial
agents and evaluate their effect on antimicrobial activities.
A series of benzimidazole amines including alkyl com-
pound 4, aralkyl derivatives involving halobenzyl com-
pound 5, coumarin compound 11, and carbazole-derivative
compound 14, as well as their corresponding salts 6 and 7,
were prepared for consideration from commercially availa-
ble o-phenylene diamine and chloroacetic acid according to
the synthetic routes shown in Schemes 1 and 2. These new-
ly synthesized compounds were evaluated for antibacterial
and antifungal activities. In addition, the synergistic effects
of some representative benzimidazole compounds with
clinical antibacterials Chloromycin and Norfloxacin, and
antifungal Fluconazole, were evaluated in vitro against four
Gram-positive bacteria, four Gram-negative bacteria, and
five fungi. Additionally, in order to further elucidate the
action mechanisms, the interaction of a strong active com-
pound with calf thymus DNA was also studied [35, 36].
2 Experimental
2.1 Materials and measurements
Melting points were recorded on an X-6 melting point ap-
paratus (Beijing Focus Instrument CO., Ltd) and left uncor-
rected. TLC analysis was done using precoated silica gel
plates. FT-IR spectra were carried out on a Bruker
RFS100/S spectrophotometer (Bio-Rad, Cambridge, MA,
1
USA) using KBr pellets in the 400–4000 cm1 range. H
NMR and 13C NMR spectra were recorded on a Bruker AV
300 spectrometer (Bruker BioSpin AG Ltd., Beijing, China)
using TMS as an internal standard. The following abbrevia-
tions were used to designate aryl groups: Bim = benzimid-
azolyl, Ph = phenyl. The chemical shifts are reported in
parts per million (ppm), the coupling constants (J) are ex-
pressed in hertz (Hz), and signals are described as singlet (s),
Figure 1 Structures of antimicrobial benzimidazoles.

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
3
Scheme 1 Synthesis of benzimidazole compounds 47. Conditions and reagents: (i) chloroacetic acid, 5 mol/L hydrochloric acid, reflux; (ii) anilines, eth-
anol, reflux; (iii) alkyl bromide, potassium carbonate, acetonitrile, 70 ºC; (iv) substituted halobenzyl halide, potassium carbonate, acetonitrile, 70 ºC; (v) 4
mol/L hydrochloric acid, ethyl ether, rt.
Scheme 2 Synthesis of benzimidazole-based coumarin 11 and carbazole 14. Conditions and reagents: (vi) ethyl acetoacetate, oxalic acid, 90–100 ºC; (vii)
ethyl acetoacetate, piperidine, 530 ºC; (viii) 1,6-dibromohexane, potassium carbonate, acetone, reflux; (ix) 1,4-bis(bromomethyl)benzene, potassium car-
bonate, acetone, reflux; (x) compounds 3c and 3f, potassium carbonate, acetonitrile, 70 ºC; (xi) 1,4-dibromobutane, sodium hydride, tetrahydrofuran; (xii)
compound 3a, potassium carbonate, acetonitrile, 70 ºC.
doublet (d), triplet (t), and multiplet (m). The mass spectra
were recorded on an LCMS-2010A (Thermo Electron Cor-
poration, Bremen, Germany) and an HRMS (Bruker Dal-
tonics, MA, USA). All of the chemicals and solvents were
commercially available and used without further purifica-
tion.
2.2 Synthesis
2-(Chloromethyl)-1H-benzo[d]imidazole (2)
Compound 2 was prepared according to the procedure de-
scribed in the literature [37], starting with o-phenylene dia-
mine (5.412 g, 0.05 mol) and chloroacetic acid (7.215 g,

4
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
0.075 mol). Yield: 90.7%; mp: 217–218 ºC. (literature [37]
mp: 218–219 ºC).
sodium carbonate (2.565 g, 0.024 mol). The pure product 3g
was obtained as yellow solid. Yield: 79.6%; mp: 165−
167 ºC (literature [16] mp: 166–167 ºC).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-2-fluoroaniline (3a)
N-((1H-Benzo[d]imidazol-2-yl)methyl)-2,4-dichloroaniline
(3h)
3a was prepared according to the procedure described in the
literature [16], starting with compound 2 (3.335 g, 0.02
mol), 2-fluoroaniline (2.226 g, 0.02 mol), and sodium car-
bonate (2.546 g, 0.024 mol). Yield: 73.2%; mp: 164–166 ºC
(literature mp: 164−165 ºC).
Compound 3h was prepared according to the procedure
described for compound 3a, starting with compound 2
(3.337 g, 0.02 mol), 2,4-dichoroaniline (3.228 g, 0.02 mol),
and sodium carbonate (2.546 g, 0.024 mol). The pure prod-
uct 3h was obtained as yellow solid. Yield: 72.3%; mp:
163−165 ºC (literature [16] mp: 163–164 ºC).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-3-(trifluoromethyl)-
aniline (3b)
Compound 3b was prepared according to the procedure
described for compound 3a, starting with compound 2
(3.334 g, 0.02 mol), 3-(trifluoromethyl)aniline (3.214 g,
0.02 mol), and sodium carbonate (2.547 g, 0.024 mol). The
pure product 3b was obtained as yellow solid. Yield: 71.3%;
mp: 155−156 ºC (literature [16] mp: 155−156 ºC).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-3,5-bis(trifluoro-
methyl)aniline (3i)
Compound 3i was prepared according to the procedure de-
scribed for compound 3a, starting with compound 2 (3.326
g, 0.02 mol), 3,5-bis(trifluoromethyl)aniline (4.467 g, 0.02
mol), and sodium carbonate (2.559 g, 0.024 mol). The pure
product 3i was obtained as white solid. Yield: 83.2%; mp:
211−212 ºC (literature [16] mp: 212–214 ºC).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-4-fluoroaniline (3c)
Compound 3c was prepared in the same way as the general
procedure described for compound 3a, starting with com-
pound 2 (3.340 g, 0.02 mol), 4-fluoroaniline (2.233 g, 0.02
mol), and sodium carbonate (2.544 g, 0.024 mol). The pure
product 3c was obtained as yellow solid. Yield: 74.0%; mp:
168−170 ºC (literature [16] mp: 168−169 ºC).
4-Methyl-N-((1-propyl-1H-benzo[d]imidazol-2-yl)methyl)-
aniline (4a)
To a solution of compound 3f (0.475 g, 0.002 mol) in ace-
tonitrile (15 mL) were added potassium carbonate (0.410 g,
0.003 mol) and 1-bromopropane (0.247 g, 0.002 mol). The
mixture was stirred at 70 ºC. After the reaction ended (mon-
itored by TLC, eluent: petroleum ether/ethyl acetate (4/1,
v/v)), the solvent was removed and the residue was exacted
with ethyl acetate (3  15 mL), dried over anhydrous sodi-
um sulfate, and purified by silica gel column chromatog-
raphy (eluent: petroleum ether/ethyl acetate (5/1, v/v)) to
afford compound 4a. The N-propyl benzimidazole amine 4a
was obtained as yellow oil. Yield: 58.7%; IR (KBr) ν: 3370
(NH), 3030 (aromatic CH), 2918, 2850 (CH2, CH3), 1608
(C=N), 1598, 1500, 1457 (aromatic frame), 1381, 1379
(CH3), 815 cm–1; 1H NMR (300 MHz, CDCl3) δ: 1.10−1.08
(t, 3H, J = 6.0 Hz, CH2CH2CH3), 1.34−1.32 (m, 2H,
CH2CH2CH3), 2.23 (s, 3H, 4-CH3Ph), 4.19−4.16 (t, 2H, J =
9.0 Hz, CH2CH2CH3), 4.53 (s, 2H, Bim-CH2), 6.69 (d, 2H, J
= 9.0 Hz, 4-CH3Ph-2,6-H), 7.04 (d, 2H, J = 9.0 Hz,
4-CH3Ph-3,5-H), 7.36−7.26 (m, 4H, Bim-5,6,7,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 11.4, 20.2, 21.9, 41.8, 47.1,
111.3, 114.9, 118.7, 122.7, 128.6, 128.7, 132.7, 140.2,
142.2, 151.1 ppm; MS (m/z): 280 [M + H]+; HRMS (ESI)
calcd for C18H22N3 [M + H]+, 280.1814; found, 280.1815.
N-((1H-Benzo[d]imidazol-2-yl)methyl)-4-chloroaniline (3d)
Compound 3d was prepared according to the procedure
described for compound 3a, starting with compound 2
(3.334 g, 0.02 mol), 4-chloroaniline (2.623 g, 0.02 mol),
and sodium carbonate (2.560 g, 0.024 mol). The pure prod-
uct 3d was obtained as yellow solid. Yield: 75.6%; mp:
208−209 ºC (literature [16] mp: 208−210 ºC).
N-((1H-Benzo[d]imidazol-2-yl)methyl)-4-iodoaniline (3e)
Compound 3e was prepared according to the procedure de-
scribed for compound 3a, starting with compound 2 (3.308
g, 0.02 mol), 4-iodoaniline (4.157 g, 0.02 mol), and sodium
carbonate (2.545 g, 0.024 mol). The pure product 3e was
obtained as yellow syrup. Yield: 70.5%.
N-((1H-Benzo[d]imidazol-2-yl)methyl)-4-methylaniline (3f)
Compound 3f was prepared according to the experimental
procedure described for compound 3a, starting with com-
pound 2 (3.319 g, 0.02 mol), 4-methylaniline (2.565 g, 0.02
mol), and sodium carbonate (2.543 g, 0.024 mol). The pure
product 3f was obtained as yellow syrup. Yield: 83.8%; mp:
199−200 ºC (literature [38] mp: 175–177 ºC).
N-((1-Hexyl-1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-
aniline (4b)
N-((1H-Benzo[d]imidazol-2-yl)methyl)-2,4-difluoroaniline
Pure compound 4b was obtained in the process of synthe-
sizing compound 4a, as yellow oil. Yield: 59.8%; IR (KBr)
ν: 3367 (NH), 3028 (aromatic CH), 2916, 2849 (CH2,
CH3), 1607 (C=N), 1593, 1569, 1454 (aromatic frame),
1380, 1378 (CH3), 814 cm–1; 1H NMR (300 MHz, CDCl3) δ:
(3g)
Compound 3g was prepared according to the procedure de-
scribed for compound 3a, starting with compound 2 (3.329
g, 0.02 mol), 2,4-difluoroaniline (2.582 g, 0.02 mol), and

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
5
0.87−0.86 (t, 3H, J = 3.0 Hz, CH2(CH2)4CH3), 1.30−1.26 (m,
4H, (CH2)3(CH2)2CH3), 1.82−1.80 (m, 4H, CH2(CH2)2-
(CH2)2CH3), 2.25 (s, 3H, 4-CH3Ph), 4.17−4.13 (t, 2H, J =
6.0 Hz, CH2(CH2)4CH3), 4.52 (s, 2H, Bim-CH2), 6.68 (d,
2H, J = 9.0 Hz, 4-CH3Ph-2,6-H), 7.04 (d, 2H, J = 9.0 Hz,
4-CH3Ph-3,5-H), 7.34−7.26 (m, 4H, Bim-5,6,7,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 13.9, 20.2, 21.3, 26.6, 28.5,
31.0, 41.7, 47.2, 111.3, 114.9, 118.7, 122.7, 128.7, 132.7,
140.2, 142.2, 151.2 ppm; MS (m/z): 322 [M + H]+; HRMS
(ESI) calcd for C21H28N3 [M + H]+, 322.2283; found,
322.2280.
CH3), 1607 (C=N), 1599, 1509, 1459 (aromatic frame),
1380, 1377 (CH3), 814 cm1; 1H NMR (300 MHz, CDCl3) δ:
0.88−0.86 (t, 3H, J = 6.0 Hz, (CH2)11CH3), 1.32−1.28 (m,
16H, (CH2)3(CH2)8CH3), 1.82−1.79 (m, 4H, CH2(CH2)2-
(CH2)8CH3), 2.24 (s, 3H, 4-CH3Ph), 4.16−4.14 (t, 2H, J =
6.0 Hz, CH2(CH2)10CH3), 4.53 (s, 2H, Bim-CH2), 6.69 (d,
2H, J = 9.0 Hz, 4-CH3Ph-2,6-H), 7.04 (d, 2H, J = 9.0 Hz,
4-CH3Ph-3,5-H), 7.35−7.26 (m, 4H, Bim-5,6,7,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 13.9, 20.2, 21.3, 26.9, 28.3,
28.4, 28.6, 31.2, 41.8, 47.2, 111.4, 114.9, 118.8, 122.8,
128.8, 132.7, 140.3, 142.2, 151.2 ppm; MS (m/z): 406 [M +
H]+; HRMS (ESI) calcd for C27H39N3 [M + H]+, 406.3222;
found, 406.3225.
4-Methyl-N-((1-octyl-1H-benzo[d]imidazol-2-yl)methyl)-
aniline (4c)
4-Methyl-N-((1-octadecyl-1H-benzo[d]imidazol-2-yl)methyl)-
aniline (4f)
Pure compound 4c was obtained in the process of synthe-
sizing compound 4a, as yellow oil. Yield: 60.3%; IR (KBr)
ν: 3366 (NH), 3028 (aromatic CH), 2915, 2849 (CH2,
CH3), 1606 (C=N), 1594, 1454 (aromatic frame), 1381,
Compound 4f was obtained as brown syrup. Yield: 49.4%;
IR (KBr) ν: 3365 (NH), 3033 (aromatic CH), 2911, 2839
(CH2, CH3), 1605 (C=N), 1599, 1573, 1458 (aromatic
1
1379 (CH3), 814 cm–1; H NMR (300 MHz, CDCl3) δ:
1
frame), 1381, 1378 (CH3), 813 cm–1; H NMR (300 MHz,
0.88−0.86 (t, 3H, J = 6.0 Hz, (CH2)7CH3), 1.31−1.27 (m, 8H,
(CH2)3(CH2)4CH3), 1.82−1.80 (m, 4H, CH2(CH2)2(CH2)4-
CH3), 2.24 (s, 3H, 4-CH3Ph), 4.15−4.13 (t, 2H, J = 6.0 Hz,
CH2(CH2)6CH3), 4.53 (s, 2H, Bim-CH2), 6.69 (d, 2H, J =
9.0 Hz, 4-CH3Ph-2,6-H), 7.05 (d, 2H, J = 9.0 Hz,
4-CH3Ph-3,5-H), 7.32−7.24 (m, 4H, Bim-5,6,7,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 13.9, 20.2, 21.3, 26.8, 28.3,
28.5, 31.2, 41.7, 47.2, 111.4, 114.9, 118.8, 122.7, 128.8,
132.7, 140.2, 142.2, 151.2 ppm; MS (m/z): 350 [M + H]+;
HRMS (ESI) calcd for C23H31N3 [M + H]+, 350.2596; found,
350.3599.
CDCl3) δ: 0.89−0.86 (t, 3H, J = 9.0 Hz, (CH2)17CH3),
1.33−1.27 (m, 28H, (CH2)3(CH2)14CH3), 1.83−1.79 (m, 4H,
CH2(CH2)2(CH2)14CH3), 2.23 (s, 3H, 4-CH3Ph), 4.17−4.14
(t, 2H, J = 9.0 Hz, CH2(CH2)10CH3), 4.54 (s, 2H, Bim-CH2),
6.68 (d, 2H, J = 9.0 Hz, 4-CH3Ph-2,6-H), 7.05 (d, 2H, J =
9.0 Hz, 4-CH3Ph-3,5-H), 7.34−7.23 (m, 4H, Bim-5,6,7,8-H)
ppm; 13C NMR (75 MHz, CDCl3) δ: 13.9, 20.2, 21.3, 26.9,
28.3, 28.4, 28.6, 31.2, 41.8, 47.2, 111.4, 115.0, 118.8, 122.8,
128.8, 132.7, 140.3, 142.2, 151.2 ppm; MS (m/z): 490 [M +
H]+; HRMS (ESI) calcd for C33H51N3 [M + H]+, 490.4161;
found, 490.4158.
N-((1-Decyl-1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-
N-((1-(3,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
aniline (4d)
methyl)-2-fluoroaniline (5a)
Pure compound 4d was obtained in the process of synthe-
sizing compound 4a, as yellow oil. Yield: 57.7%; IR (KBr)
ν: 3366 (NH), 3029 (aromatic CH), 2915, 2847 (CH2,
CH3), 1606 (C=N), 1594, 1573, 1459 (aromatic frame),
1382, 1379 (CH3), 815 cm–1; 1H NMR (300 MHz, CDCl3) δ:
0.87−0.86 (t, 3H, J = 3.0 Hz, (CH2)9CH3), 1.30−1.28 (m,
12H, (CH2)3(CH2)6CH3), 1.82−1.80 (m, 4H, CH2(CH2)2-
(CH2)6CH3), 2.23 (s, 3H, 4-CH3Ph), 4.15−4.14 (t, 2H, J =
3.0 Hz, CH2(CH2)8CH3), 4.54 (s, 2H, Bim-CH2), 6.68 (d,
2H, J = 9.0 Hz, 4-CH3Ph-2,6-H), 7.05 (d, 2H, J = 9.0 Hz,
4-CH3Ph-3,5-H), 7.34−7.24 (m, 4H, Bim-5,6,7,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 13.9, 20.2, 21.3, 26.9, 28.3,
28.4, 28.6, 31.2, 41.8, 47.2, 111.4, 114.9, 118.8, 122.7,
128.8, 132.7, 140.3, 142.2, 151.2 ppm; MS (m/z): 378 [M +
H]+; HRMS (ESI) calcd for C25H35N3 [M + H]+, 378.2909;
found, 378.2906.
To a solution of compound 3a (1.035 g, 0.004 mol) in ace-
tonitrile were added potassium carbonate and 3,4-dichloro-
1-(chloromethyl)benzene (0.832 g, 0.004 mol). The mixture
was stirred at 70 ºC. After the reaction ended (monitored by
TLC, petroleum ether/ethyl acetate (4/1, v/v)), the solvent
was removed and the residue was exacted with ethyl acetate,
dried over anhydrous sodium sulfate, and purified by silica
gel column chromatography (eluent: petroleum ether/ethyl
acetate (5/1, v/v)) to afford compound 5a as white solid.
Yield: 70.4%; mp: 134–135 ºC; IR (KBr) ν: 3370 (NH),
3025 (aromatic CH), 2908, 2859 (CH2
, CH ), 1606 (C=N),
3
1593, 1505, 1462 (aromatic frame), 813, 760 cm–1; 1H NMR
(300 MHz, CDCl3) δ: 4.57 (s, 2H, Bim-CH2), 5.42 (s, 2H,
3,4-Cl2Ph-CH2), 6.73−6.66 (m, 1H, 2-FPh-6-H), 6.84−6.79
(t, 2H, J = 6.0 Hz, 2-FPh-4,5-H), 7.00−6.93 (m, 2H,
2-FPh-3-H, 3,4-Cl2Ph-6-H), 7.13 (s, 1H, 3,4-Cl2Ph- 2-H),
7.33−7.20 (m, 4H, Bim-5,6,7,8-H), 7.84 (d, 1H, J = 6.0 Hz,
3,4-Cl2Ph-5-H) ppm; 13C NMR (75 MHz, CDCl3) δ: 41.7,
46.2, 109.5, 112.7, 114.5, 114.8, 118.1, 118.3, 120.0, 122.8,
123.4, 124.6, 125.3, 128.1, 130.9, 132.2, 133.2, 135.6,
135.8, 142.1, 151.1 ppm; MS (m/z): 400 [M+H]+; HRMS
N-((1-Dodecyl-1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-
aniline (4e)
Pure compound 4e was obtained in the process of synthe-
sizing compound 4a, as yellow oil. Yield: 53.4%; IR (KBr)
ν: 3365 (NH), 3030 (aromatic CH), 2913, 2842 (CH2,

6
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
(ESI) calcd for C21H16Cl2FN3 [M + H]+, 400.0784; found,
400.0782.
(0.502 g) as white solid. Yield: 68.7%; mp: 146–148 ºC; IR
(KBr) ν: 3371 (NH), 3019 (aromatic CH), 2908, 2860
(CH2, CH3), 1605 (C=N), 1592, 1568, 1455 (aromatic
frame), 825, 753 cm–1; 1H NMR (300 MHz, CDCl3) δ: 4.43
(s, 2H, Bim-CH2), 5.37 (s, 2H, 4-FPh-CH2), 6.60 (d, 2H,
4-ClPh-2,6-H), 7.00 (d, 2H, 4-FPh-3,5-H), 7.01 (d, 2H,
4-FPh-2,6-H), 7.13 (d, 2H, 4-ClPh-3,5-H), 7.33−7.29 (m,
4H, Bim-5,6,7,8-H) ppm; 13C NMR (75 MHz, CDCl3) δ:
41.7, 46.4, 109.6, 114.3, 115.9, 116.2, 119.7, 122.5, 123.2,
127.8, 129.1, 131.2, 135.7, 142.1, 145.7, 151.1 ppm; MS
(m/z): 366 [M + H]+; HRMS (ESI) calcd for C20H20ClN3
[M + H]+, 365.1095; found, 365.1092.
N-((1-(2,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-3-(trifluoromethyl) aniline (5b)
Compound 5b was synthesized starting with compound 3b
(1.179 g, 0.004 mol) and 2,4-dichloro-1-(chloromethyl)
benzene (0.832 g, 0.004 mol). The crude product was ob-
tained and purified via silica gel column chromatography
(eluent: petroleum ether/ethyl acetate (5/1, v/v)) to give pure
compound 5b (0.522 g) as yellow solid. Yield: 76.5%; mp:
137–138 ºC; IR (KBr) ν: 3373 (NH), 3024 (aromatic CH),
2912, 2862 (CH2, CH3), 1608 (C=N), 1595, 1572, 1464
1
(aromatic frame), 832, 791, 692 cm–1; H NMR (300 MHz,
N-((1-(2-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl)-
4-methylaniline (5e)
CDCl3) δ: 4.51 (s, 2H, Bim-CH2), 5.44 (s, 2H, 2,4-Cl2Ph-
CH2), 6.38 (d, 1H, J = 9.0 Hz, 3-CF3Ph-6-H), 6.85 (d, 2H,
3-CF3Ph-2,4-H), 7.03−6.96 (m, 2H, 3-CF3Ph- 5-H,
2,4-Cl2Ph-6-H), 7.36−7.19 (m, 4H, Bim-5,6,7,8-H), 7.48 (d,
1H, J = 3.0 Hz, 2,4-Cl2Ph-5-H), 7.84 (d, 1H, J = 9.0 Hz,
2,4-Cl2Ph-3-H) ppm; 13C NMR (75 MHz, CDCl3) δ: 41.4,
44.5, 109.2, 109.5, 114.7, 116.1, 119.9, 122.9, 123.5, 127.7,
127.8, 129.7, 131.5, 131.7, 132.8, 134.6, 135.5, 142.0,
147.2, 151.1 ppm; MS (m/z): 450 [M + H]+; HRMS (ESI)
calcd for C22H16Cl2F3N3 [M + H]+, 450.0752; found,
450.0751.
Compound 5e was synthesized according to the experi-
mental procedure, starting with compound 3f (0.234 g,
0.001 mol) and 1-chloro-3-(chloromethyl)benzene (0.174 g,
0.001 mol). The crude product was obtained and purified
via silica gel column chromatography (eluent: petroleum
ether/ethyl acetate (5/1, v/v)) to give pure compound 5e
(0.234 g) as white solid. Yield: 65.2%; mp: 169–171 ºC; IR
(KBr) ν: 3368 (NH), 3017 (aromatic CH), 2905, 2853
(CH2, CH3), 1605 (C=N), 1592, 1565, 1452 (aromatic
frame), 1386 (CH3), 820, 751 cm–1; H NMR (300 MHz,
1
CDCl3) δ: 2.24 (s, 3H, CH3), 4.53 (s, 2H, Bim-CH2), 5.41 (s,
2H, 2-ClPh-CH2), 6.63 (d, 2H, J = 6.0 Hz, 4-CH3Ph-2,6-H),
6.98 (m, 4H, 4-CH3Ph-3,5-H, 4-ClPh-2,6-H), 7.26−7.24 (m,
2H, Bim-5,6,7,8-H, 4-ClPh-3,5-H) ppm; 13C NMR (75 MHz,
CDCl3) δ: 20.4, 42.2, 46.4, 109.6, 113.5, 119.8, 122.5,
123.1, 127.6, 127.7, 129.2, 129.8, 133.8, 134.3, 135.7,
142.3, 145.0, 151.9 ppm; MS (m/z): 362 [M+H]+; HRMS
(ESI) calcd for C20H20ClN3 [M + H]+, 362.1424; found,
362.1423.
N-((1-(2,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-4-fluoroaniline (5c)
Prepared according to the general procedure, starting with
compound 3c (0.970 g, 0.004 mol) and 2,4-dichloro-1-
(chloromethyl)benzene (0.804 g, 0.004 mol), pure com-
pound 5c (0.482 g) was obtained as yellow solid. Yield:
70.1%; mp: 134–135 ºC; IR (KBr) ν: 3369 (NH), 3020
(aromatic CH), 2911, 2862 (CH2, CH3), 1607 (C=N), 1593,
1510, 1458 (aromatic frame), 831, 756 cm–1; 1H NMR (300
MHz, CDCl3) δ: 4.46 (s, 2H, Bim-CH2), 5.46 (s, 2H,
2,4-Cl2Ph-CH2), 6.36 (d, 1H, J = 9.0 Hz, 2,4-Cl2Ph-6-H),
6.62−6.58 (m, 2H, 4-FPh-2,6-H), 6.89−6.83 (m, 2H,
4-FPh-3,5-2H), 7.01 (d, 1H, J = 9.0 Hz, 2,4-Cl2Ph-5-H),
7.35−7.18 (m, 4H, Bim-5,6,7,8-H), 7.84 (d, 1H, J = 9.0 Hz,
2,4-Cl2Ph-3-H) ppm; 13C NMR (75 MHz, CDCl3) δ: 42.3,
44.2, 109.5, 114.1, 114.2, 115.5, 115.8, 119.8, 122.7, 123.4,
127.7, 127.8, 129.5, 131.7, 142.0, 134.4, 135.4, 143.4,
151.6 ppm; MS (m/z): 400 [M + H]+; HRMS (ESI) calcd for
C21H16Cl2FN3 [M + H]+, 400.0784; found, 400.0783.
N-((1-(4-Chlorobenzyl)-1H-benzo[d]imidazol-2-yl)methyl)-
4-methylaniline (5f)
Compound 5f was prepared according to the experimental
procedure, starting with compound 3f (0.158 g, 0.7 mmol)
and 1-chloro-4-(chloromethyl)benzene (0.130 g, 0.7 mmol).
The desired pure compound 5f (0.169 g) was obtained as
white solid. Yield: 66.7%; mp: 103–105 ºC; IR (KBr) ν:
3367 (NH), 3018 (aromatic CH), 2900, 2852 (CH2, CH3),
1605 (C=N), 1593, 1506, 1453 (aromatic frame), 1387
1
(CH3), 821, 752 cm1; H NMR (300 MHz, CDCl3) δ: 2.24
(s, 3H, CH3), 4.46 (s, 2H, Bim-CH2), 5.40 (s, 2H,
4-ClPh-CH2), 6.62 (d, 2H, J = 6.0 Hz, 4-CH3Ph-2,6-H),
6.98 (m, 4H, 4-CH3Ph-3,5-H, 4-ClPh-2,6-H), 7.27−7.24 (m,
2H, Bim-5,6,7,8-H, 4-ClPh-3,5-H) ppm; 13C NMR (75 MHz,
CDCl3) δ: 20.4, 42.2, 49.3, 109.6, 113.5, 119.8, 122.5,
127.6, 129.3, 129.8, 133.9, 134.3, 136.2, 142.3, 145.0,
151.9 ppm; MS (m/z): 362 [M + H]+; HRMS (ESI) calcd for
C22H20ClN3 [M + H]+, 362.1424; found, 362.1421.
4-Chloro-N-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)methyl)aniline (5d)
Compound 5d was synthesized according to the experi-
mental procedure, starting with compound 3d (0.519 g,
0.002 mol) and 1-chloro-3-(chloromethyl)benzene (0.312 g,
0.002 mol). The crude product was obtained and purified
via silica gel column chromatography (eluent: petroleum
ether/ethyl acetate (5/1, v/v)) to give pure compound 5d

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
7
N-((1-(2,4-Difluorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-4-methylaniline (5g)
4H, Bim-5,6,7,8-H), 7.83 (d, 1H, J = 9.0 Hz, 3,4-Cl2Ph-5-H)
ppm; 13C NMR (75 MHz, CDCl3) δ: 20.4, 42.2, 48.9, 109.4,
113.4, 119.8, 122.7, 123.3, 127.5, 127.6, 127.7, 127.8,
127.9, 129.8, 134.3, 135.4, 142.2, 144.9, 152.0 ppm; MS
(m/z): 396 [M + H]+; HRMS (ESI) calcd for C22H19Cl2N3
[M + H]+, 396.1034; found, 396.1035.
Compound 5g was prepared according to the experimental
procedure, starting with compound 3f (0.330 g, 1.4 mmol)
and 1-(bromomethyl)-2,4-difluorobenzene (0.302 g, 1.5
mmol). The pure product 5g was obtained as white solid.
Yield: 63.5%; mp: 117–119 ºC; IR (KBr) ν: 3378 (NH),
3033 (aromatic CH), 2925, 2855 (CH2, CH3), 1616 (C=N),
1599, 1500, 1459 (aromatic frame), 1391 (CH3), 848, 720
N-((1-(2,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-2,4-difluoroaniline (5j)
1
cm–1; H NMR (300 MHz, CDCl3) δ: 2.23 (s, 3H, CH3),
Compound 5j was prepared according to the experimental
procedure, starting with compound 3g (1.044 g, 0.004 mol)
and 2,4-dichloro-1-(chloromethyl)benzene (0.803 g, 0.004
mol). The pure product 5j was obtained as yellow solid.
Yield: 72.5%; mp: 146–147 ºC; IR (KBr) ν: 3384 (NH),
3025 (aromatic CH), 2919, 2860 (CH2, CH3), 1610 (C=N),
1597, 1507, 1456 (aromatic frame), 835, 725 cm–1; 1H NMR
(300 MHz, CDCl3) δ: 4.53 (s, 2H, Bim-CH2), 5.49 (s, 2H,
2,4-Cl2Ph-CH2), 6.31 (d, 1H, 2,4-F2Ph-3-H), 6.77−6.70 (m,
2H, 2,4-F2Ph-5,6-H), 6.97 (d, 1H, 2,4-Cl2Ph-6-H),
7.35−7.18 (m, 4H, Bim-5,6,7,8-H), 7.46 (d, 1H, J = 3.0 Hz,
2,4-Cl2Ph-5-H), 7.85 (d, 1H, J = 3.0 Hz, 2,4-Cl2Ph-3-H)
ppm; 13C NMR (75 MHz, CDCl3) δ: 41.9, 48.4, 103.2,
103.8, 109.4, 110.4, 110.8, 112.8, 120.0, 122.8, 123.4,
127.5, 127.6, 129.5, 131.7, 132.7, 134.4, 135.5, 142.2,
151.3 ppm; MS (m/z): 418 [M + H]+; HRMS (ESI) calcd for
C21H16Cl2F2N3 [M + H]+, 418.0689; found, 418.0687.
4.53 (s, 2H, Bim-CH2), 5.44 (s, 2H, 2,4-F2Ph-CH2), 6.65 (d,
2H, J = 9.0 Hz, 4-CH3Ph-2,6-H), 6.79−6.71 (m, 2H,
2,4-F2Ph-3,5-H), 6.91−6.84 (m, 1H, 2,4-F2Ph-6-H), 7.01 (d,
2H, J = 9.0 Hz, 4-CH3Ph-3,5-H), 7.33−7.27 (m, 4H,
Bim-5,6,7,8-H) ppm; 13C NMR (75 MHz, CDCl3) δ:
20.4, 42.2, 46.0, 107.3, 109.4, 110.7, 113.4, 119.8, 120.1,
122.7, 127.7, 127.8, 130.2, 131.9, 134.2, 142.2, 145.0,
152.0, 153.9, 160.9 ppm; MS (m/z): 364 [M + H]+; HRMS
(ESI) calcd for C22H19F2N3 [M + H]+, 364.1625; found,
364.1627.
N-((1-(2,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-4-methylaniline (5h)
Compound 5h was prepared according to the experimental
procedure, starting with compound 3f (0.956 g, 4.0 mmol)
and 2,4-dichloro-1-(chloromethyl)benzene (0.902 g, 4.3
mmol). The pure product 5h was obtained as white solid.
Yield: 58.4%; mp: 187–189 ºC; IR (KBr) ν: 3380 (N–H),
3031 (aromatic C–H), 2927, 2853 (CH2, CH3), 1613 (C=N),
1597, 1506, 1455 (aromatic frame), 1389 (CH3), 844, 724
2,4-Dichloro-N-((1-(2,4-difluorobenzyl)-1H-benzo[d]-
imidazol-2-yl)methyl)aniline (5k)
Compound 5k was prepared according to the experimental
procedure, starting with compound 3h (0.475 g, 0.004 mol)
and 1-(bromomethyl)-2,4-difluorobenzene (0.621 g, 0.004
mol). The pure product 5k was obtained as yellow oil. Yield:
69.5%; mp: 136–138 ºC; IR (KBr) ν: 3386 (NH), 3027
(aromatic CH), 2921, 2862 (CH2, CH3), 1608 (C=N), 1595,
1510, 1459 (aromatic frame), 837, 830 cm–1; 1H NMR (300
MHz, CDCl3) δ: 4.52 (s, 2H, Bim-CH2), 5.48 (s, 2H,
2,4-Cl2Ph-CH2), 6.42 (d, 1H, J = 6.0 Hz, 2,4-Cl2Ph-6-H),
6.63 (m, H, 2,4-F2Ph-3-H), 7.09−7.07 (m, H, 2,4-F2Ph-5-H),
7.15 (d, 1H, J = 3.0 Hz, 2,4-Cl2Ph-5-H), 7.19 (m, 1H,
2,4-F2Ph-6-H), 7.35−7.22 (m, 4H, Bim-5,6,7,8-H), 7.79 (d,
1H, J = 3.0 Hz, 2,4-Cl2Ph-3-H) ppm; 13C NMR (75 MHz,
CDCl3) δ: 41.5, 48.5, 102.4, 103.8, 108.6, 110.4, 110.8,
111.1, 112.8, 113.2, 113.5, 120.1, 123.5, 124.4, 129.5,
134.2, 134.4, 135.5, 147.1, 156.7 ppm; MS (m/z): 418 [M +
H]+; HRMS (ESI) calcd for C21H16Cl2F2N3 [M + H]+,
418.0689; found, 418.0686.
1
cm1; H NMR (300 MHz, CDCl3) δ: 2.22 (s, 3H, CH3),
4.48 (s, 2H, Bim-CH2), 5.46 (s, 2H, 2,4-Cl2Ph-CH2), 6.37 (d,
1H, J = 9.0 Hz, 2,4-Cl2Ph-6-H), 6.61 (d, 2H, J = 9.0 Hz,
4-CH3Ph-2,6-H), 6.98 (d, 2H, J = 9.0 Hz, 4-CH3Ph-3,5-H),
7.00 (d, 1H, Bim-7-H), 7.18 (d, 1H, J = 6.0 Hz, Bim-6-H),
7.34−7.23 (m, 2H, Bim-8-H, 2,4-Cl2Ph-5-H), 7.46 (d, 1H, J
=
3.0 Hz, Bim-5-H), 7.84 (d, 1H, J = 9.0 Hz,
2,4-Cl2Ph-3-H); 13C NMR (75 MHz, CDCl3) δ: 20.4, 42.2,
44.4, 109.4, 113.4, 119.8, 122.7, 123.3, 127.7, 127.8, 127.9,
129.5, 129.7, 131.9, 132.8, 134.3, 135.5, 142.2, 144.9,
152.0 ppm; MS (m/z): 396 [M + H]+; HRMS (ESI) calcd for
C22H19Cl2N3 [M + H]+, 396.1034; found, 396.1032.
N-((1-(3,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-4-methylaniline (5i)
Pure compound 5i was obtained in general process as white
solid. Yield: 60.2%; mp: 140–141 ºC; IR (KBr) ν: 3382
(NH), 3023 (aromatic CH), 2924, 2855 (CH2, CH3), 1611
(C=N), 1596, 1508, 1453 (aromatic frame), 1387 (CH3),
N-((1-(2,4-Difluorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-3,5-bis(trifluoromethyl)aniline (5l)
1
842, 722 cm1; H NMR (300 MHz, CDCl3) δ: 2.24 (s, 3H,
CH3), 4.49 (s, 2H, Bim-CH2), 5.40 (s, 2H, 3,4-Cl2Ph-CH2),
6.62 (d, 2H, J = 6.0 Hz, 4-CH3Ph-2,6-H), 6.84 (d, 1H, J =
9.0 Hz, 3,4-Cl2Ph-6-H), 7.01 (d, 2H, J = 6.0 Hz,
4-CH3Ph-3,5-H), 7.15 (s, 1H, 3,4-Cl2Ph-2-H), 7.35−7.19 (m,
Compound 5l was prepared according to the experimental
procedure, starting with compound 3i (1.079 g, 0.003 mol)
and 1-(bromomethyl)-2,4-difluorobenzene (0.635 g, 0.003
mol). The pure product 5l was obtained as white solid.

8
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
+ H]+; HRMS (ESI) calcd for C23H32ClN3 [M – 2HCl + H]+,
350.2596; found, 350.2598.
Yield: 84.3%; mp: 146–148 ºC; IR (KBr) ν: 3389 (NH),
3035 (aromatic CH), 2927, 2853 (CH2, CH3), 1626 (C=N),
1508, 1470 (aromatic frame), 860, 820 cm–1; 1H NMR (300
MHz, CDCl3) δ: 4.56 (s, 2H, Bim-CH2), 5.40 (s, 2H,
2,4-F2Ph-CH2), 6.78−6.75 (m, 2H, 2,4-F2Ph-3,5-H),
6.93−6.87 (m, 2H, 3,5-(CF3)2Ph-2,6-H), 7.04 (s, 2H,
2,4-F2Ph-6-H, 3,5-(CF3)2Ph-4-H), 7.30−7.21 (m, 4H,
Bim-5,6,7,8-H) ppm; 13C NMR (75 MHz, CDCl3) δ: 41.9,
48.2, 103.1, 109.4, 110.8, 111.1, 112.1, 119.8, 120.3, 123.0,
123.6, 127.5, 127.6, 132.8, 141.9, 147.6, 150.1, 161.9,
166.9 ppm; MS (m/z): 486 [M + H]+; HRMS (ESI) calcd for
C23H16F8N3 [M + H]+, 486.1216; found, 486.1214.
N-((1-(2,4-Difluorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-3,5-bis(trifluoromethyl)aniline hydrochloride (7)
Compound 7 was prepared according to the procedure de-
scribed for compound 6, starting with compound 4m (101
mg, 0.2 mmol). The product 7 (85 mg) was obtained as yel-
low solid. Yield: 75.8%; mp > 250 ºC; IR (KBr) ν: 3493
(NH), 3069 (aromatic CH), 2931, 2882 (CH2, CH3), 1689
(C=N), 1605, 1579, 1517, 1464 (aromatic frame), 892, 853
cm–1; 1H NMR (300 MHz, DMSO-d6) δ: 5.16 (s, 2H,
Bim-CH2), 5.89 (s, 2H, 2,4-F2Ph-CH2), 7.09−7.04 (m, 1H,
2,4-F2Ph-3-H), 7.37−7.23 (m, 4H, 2,4-F2Ph-5,6-H, Bim-5,6-H),
7.50−7.42 (m, 1H, 3,5-(CF3)2Ph-4-H), 7.62−7.58 (m, 2H,
3,5-(CF3)2Ph-2,6-H), 7.89−7.82 (m, 1H, Bim-7,8-H) ppm;
13C NMR (75 MHz, DMSO-d6) δ: 43.1, 65.3, 104.8, 109.7,
111.5, 112.6, 113.4, 115.0, 117.9, 122.1, 125.8, 126.4,
126.6, 130.7, 131.2, 131.5, 133.0, 149.2, 153.0, 159.0,
160.9 ppm; MS (m/z): 486 [M – 2HCl + H]+; HRMS (ESI)
calcd for C23H16ClF8N3 [M – 2HCl + H]+, 486.1216; found,
486.1213.
N-((1-(2,4-Dichlorobenzyl)-1H-benzo[d]imidazol-2-yl)-
methyl)-3,5-bis(trifluoromethyl)aniline (5m)
Prepared according to the general procedure, starting with
compound 3i (1.049 g, 0.003 mol) and 2,4-dichloro-1-
(chloromethyl)benzene (0.603 g, 0.003 mol). Pure com-
pound 5m was obtained as white solid. Yield: 81.7%; mp:
178–180 ºC; IR (KBr) ν: 3391 (NH), 3036 (aromatic CH),
2925, 2859 (CH2, CH3), 1627 (C=N), 1602, 1512, 1462
(aromatic frame), 863, 822 cm–1; 1H NMR (300 MHz,
CDCl3) δ: 4.52 (s, 2H, Bim-CH2), 5.43 (s, 2H,
2,4-Cl2Ph-CH2), 6.40 (d, 1H, J = 6 Hz, 2,4-Cl2Ph-6-H), 7.00
(s, 2H, 3,5-(CF3)2Ph-2,6-H), 7.06 (d, 1H, J = 6 Hz,
2,4-Cl2Ph-5-H), 7.36−7.19 (m, 5H, Bim-5,6,7,8-H,
3,5-(CF3)2Ph-4-H), 7.49 (s, 2H, 2,4-Cl2Ph-3-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 41.9, 44.2, 109.4, 111.2,
112.1, 119.8, 123.0, 123.6, 127.5, 127.8, 129.7, 131.2,
132.1, 132.8, 134.7, 135.4, 141.9, 147.6, 150.1 ppm; MS
(m/z): 518 [M + H]+; HRMS (ESI) calcd for C23H16Cl2F6N3
[M + H]+, 518.0625; found, 518.0622.
7-Hydroxy-4-methyl-2H-chromen-2-one (9a)
Compound 9a was prepared according to the procedure de-
scribed in the literature, starting with compound 8a (11.02 g,
100.0 mmol), ethyl acetoacetate (13.61 g, 100.0 mmol), and
oxalic acid (5.24 g, 50.0 mmol). The desired compound 9a
(15.46 g) was obtained as white solid. Yield: 95.0%; mp:
191−193 ºC, in agreement with the literature [31] (mp:
192−193 ºC).
3-Acetyl-7-hydroxy-2H-chromen-2-one (9b)
4-Methyl-N-((1-octyl-1H-benzo[d]imidazol-2-yl)methyl)-
aniline hydrochloride (6)
Compound 9b was synthesized according to the experi-
mental procedure reported for compound 9a, starting with
compound 8b (13.75 g, 100.0 mmol), ethyl acetoacetate
(13.61 g, 100.0 mmol), and oxalic acid (5.21 g, 50.0 mmol).
The desired compound 9b (17.73 g) was obtained as white
solid. Yield: 86.7%; mp: 212−213 ºC (literature [31] mp:
192−193 ºC).
To a solution of compound 4c (98 mg, 0.3 mmol) in ethyl
ether (10 mL) was added hydrochloride acid (10 mL, 4
mol/L). The mixture was stirred at room temperature for
56 h. After the reaction was completed (monitored by TLC,
eluent; ethyl acetate/petroleum ether (1/1, v/v)), the formed
precipitate was filtered, washed with chloroform, and dried
by freeze-drying oven to afford hydrochloride hydrochlo-
ride 6 (80 mg) as yellow solid. Yield: 63.5%; mp: 178–180
ºC; IR (KBr) ν: 3487 (NH), 3057 (aromatic CH), 2930,
2853 (CH2, CH3), 1687 (C=N), 1601, 1582, 1521, 1469
7-(6-Bromohexyloxy)-4-methyl-2H-chromen-2-one (10a)
Compound 10a was prepared according to the procedure
described in the literature, starting with compound 9a (1.62
g, 10.0 mmol), 1,4-dibromohexane (2.91 g, 12.0 mmol), and
potassium carbonate (2.14 g, 15.1 mmol). The desired
compound 10a (2.92 g) was obtained as white solid. Yield:
84.1%; mp: 56−58 ºC, in agreement with the literature [31]
(mp: 56−57 ºC).
1
(aromatic frame), 1383, 1379 (CH3), 825 cm–1; H NMR
(300 MHz, CDCl3) δ: 0.91−0.89 (t, 3H, J = 3.0 Hz,
CH2CH3), 1.53−1.51 (m, 12H, (CH2)6CH3), 2.33 (s, 3H,
4-CH3Ph-CH3), 5.07−5.05 (t, 2H,
J
=
3.0 Hz,
CH2(CH2)6CH3), 5.11 (s, 2H, Bim-CH2), 7.25−7.23 (m, 2H,
4-CH3Ph-3,5-H), 7.54−7.51 (m, 2H, Bim-6,7-H), 7.74 (m,
2H, 4-CH3Ph-2,6-H), 7.91−7.89 (m, 2H, Bim-5,8-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 14.0, 20.7, 21.8, 27.3, 28.9,
29.0, 31.8, 42.4, 64.4, 113.5, 115.6, 119.3, 123.2, 129.1,
133.2, 151.5, 151.9, 159.9 ppm; MS (m/z): 350 [M – 2HCl
3-Acetyl-7-(4-(bromomethyl)benzyloxy-2H-chromen-2-one
(10b)
Compound 10b was prepared according to the experimental
procedure reported for compound 10a, starting with com-
pound 9b (2.07 g, 10.0 mmol), 1,4-bis(bromomethyl)

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
9
benzene (3.18 g, 12.0 mmol), and potassium carbonate
N-((1-(4-(9H-Carbazol-9-yl)butyl)-1H-benzo[d]imidazol-2-
(2.15 g, 15.1 mmol). The desired compound 10b (2.76 g)
was obtained as white solid. Yield: 71.2%; mp: 68−69 ºC,
in agreement with the literature [31] (mp: 66−67 ºC).
yl)methyl)-2-fluoroaniline (14)
Compound 14 was obtained as yellow solid. Yield: 62.5%;
mp: 143–145 ºC; IR (KBr) ν: 3382 (NH), 3053 (aromatic
CH), 2964, 2863 (CH2, CH3), 1619 (C=N), 1592, 1511,
7-(6-(2-((4-Fluorophenylamino)methyl)-1H-benzo[d]-
imidazol-1-yl)hexyloxy)-4-methyl-2H-chromen-2-one (11a)
1453 (aromatic frame), 848, 751, 722 cm–1; H NMR (300
1
MHz, CDCl3) δ: 1.94−1.84 (m, 4H, carbazole-CH2(CH2)2),
4.02 (m, 2H, carbazole-(CH2)3CH2), 4.27 (m, 2H, carba-
zole-CH2), 4.43 (s, 2H, Bim-CH2), 6.72−6.66 (m, 1H,
2-FPh-4-H), 6.84−6.78 (t, 1H, J = 9.0 Hz, 2-FPh-6-H),
6.99−6.96 (m, 2H, 2-FPh-3,5-H), 7.16 (d, 1H, J = 9.0 Hz,
Bim-7-H), 7.32−7.24 (m, 6H, Bim-6,8-H, carbazole-
4,5,10,11-H), 7.47−7.42 (t, 2H, J = 7.5 Hz, Bim-5-H, car-
bazole-12-H), 7.76−7.73 (m, 1H, carbazole-3-H), 8.11−8.09
(d, 2H, J = 6.0 Hz, carbazole-6,9-H) ppm; 13C NMR (75
MHz, CDCl3) δ: 26.5, 27.5, 41.4, 42.5, 43.6, 108.5, 109.5,
112.7, 112.8, 114.5, 114.7, 117.9, 118.0, 119.1, 119.7,
120.5, 122.2, 122.8, 124.7, 124.8, 125.8, 135.3, 140.2,
150.8 ppm; MS (m/z): 463 [M + H]+; HRMS (ESI) calcd for
C30H27FN4 [M + H]+, 463.2298; found, 463.2296.
Compound 11a was obtained as yellow solid. Yield: 51.3%;
mp: 123–125 ºC; IR (KBr) ν: 3383 (NH), 3034 (aromatic
CH), 2938, 2860 (CH2, CH3), 1624 (C=N), 1599, 1510,
1464 (aromatic frame), 1390 (CH3), 837, 746, 637 cm–1;
1H NMR (300 MHz, CDCl3) δ: 1.53−1.49 (m, 4H, couma-
rin-O(CH2)2(CH2)2(CH2)2), 1.83−1.80 (m, 4H, couma-
rin-OCH2CH2(CH2)2CH2), 2.39 (s, 3H, coumarin-CH3),
4.04−3.95 (m, 2H, coumarin-O(CH2)5CH2), 4.41−4.38 (t,
2H, J = 9.0 Hz, coumarin-OCH2), 4.51 (s, 2H, Bim-CH2),
6.14 (s, 1H, coumarin-3-H), 6.82−6.77 (m, 2H,
4-FPh-2,6-H), 6.99−6.94 (m, 2H, coumarin-7,9-H),
7.14−7.09 (m, 2H, 4-FPh-3,5-H), 7.34−7.27 (m, 4H,
Bim-5,6,7,8-H), 7.73 (d, 1H, J = 9.0 Hz, coumarin-6-H)
ppm; 13C NMR (75 MHz, CDCl3) δ: 19.0, 24.3, 26.4, 28.4,
28.5, 41.5, 47.2, 48.8, 109.3, 114.2, 114.3, 114.4, 115.1,
115.2, 115.3, 116.6, 123.1, 125.4, 138.3, 145.6, 149.2,
154.2, 155.4, 156.6, 156.8, 158.0 ppm; MS (m/z): 500 [M +
H]+; HRMS (ESI) calcd for C30H30FN3O3 [M + H]+,
500.2349; found, 500.2347.
2.3 Biological assays
The in vitro minimal inhibitory concentrations (MICs) of
the target compounds were determined using the twofold
serial dilution technique in 96-well microtest plates, ac-
cording to the National Committee for Clinical Laboratory
Standards (NCCLS) [39, 40]. The tested microorganism
strains were provided by the School of Pharmaceutical Sci-
ences, Southwest University and the College of Pharmacy,
Third Military Medical University, China. Orbifloxacin,
Chloromycin, and Fluconazole were used as standard drugs.
3-Acetyl-7-(4-((2-((p-tolylamino)methyl)-1H-benzo[d]-
imidazol-1-yl)methyl)benzyloxy)-2H-chromen-2-one (11b)
Compound 11b was obtained as brown syrup. Yield: 56.3%;
IR (KBr) ν: 3385 (NH), 3035 (aromatic CH), 2921, 2854
(CH2, CH3), 1619 (C=N), 1597, 1574, 1462 (aromatic
1
frame), 1386 (CH3), 839, 749, 638 cm–1; H NMR (300
Antibacterial assays
MHz, CDCl3) δ: 2.17 (s, 3H, coumarin-CH3), 2.24 (s, 3H,
4-CH3Ph-CH3), 4.50 (s, 2H, Bim-CH2), 5.20 (s, 2H, couma-
rin-OCH2), 5.39 (s, 2H, Ph-CH2), 6.61 (d, 2H, J = 9.0 Hz,
4-CH3Ph-2,6-H), 7.06−7.03 (m, 2H, coumarin-6,8-H), 7.10
(d, 2H, J = 9.0 Hz, 4-CH3Ph-3,5-H), 7.19−7.13 (m, 4H,
Ph-H), 7.32−7.25 (m, 2H, Bim-5,6,7,8-H), 7.83 (d, 1H, J =
9.0 Hz, coumarin-5-H), 8.92 (s, 1H, coumarin-4-H) ppm;
13C NMR (75 MHz, CDCl3) δ: 20.2, 28.6, 41.5, 46.3, 69.1,
109.3, 114.0, 114.1, 114.2, 115.1, 122.7, 125.8, 128.5,
128.9, 130.7, 131.1, 134.8, 136.9, 137.0, 138.2, 145.7,
152.1, 157.1, 157.9, 158.8, 185.3 ppm; MS (m/z): 544 [M +
H]+; HRMS (ESI) calcd for C34H29N3O4 [M + H]+, 544.2236;
found, 544.2238.
The prepared compounds 4–7, 11, and 14 were evaluated
for their antibacterial activities against Gram-positive bacte-
ria (S. aureus ATCC 6538, Methicillin-resistant Staphylo-
coccus aureus N315 (MRSA), B. subtilis ATCC 21216, and
M. luteus), Gram-negative bacteria (E. coli ATCC 8099, P.
aeruginosa ATCC 27853, B. proteus ATCC 13315, and B.
typhi). The bacterial suspension was adjusted with sterile
saline to a concentration of 1 × 105 CFU. The tested com-
pounds were dissolved in DMSO to prepare the stock solu-
tions. The tested compounds and reference drugs were pre-
pared in Mueller-Hinton broth (Guangdong Huaikai Micro-
bial Sci & Tech Co., Ltd., Guangzhou, Guangdong, China)
by two fold serial dilution to obtain the required concentra-
tions of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 µg/mL.
These dilutions were inoculated and incubated at 37 ºC for
24 h. To ensure that the solvent had no effect on bacterial
growth, a control test was performed with test medium sup-
plemented with DMSO at the same dilutions used in the
experiment. The minimum inhibitory concentration values
(MICs) (in µg/mL) for 4–7, 11, and 14 are summarized in
Table 1.
9-(4-Bromobutyl)-9H-carbazole (13)
Compound 13 was prepared according to the procedure de-
scribed in the literature, starting with 9H-carbazole (4.99 g,
30.0 mmol), 1,4-dibromobutane (6.03 g, 30.1 mmol), and
sodium hydride (1.22 g, 50.0 mmol). The desired compound
13 (7.05 g) was obtained as white solid. Yield: 81.5%; mp:
102−103 ºC, in agreement with the literature [34] (mp:
103−104 ºC).

10
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
Table 1 ClogP values and antibacterial data as MIC (μg/mL) for compounds 4–14 a, b, c)
Gram-positive bacteria
Gram-negative bacteria
Compounds
ClogP
S. aureus
16
MRSA
256
256
128
64
B. subtilis
64
M. luteus
64
B. proteus
128
64
E. coli
32
P. aeruginosa
B. typhi
64
4a
4.09
5.67
6.73
7.79
8.85
12.02
6.05
7.13
6.17
5.45
5.51
5.51
5.01
6.22
6.10
6.10
6.42
7.06
8.20
7.87
7.48
6.64
5.66
7.32
–1.09
0.58
16
64
16
128
16
64
64
32
64
32
128
64
32
32
64
128
64
16
32
128
16
16
128
64
16
1
4b
128
32
64
256
128
128
128
256
64
256
128
128
128
256
32
64
4c
16
64
64
4d
128
32
64
256
64
128
32
4e
128
128
128
32
16
4f
128
64
64
64
64
5a
16
64
128
32
5b
32
64
32
64
64
5c
32
64
64
64
32
128
32
64
5d
64
128
128
64
64
128
128
128
128
64
32
64
5e
64
64
128
256
64
32
128
64
5f
128
32
128
64
128
32
5g
64
32
5h
128
64
128
64
64
64
128
32
128
64
5i
32
128
64
128
128
32
5j
32
64
128
64
64
64
5k
64
128
32
64
32
128
32
5l
16
16
16
16
8
5m
32
64
16
64
16
64
32
6
16
64
16
64
64
128
32
64
7
11a
8
16
8
8
16
16
32
128
64
16
128
64
64
64
64
11b
32
64
256
64
128
128
16
64
14
64
64
64
128
8
64
Chloromycin
Norfloxacin
8
16
32
32
32
8
1
2
1
4
1
1
a) Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates. b) S. aureus, Staphylococcus aureus
(ATCC25923); MRSA, Methicillin-Resistant Staphylococcus aureus (N315); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus (ATCC4698); B.
proteus, Bacillus proteus (ATCC13315); E. coli, Escherichia coli (JM109); P. aeruginosa, Pseudomonas aeruginosa; B. typhi, Bacillus typhi; C. albicans,
Candida albicans (ATCC76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisia, Saccharomyces cerevisia; A. flavus, Aspergil-
lus flavus. C) ClogP values were calculated by a ChemDraw Ultra 10.0 (Cambridge Software Corporation, Hopkinton, MA, USA).
Antifungal assays
3 Results and discussion
The newly synthesized compounds 4–7, 11, and 14 were
evaluated for their antifungal activities against C. albicans
ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cere-
visia, and A. flavus. A spore suspension in sterile distilled
water was prepared from a culture of the fungi growing on
Sabouraud agar (SA) media, aged 1 d. The final spore con-
centration was 1 × 1035 × 103 spore mL1. From the stock
solutions of the tested compounds and reference antifungal
drug Fluconazole, dilutions in sterile RPMI 1640 medium
(Neuronbc Laboraton Technology CO., Ltd., Beijing, China)
were made, resulting in eleven desired concentrations (0.5
to 512 μg/mL) of each tested compound. These dilutions
were inoculated and incubated at 35 ºC for 24 h. The mini-
mum inhibitory concentration values (MICs) (in µg/mL) are
summarized in Table 2.
3.1 Effect of clogP values on antimicrobial activities
Hydrophobic/lipophilic properties played an important role
in exerting biological activities [41, 42]. The clogP values
have been widely employed to predict bioactivity. As seen
in Table 1, the antimicrobial activities of benzimidazoles
4a–c were generally enhanced as the clogP values increased,
but the bioactivities were decreased in compounds 4d–f.
These results might be explained by lipophilic ability that
was favorable for delivering the compounds to the binding
sites in organisms, and indicative of the significant role of
preferable lipophilicity in drug design.
3.2 Biological activity
The in vitro antimicrobial screening for all of the synthesized

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
Table 2 Antifungal data as MIC (μg/mL) for compounds 4–14 a, b, c)
11
Compounds
C. mycoderma
C. albicans
128
128
32
C. utilis
64
128
32
64
32
64
32
32
64
32
64
64
64
32
64
64
32
8
S. cerevisiae
A. flavus
128
64
4a
128
64
16
64
64
32
32
64
32
64
32
64
128
32
64
128
128
16
32
64
8
64
64
16
32
64
64
64
32
64
64
32
128
64
64
64
128
32
16
32
64
8
4b
4c
32
4d
128
16
64
4e
64
4f
32
128
64
5a
32
5b
32
64
5c
64
64
5d
32
32
5e
32
64
5f
32
64
5g
64
128
32
5h
32
5i
128
32
128
128
64
5j
5k
64
5l
32
16
5m
32
64
64
16
16
64
32
8
32
6
128
16
64
7
11a
8
16
64
32
1
32
32
32
64
16
32
11b
64
32
14
64
64
Fluconazole
4
256
compounds were evaluated for four Gram-positive bacteria
(Staphylococcus aureus ATCC 6538, Methicillin-resistant
Staphylococcus aureus N315 (MRSA), Micrococcus luteus
ATCC 4698, and Bacillus subtilis ATCC 21216); four
Gram-negative bacteria (Escherichia coli ATCC 8099,
Pseudomonas aeruginosa ATCC 27853, Bacillus typhi, and
Bacillus proteus ATCC 13315); and five fungi (Candida
albicans ATCC 76615, Candida mycoderma, Candida utilis,
Saccharomyces cerevisia, and Aspergillus flavus) using the
twofold serial dilution technique recommended by the Na-
tional Committee for Clinical Laboratory Standards
(NCCLS), with positive controls of the clinically antimicro-
bial drugs Chloromycin, Norfloxacin, and Fluconazole [43].
The combination studies were screened by the microdilution
checkerboard method as described by Fivelman and col-
leagues (FIC (fractional inhibitory concentration) = MIC of
compound A in mixture/MIC of compound A alone + MIC
of compound B in mixture/MIC of compound B alone; FIC
 1 represents synergistic effect; FIC  1 and < 2 represents
additive interaction; and FIC  2 represents antagonistic
effect) [4446]. The values of clogP, a partition coefficient
as a kind of measurement of hydrophobicity/lipophilicity,
were calculated using ChemDraw Ultra 10.0 software inte-
grated with Cambridgesoft software (Cambridge Software
Corporation, Hopkinton, MA, USA). The antibacterial and
antifungal data as well as clogP values are depicted in Ta-
bles 15.
Antibacterial activity
The antibacterial test indicated that the newly prepared tar-
get compounds could, to some extent, effectively inhibit the
growth of all the tested microorganisms in vitro (i.e., they
exhibited potent antibacterial activity and broad spectra).
Results (Table 1) showed that the types of N-alkylated
moieties and substituents on aniline rings exerted significant
effects on the biological activity. It could be clearly seen
that most of the alkyl benzimidazole derivatives 4af gave
moderate bioactivity against all of the tested bacteria and
were relatively more sensitive to B. subtilis than other bac-
teria. N-Octyl benzimidazole 4c displayed the best anti-B.
subtilis activity with an MIC value of 16 μg/mL, which was
twice as potent as Chloromycin (MIC = 32 μg/mL) and
showed equivalent potency against P. aeruginosa as Chlo-
romycin at the concentration of 16 μg/mL. The substitution
of butyl moiety for the octyl group, which produced com-
pound 4b, led to lower ability against the tested strains, with
MIC values ranging from 64 to 256 μg/mL. Additionally,
benzimidazole derivatives 4df, all decyl-, dodecyl-, and

12
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
Table 3 Synergistic effects of compounds 5l and 7 with antibacterial Chloromycin a, b, c)
Bacteria
Compounds
MIC
1
FIC index
0.250
Effect
Bacteria
Compounds
MIC
1
FIC index
0.375
Effect
Chloromycin
Chloromycin
S. aureus
Synergistic
S. aureus
Synergistic
5l
2
7
2
Chloromycin
2
Chloromycin
2
MRSA
B. subtilis
M. luteus
B. proteus
E. coli
0.375
0.375
0.500
0.250
0.250
0.375
0.250
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
MRSA
B. subtilis
M. luteus
B. proteus
E. coli
0.375
0.500
0.375
0.375
0.500
0.375
0.375
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
5l
8
7
4
Chloromycin
4
Chloromycin
8
5l
4
7
4
Chloromycin
2
Chloromycin
1
5l
4
7
2
Chloromycin
4
Chloromycin
4
5l
2
7
4
Chloromycin
2
Chloromycin
4
5l
1
7
8
Chloromycin
2
Chloromycin
2
P. aeruginosa
B. typhi
P. aeruginosa
B. typhi
5l
Chloromycin
5l
4
7
4
4
Chloromycin
4
4
7
4
Table 4 Synergistic effects of compounds 5l and 7 with antibacterial Norfloxacin a, b, c)
Bacteria
Compounds
MIC
1
FIC index
0.250
Effect
Bacteria
Compounds
MIC
1
FIC index
0.250
Effect
Norfloxacin
Norfloxacin
S. aureus
Synergistic
S. aureus
Synergistic
5l
2
7
1
Norfloxacin
0.25
8
Norfloxacin
0.25
2
MRSA
B. subtilis
M. luteus
B. proteus
E. coli
0.500
0.250
0.375
0.250
0.375
0.375
0.375
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
MRSA
B. subtilis
M. luteus
B. proteus
E. coli
0.375
0.250
0.250
0.250
0.375
0.375
0.375
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
5l
7
Norfloxacin
0.25
2
Norfloxacin
0.25
2
5l
7
Norfloxacin
0.125
4
Norfloxacin
0.125
1
5l
7
Norfloxacin
0.5
2
Norfloxacin
0.5
2
5l
7
Norfloxacin
0.25
2
Norfloxacin
0.25
4
5l
Norfloxacin
5l
7
0.25
2
Norfloxacin
0.125
4
P. aeruginosa
B. typhi
P. aeruginosa
B. typhi
7
Norfloxacin
5l
0.125
8
Norfloxacin
0.25
2
7
Table 5 Synergistic effects of compounds 5l and 7 with antifungal Fluconazole a, b, c)
MIC (μg/mL)
MIC (μg/mL)
Fungi
Compounds
FIC index
0.500
Effect
Fungi
Compounds
FIC index
0.375
Effect
Fluconazole
0.25
4
Fluconazole
0.25
1
C. mycoderma
Synergistic
C. mycoderma
Synergistic
5l
7
Fluconazole
1
Fluconazole
0.5
2
C. albicans
C. utilis
0.375
0.375
0.375
0.375
Synergistic
Synergistic
Synergistic
Synergistic
C. albicans
C. utilis
0.250
0.375
0.375
0.375
Synergistic
Synergistic
Synergistic
Synergistic
5l
4
7
Fluconazole
2
Fluconazole
1
5l
Fluconazole
5l
1
7
4
2
Fluconazole
2
S. cerevisiae
A. flavus
S. cerevisiae
A. flavus
4
7
2
Fluconazole
5l
32
4
Fluconazole
32
2
7
octadecyl-alkylated compounds, exhibited relatively weaker
inhibitory activity. As mentioned above, the length of the
alkyl chain had remarkable effects on biological activities,
and that the suitable length of the alkyl chains with con-
formable lipophilicity in the target compounds might make
them favorable for delivery to the binding sites.

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
13
fungal activity, with MIC values ranging from 8 to 32
μg/mL among the aralkyl benzimidazoles. It gave especially
good activity toward A. flavus fungi, with MIC value of 16
μg/mL, which was sixteen times more potent than Flucona-
zole. Moreover, hydrochlorides 6 and 7 displayed higher
bioactivity, with low MIC value of 8128 μg/mL, than cor-
responding precursors; this result was attributed to the im-
proved water solubility.
Compared with alkyl benzimidazole compounds 4af,
most of the halobenzyl benzimidazole derivatives 5am
gave relatively better bioactivities in inhibiting the growth
of the tested strains. Compound 5l with 2,4-difluorobenzyl
group exerted the best efficiency, in MIC values of 8–32
μg/mL, toward the corresponding bacteria. Notably, its
anti-B. subtilis, anti-B. proteus, and anti-E. coli activities
(MIC = 16, 16, and 8 μg/mL respectively) were twice as
potent as the reference drug Chloromycin. However, sub-
stitution of 2,4-difluorobenzyl group with a 2,4-dichloro-
benzyl fragment, which yielded compound 5m, resulted in
weaker antibacterial efficacy at the concentrations of 16–64
μg/mL. The 3-trifluoromethyl aniline containing benzimid-
azole 5b displayed moderate antibacterial activity, with
MIC values ranging from 32 to 64 μg/mL.
As reported, the introduction of coumarin or carbazole
ring was beneficial to the improvement of antimicrobial
potency [30, 32, 33]. In our research, the coumarin benzim-
idazole 11a gave stronger antibacterial activity against some
of the tested strains, such as B. subtilis (MIC = 16 μg/mL)
and P. aeruginosa (MIC = 16 μg/mL), than the correspond-
ing compound 4b. Moreover, the replacement of the alkyl
chain (CH2)6 with a phenylene spacer, which yielded com-
pound 11b, improved the antibacterial efficacy to some ex-
tent. A similar phenomenon was observed in carbazole ben-
zimidazole derivative 14 (MIC = 64128 μg/mL).
In addition, some strongly active compounds were cho-
sen to transform into water-soluble hydrochlorides 6 and 7,
to enhance the bioactivity. As was expected, salts 6 and 7
exhibited two to four times more potent inhibitory activity
than their corresponding precursors. Among all target ben-
zimidazole derivatives, including alkyl compound 4, aralkyl
derivative 5, coumarin compound 11, and carbazole deriva-
tive 14, as well as their corresponding salts, compound 7
displayed the best antibacterial activity, at the concentra-
tions of 832 μg/mL. It was especially more sensitive to the
S. aureus, B. subtilis, and M. luteus species, with an MIC
value of 8 μg/mL, which was almost equipotent or even
superior to the reference drug Chloromycin.
3.3 Synergistic effects
The combination of target benzimidazole derivatives with
antibacterial Chloromycin or Norfloxacin against microor-
ganisms are described in Tables 3 and 4. The combinations
of the representative benzimidazoles with clinical drugs
gave excellent antibacterial efficacies with lower dosages
and broader antimicrobial spectra; in addition, all the FIC
indexes were less than 0.5. Therefore, these combined sys-
tems of benzimidazoles with Chloromycin or Norfloxacin
had excellent synergistic effects. Notably, the combination
of compounds 5l or 7 with Chloromycin (2 μg/mL) or Nor-
floxacin (0.25 μg/mL) was eight or four times, respectively,
more potent against MRSA than the uncombined com-
pounds alone.
As shown in Table 5, combinations of Fluconazole with
compounds 5l or 7 also manifested good activity against all
the tested fungi with low MIC values of 14 μg/mL. Intri-
guingly, these combinations displayed excellent activity
against Fluconazole-insensitive A. flavus in comparison
with Fluconazole employed alone (MIC = 256 μg/mL). All
of these results demonstrated that these combinations could
highly enhance antimicrobial activity, overcome drug re-
sistance and broaden spectra, and these results might be
attributed to the different binding sites of these compounds
toward the tested microorganism.
3.4 Interaction with calf thymus DNA
DNA as the informational molecule encoding the genetic
instructions was widely employed in the development and
function of almost all known living organisms. Recently,
DNA as the therapeutic target has attracted considerable
attention in biomedical field. The binding studies of small
molecules with DNA are very important for the rational
design and construction of novel and efficient drugs which
could target to DNA. Calf thymus DNA was always select-
ed as DNA model due to its medical importance, low cost
and ready available properties. To explore the possible an-
timicrobial action mechanism, the binding behavior of
compound 5l with calf thymus DNA was researched on
molecular level in vitro using neutral red (NR) dye as a
spectral probe by UV-Vis spectroscopic methods [47].
Antifungal activity
The antifungal evaluation in vitro revealed that all target
benzimidazole derivatives gave better activity against Flu-
conazole-insensitive A. flavus than clinical Fluconazole
(MIC = 256 μg/mL). Compound 5m displayed comparable
efficacy to Fluconazole against C. utilis and S. cerevisiae,
with MIC values of 8 and 16 μg/mL respectively, whereas
compound 4c showed equivalent anti-S. cerevisiae activity
to the clinical drug. However, all the prepared compounds
gave weaker activity toward other fungi than the reference
drug. As depicted, benzimidazole 4c with alkyl chain
(CH2)8 was observed to be the suitable length and possessed
the best antifungal efficacy in series of alkyl benzimidazole
derivatives 4af. Similar to the antibacterial results,
2,4-diflurobenzyl derivative 5l manifested the highest anti-
Absorption spectra of DNA in the presence of compound 5l
The application of absorption spectroscopy is one of the

14
Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
fundamental techniques in DNA-binding studies. Generally,
hypochromism and hyperchromism are regarded as im-
portant spectral features to distinguish the change of DNA
double-helical structure. The hyperchromism originates
from the breakage of the DNA duplex secondary structure;
while the hypochromism generates from the stabilization of
the DNA duplex by either the intercalation binding mode or
the electrostatic effect of small molecules [48]. The ob-
served large hypochromism strongly suggested a close
proximity of the aromatic chromophore to the DNA bases,
which might be attributed to the strong interaction between
the electronic states of intercalating chromophore and that
of the DNA base. With a fixed concentration of DNA,
UV-Vis absorption spectra were recorded with the increas-
ing amount of compound 5l. As shown in Figure 2, UV-Vis
spectra showed that the maximum absorption of DNA at
260 nm displayed proportional increase with the increasing
concentration of compound 5l. Simultaneously, the absorp-
tion value of simply sum of free DNA and free compound
5l was a little greater than the measured value of 5l-DNA
complex, which was observed in the inset of Figure 2. The-
se indicated that a weak hypochromic effect existed be-
tween DNA and compound 5l. Moreover, the intercalation
of the aromatic chromophore of compound 5l into the helix
and the strong overlap of π-π* states in the large π-conju-
gated system with the electronic states of DNA bases were
consistent with the observed spectral changes [35, 49].
On the basis of the variations in the absorption spectra of
DNA upon binding to 5l, Eq. (1) can be utilized to calculate
the binding constant (K).
absorption coefficients of compound 5l and 5l-DNA com-
plex, respectively. The plot of A0/(A-A0) versus
1/[compound 5l] is constructed by using the absorption ti-
tration data and linear fitting (Figure 3), yielding the bind-
ing constant, K = 1.54 × 104 L/mol, R = 0.998, SD = 0.16
(R is the correlation coefficient; SD is standard deviation).
Absorption spectra of NR interaction with DNA
Neutral Red (NR) is a planar phenazine dye and is structurally
similar to other planar dyes, for example, those of the acri-
dine, thiazine and xanthene kind. Recently, it has been
demonstrated that the binding of NR with DNA is an inter-
calation binding. Therefore, NR was employed as a spectral
probe to investigate the binding mode of 5l with DNA in the
present work.
The absorption spectra of the NR dye upon the addition
of DNA were showed in Figure 4. It was apparent that the
absorption peak of the NR at around 460 nm showed gradu-
al decrease with the increasing concentration of DNA, and a
new band at around 530 nm developed. This was attributed
to the formation of the new DNA-NR complex. An isosbes-
tic point at 504 nm provided evidence of DNA-NR complex
formation.
A0
1
C
C



(1)
A A0 DC C DC C K[Q]
A0 and A represent the absorbance of DNA in the absence
and presence of compound 5l at 260 nm, ξC and ξD-C are the
Figure 3 The plot of A0/(A-A0) versus 1/[compound 5l].
Figure 2 UV absorption spectra of DNA with different concentrations of
compound 5l (pH = 7.4, T = 303 K). Inset: comparison of absorption at 260
nm between the 5l-DNA complex and the sum values of free DNA and free
compound 5l. c(DNA) = 9.78 × 105 mol/L, and c(compound 5l) = 0–1.2 ×
105 mol/L for curves a–g respectively at increment 0.2 × 105 mol/L.
Figure 4 UV absorption spectra of NR in the presence of DNA at pH 7.4
and room temperature. c(NR) = 2 × 105 mol/L, and c(DNA) = 0–3.81 ×
105 mol/L for curves a–i respectively at increment 0.48 × 105 mol/L.

Zhang HZ, et al. Sci China Chem May (2014) Vol.57 No.5
15
Absorption spectra of competitive interaction of compound
5l and NR with DNA
stronger) with less dosage as well as broader antimicrobial
spectrum than the separated use of them alone. Intriguingly,
the combined systems were more sensitive to Flucona-
zole-insensitive A. flavus and methicillin-resistant MRSA.
The specific interaction of compound 5l with calf thymus
DNA by UV-Vis absorption spectroscopy manifested that
Compound 5l can intercalate into DNA to form 5l-DNA
complex, which might further block DNA replication and
exert powerful antibacterial and antifungal activities.
As shown in Figure 5, the competitive binding between NR
and 5l with DNA was observed in the absorption spectra.
With the increasing concentration of compound 5l, an ap-
parent intensity increase was observed around 276 nm.
Compared with the absorption around 276 nm of NR-DNA
complex, the absorbance at the same wavelength exhibited
the reverse process (inset of Figure 5). These various spec-
tral changes were consistent with the intercalation of com-
pound 5l into DNA by substituting for NR in the DNA-NR
complex.
This work was partly supported by the National Natural Science Founda-
tion of China (21172181, 21372186), the Research Fellowship for Interna-
tional Young Scientists from the International (Regional) Cooperation and
Exchange Program (81250110554, 81350110338, 81350110523), the key
program from Natural Science Foundation of Chongqing (CSTC2012-
jjB10026), and the Specialized Research Fund for the Doctoral Program of
Higher Education of China (20110182110007).
4 Conclusions
In summary, a novel series of benzimidazole derivatives
have been successfully synthesized starting with commer-
cial o-phenylene diamine, chloroacetic acid and anilines.
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All new compounds were confirmed by IR, H NMR, 13C
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activities revealed that most title compounds showed mod-
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some displayed equipotent or superior activities to the clin-
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