Microwave synthesis, biological evaluation and docking studies of 2-substituted methyl 1-(4-fluorophenyl)-1H-benzimidazole-5-carboxylates

Article abstract of DOI:10.1007/s00044-016-1763-z

A library of 22 novel 2-substituted fluorinated benzimidazoles (5a-v) was synthesized under microwave conditions in yields of between 85–96% and tested for their antimicrobial and antioxidant activity. Two trioxygenated derivatives 5p and 5r had minimum bactericidal concentration values ranging between 14.5–115.7 μM (5p) and 25.6–74.3 μM (5r) against S. aureus, E. coli, P. aeruginosa and K. pneumoniae. The benzimidazole 5e with a CF₃ substituent had the best antifungal activity at 94.3 μM against C. albicans. Compounds 5p and 5r also showed good antioxidant activities of 386.6 and 306.7 μM respectively, comparable to that of ascorbic acid. Docking studies of 5 h and 5r into the active site of topoisomerase II DNA-gyrase indicated that interaction with the Mn²⁺ ion in the active site of the enzyme was crucial for antibacterial activity.

Full text of DOI:10.1007/s00044-016-1763-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1763-z
ORIGINAL RESEARCH
Microwave synthesis, biological evaluation and docking studies of
2-substituted methyl 1-(4-fluorophenyl)-1H-benzimidazole-5-
carboxylates
Suhas A. Shintre1 Deresh Ramjugernath2 Parvesh Singh1 Chunderika Mocktar3
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●
●
●
1
Neil A. Koorbanally
Received: 7 September 2016 / Accepted: 19 December 2016
© Springer Science+Business Media New York 2016
Abstract A library of 22 novel 2-substituted fluorinated
benzimidazoles (5a-v) was synthesized under microwave
conditions in yields of between 85–96% and tested for their
antimicrobial and antioxidant activity. Two trioxygenated
derivatives 5p and 5r had minimum bactericidal con-
centration values ranging between 14.5–115.7 μM (5p) and
25.6–74.3 μM (5r) against S. aureus, E. coli, P. aeruginosa
and K. pneumoniae. The benzimidazole 5e with a CF3
substituent had the best antifungal activity at 94.3 µM
against C. albicans. Compounds 5p and 5r also showed
good antioxidant activities of 386.6 and 306.7 µM respec-
tively, comparable to that of ascorbic acid. Docking studies
of 5 h and 5r into the active site of topoisomerase II DNA-
gyrase indicated that interaction with the Mn2+ ion in the
active site of the enzyme was crucial for antibacterial
activity.
Introduction
Resistance to antibiotics is one of the major health concerns
plaguing mankind in the 21st century. There is a constant
need for new and improved antibiotics to fight of strains of
bacteria that have become resistant to currently prescribed
antibiotics. The benzimidazole moiety has gained impor-
tance in recent years having antibacterial, antifungal, anti-
viral, anticancer, antioxidant, anthelmintic, antiparasitic,
antimycobacterial, antidiabetic, antihypertensive, analgesic,
antipsychotic, anticoagulant, cardiovascular, and anti-
inflammatory properties (Navarrete-Vázquez et al. 2001;
Tomic et al. 2004; Biron 2006; Kuş et al. 2009; Narasimhan
et al. 2010; Vyas and Ghate 2010; Arora 2011; Kalyankar
et al. 2012; Barot et al. 2013; Gurvinder et al. 2013; Jain
et al. 2013; Keri et al. 2015; Yoon et al. 2014; Singla et al.
2015). Synthesizing novel compounds with the benzimi-
dazole moiety can therefore produce lead compounds for
the pharmaceutical industry.
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Keywords Benzimidazole Microwave Thermal
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Antimicrobial Antioxidant Docking
Benzimidazoles are generally synthesized by coupling
reactions between o-phenylenediamines with carboxylic
acids, carboxylic acid chlorides or aldehydes and in some
cases esters and amides (Khanna et al. 2012; Panda et al.
2012; Keri et al. 2015; Prajapti et al. 2015; Azizian et al.
2016; Carvalho et al. 2015; Kattimani et al. 2015; Rithe
et al. 2015; Saberi 2015; Saleh et al. 2015). In most cases,
these reactions are carried out in the presence of acid cat-
alysts containing Sn, Ti, Zr, Bi, In, Co, Ce, B, Zn, and Hf
(Zhang et al. 2007), Ir (Tateyama et al. 2016), La (Kamal
et al. 2014), acetic acid (Jain et al. 2013) and p-toluene-
sulfonic acid (Xiangming et al. 2007; Funel et al. 2014),
basic catalysts such as NaOH (Rajasekhar et al. 2010),
KOH (Al-Mohammed et al. 2013), or inorganic salts for
example NaHSO3 (Jain et al. 2013), and Na2S2O5 (Yoon
et al. 2015). There have been greener synthetic procedures
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1763-z) contains supplementary material,
which is available to authorized users.
* Neil A. Koorbanally
Koorbanally@ukzn.ac.za
1
School of Chemistry and Physics, University of KwaZulu-Natal,
Private Bag X54001, Durban 4000, South Africa
2
School of Engineering, University of KwaZulu-Natal, Durban
4041, South Africa
3
Discipline of Pharmaceutical Sciences, School of Health Sciences,
University of KwaZulu-Natal, Private Bag X54001, Durban 4000,
South Africa

Med Chem Res
reported recently, which made use of solvents such as
methanol and water (Borhade et al. 2012; Chen et al. 2012;
Rao et al. 2014), while other methods have been employed
such as the grinding method (Banerjee et al. 2014), ultra-
sound (Patil et al. 2014) and visible light (Park et al. 2014).
In recent years, there has been much interest in microwave
syntheses (Das et al. 2012; Jacob 2012; Gawande et al.
2014). Using microwave synthesis, benzimidazoles were
synthesized without the use of catalysts and organic sol-
vents (Abdullah et al. 2012; Eren and Bekdemir 2014). Its
advantages are, therefore, that it is a green technique,
cheaper, carried out in shorter times and uses less energy
than conventional synthesis.
1H; 39.5 ppm for 13C). High-resolution mass data were
obtained using a Bruker micro TOF-Q II ESI instrument
operating at ambient temperature. The purity of the com-
pounds were determined by analytical high performance
liquid chromatography on a Shimadzu-20A5 fitted with a
C8 (150 mm × 5 μm) column using a mobile phase (A) of
0.1M KHPO4 buffer and (B) methanol, with a linear
gradient of 0 to 70% over a period of 60 min at a flow rate
of 1 mL min−1.
General procedure for the preparation of methyl
4-fluoro-3-nitrobenzoate (2)
In our study, we have synthesized fluorinated benzimi-
dazole derivatives from o-phenyldiamine (methyl 3-amino-
4-(4-fluoroaniline)benzoate) and various substituted alde-
hydes using three methods, conventional organic synthesis
with a sodium metabisulphite catalyst and ethanol as sol-
vent, the grinding method with iodine as a catalyst and
under microwave conditions with no catalyst or solvent, and
compared the yields and reaction times of these methods.
We synthesized a total of 22 2-substituted fluorinated ben-
zimidazoles (5a–5v) and tested them for their antimicrobial
and antioxidant activity.
4-Fluoro-3-nitrobenzoic acid (0.5 g, 2.7 mmol) was dis-
solved in methanol (5 mL) and conc. H2SO4 (0.5 mL) at
room temperature. The reaction mixture was heated with
stirring in a 10 mL microwave process vial for 10 min at
80 °C. After completion of the reaction (as evident from
thin layer chromatography (TLC)), the solvent was evapo-
rated under reduced pressure, the reaction mixture basified
by sodium bicarbonate and the aqueous layer extracted into
ethyl acetate (3 × 5 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pres-
sure to yield 2 as a cream-colored powder (95%).
1H NMR (CDCl3, 400 MHz) δ 8.73 (1H, dd, J = 7.2, 2.2
Hz, H-2), 8.31 (1H, m, H-6), 7.38 (1H, dd, J = 10.2, 8.8 Hz,
H-5), 3.95 (3H, s, H-8); 13C NMR (CDCl3, 100 MHz) δ
164.1 (C-7), 158.1 (d, JCF = 270.0 Hz, C-4), 137.3 (C-3),
136.5 (d, J = 9.7 Hz, C-6), 127.8 (C-2), 127.2 (d, J = 4.2 Hz,
C-1), 118.8 (d, J = 21.2 Hz, C-5), 52.9 (C-8); 19F NMR
(CDCl3, 376.5 MHz) δ −110.55.
Materials and methods
General experimental procedures
All chemicals were supplied by Sigma-Aldrich via Capital
Lab, South Africa. Organic solvents were redistilled and
dried according to standard procedures. Silica gel 60 F254
plates (Merck) were used for thin layer chromatography.
Crude compounds were purified by column chromato-
graphy using silica gel (60–120 mesh) and a mobile phase
of varying ratios of EtOAc:Hexane. Melting points were
recorded using a Stuart Scientific SMP3 apparatus. UV
spectra were obtained on a Varian Cary UV-vis spectro-
photometer in MeOH. Infrared (IR) spectra were recorded
on a Perkin Elmer 100 Fourier transform infrared spectro-
photometer with universal attenuated total reflectance
sampling accessory.
General procedure for the preparation of methyl 4-(4-
fluoroaniline)-3-nitrobenzoate (3)
Methyl-4-fluoro-3-nitrobenzoate 2 (0.5 g, 2.34 mmol) and
4-fluorophenyl aniline (0.36 mL, 2.34 mmol) were mixed in
dimethylformamide (2 mL). The reaction mixture was
subject to microwave irradiation with stirring in a 10 mL
microwave process vial for 5 min at 80 °C. After completion
of the reaction (monitored by TLC), the reaction mixture
was washed with water (2 × 10 mL) followed by 10%
Na2CO3 (10 mL). The organic layer was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure to
afford 3 as a crude product. The solid was purified by
column chromatography (30% ethyl acetate in hexane) to
afford the desired product as a reddish solid in a yield of
90%.
Microwave assisted reaction: All reactions which
involved microwave irradiation were performed using a
1
CEM Discover, Explorer-12 Hybrid microwave. H, 13C
and all two-dimensional nuclear magnetic resonance (2D
NMR) spectra were recorded on a Bruker Avance instru-
ment operating at 400 MHz. Chemical shifts are reported in
δ values (ppm) relative to an internal standard of tetra-
methylsilane and referenced to the solvent line of CDCl3
(7.24 ppm for 1H; 77.0 ppm for 13C), CD3OD (3.31 ppm for
1H; 49.0 ppm for 13C) or DMSO-d6 (2.5 ppm for
1H NMR (CDCl3, 400 MHz) δ 9.67 (1H, s, NH-10), 8.90
(1H, d, J = 2.0 Hz, H-2), 7.95 (1H, dd, J = 9.0, 2.0 Hz, H-6),
7.29 (2H, dd, J = 8.8, 4.7 Hz, H-2a/6a), 7.18 (2H, t, J = 8.8 Hz,
H-3a/5a), 7.03 (1H, d, J = 9.0 Hz, H-5), 3.89 (3H, s, H-8);
13C NMR (CDCl3, 100 MHz) δ 165.3 (C-7), 161.2

Med Chem Res
(d, JCF = 245.8 Hz, C-4a), 146.4 (C-4), 136.0 (C-2), 133.5
(d, J = 3.3 Hz, C-1a), 132.1 (C-3), 129.24 (C-6), 127.5 (d,
J = 8.5 Hz, 2C, C-2a/6a), 119.2 (C-1), 116.9 (d, J = 22.7 Hz,
2C, C-3a/5a), 115.3 (C-5), 52.2 (C-8); 19F NMR (CDCl3,
376.5 MHz) δ −114.14.
at room temperature between 5–45 min (Table 1). After
completion of the reaction (confirmed by TLC), the mixture
was treated with aqueous Na2S2O3. The aqueous layer was
extracted with ethyl acetate (3 × 5 mL), and the organic
layer dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The solid obtained was purified by col-
umn chromatography (hexane/ethyl acetate) to afford the
desired product (5a–5v) in moderate to good yields
(10–56%).
General procedure for the preparation of methyl 3-
amino-4-(4-fluoroaniline) benzoate (4)
Methyl 4-(4-fluoroaniline)-3-nitrobenzoate
3
(0.5 g,
1 mmol), Zn (0.06 g, 0.5 mmol), and ammonium formate
(0.54 g, 5 mmol) were added to methanol (5 mL). The
reaction mixture was stirred at room temperature for 10 min.
Upon completion of the reaction (monitored by TLC), it
was filtered through Celite 545 to remove the zinc. The
filtrate was evaporated under reduced pressure and the solid
purified by column chromatography (30% ethyl acetate in
hexane) to afford the desired product as a brown solid with
a yield of 92%.
Conventional method
3-Amino-4-(4-fluoroaniline) benzoate 4 (1 mmol), various
aldehydes (1 mmol) and Na2S2O5 (20 mol%) was refluxed
in ethanol (20 mL) between 8–18 h. After completion of the
reaction (as evident from TLC), the solvent was evaporated
under reduced pressure and the crude mass washed with
water (2 × 10 mL). The aqueous layer was extracted with
ethyl acetate (3 × 25 mL), and the organic layer dried over
anhydrous Na2SO4 and concentrated under reduced pres-
sure to afford 5a–5v as crude products. The solid obtained
was purified by column chromatography (hexane/ethyl
acetate) to afford the desired product in good yields
(60–90%).
1H NMR (CDCl3, 400 MHz) δ 7.50 (1H, d, J = 1.7 Hz,
H-2), 7.47 (1H, dd, J = 8.2, 1.7 Hz, H-6), 7.03 (1H, d,
J = 8.2 Hz, H-5), 6.99 (d, J = 8.3 Hz, 2H, H-2a/6a), 6.95
(2H, dd, J = 9.0, 8.3 Hz, H-3a/5a), 5.55 (s, 1H, NH-10),
3.85 (3H, s, H-8), 3.16 (2H, s, NH-9); 13C NMR (CDCl3,
100 MHz) δ 167.0 (C-7), 158.3 (d, JCF = 239.4 Hz, C-4a),
138.3 (C-1a), 137.1 (C-4), 136.0 (C-3), 123.6 (C-1), 122.6
(C-6), 120.6 (d, J = 7.7 Hz, 2C, C-2a/6a), 118.4 (C-2), 116.7
(C-5), 116.1 (d, J = 22.5 Hz, 2C, C-3a/5a), 51.8 (C-8); 19F
NMR (CDCl3, 376.5 MHz) δ −121.46.
Spectral data
Methyl
1-(4-fluorophenyl)-2-phenyl-1H-benzo[d]imida-
zole-5-carboxylate (5a) White solid (90% yield, 99%
purity); mp 160–162 °C; UV λmax (MeOH) nm (log ε) 247
(2.63), 302 (2.19); IR υmax 3069 (CH), 1713 (C=O), 1357
General procedure for the preparation of fluorinated
benzimidazoles (5a–5v)
(C-F), 1185 (C-O) cm−1; H NMR (CDCl3, 400 MHz) δ
1
8.58 (1H, bs, H-4), 7.98 (1H, d, J = 8.6 Hz, H-6), 7.54 (2H,
d, J = 7.4 Hz, H-2b/6b), 7.38 (1H, m, H-4b), 7.32 (2H, m,
H-2a/6a), 7.29 (2H, m, H-3b/5b), 7.23 (1H, m, H-7), 7.20
(2H, m, H-3a/5a), 3.94 (3H, s, H-11); 13C NMR (CDCl3,
100 MHz) δ 167.4 (C-10), 162.4 (d, JCF = 248.6 Hz, C-4a),
154.0 (C-2), 142.2 (C-8), 140.2 (C-9), 132.2 (d, J = 3.3 Hz,
C-1a), 130.0 (C-4b), 129.2 (C-6b), 129.1 (d, J = 8.7 Hz, 2C,
C-2a/6a), 129.07 (C-2b), 129.0 (C-1b), 128.5 (2C, C-3b/
5b), 125.4 (C-5), 125.1 (C-6), 122.2 (C-4), 117.1 (d, J =
22.7 Hz, 2C, C-3a/5a), 109.3 (C-7), 52.1 (C-11); 19F NMR
(CDCl3, 376.5 MHz) δ −108.08; HRESIMS m/z (pos):
347.1198 C21H16FN2O2 (calcd. 347.1196).
Microwave method
The mixture of methyl 3-amino-4-(4-fluoroaniline) benzoate
4 (1 mmol) and various aldehydes (1 mmol) were heated in
a 5 mL microwave process vial for 5–10 min at 110 °C to
obtain compounds (5a–5v). After completion of the reaction
(monitored by TLC), the reaction mixture was washed with
water (2 × 5 mL) followed by brine solution (10 mL). The
aqueous layer was extracted into ethyl acetate (3 × 5 mL).
The organic layer was dried over anhydrous Na2SO4, con-
centrated under reduced pressure and the obtained solid
purified by column chromatography (30–50% ethyl acetate
in hexane) to afford the desired products in good yields
(85–96%).
Methyl 1,2-bis(4-fluorophenyl)-1H-benzo[d]imidazole-5-
carboxylate (5b) Yellow solid (90% yield, 98% purity);
mp 130–132 °C; UV λmax (MeOH) nm (log ε) 247 (2.7),
301 (2.4); IR υmax 3071 (CH), 1711 (C=O), 1379 (C–F),
1157 (C-O) cm−1; 1H NMR (CDCl3, 400 MHz) δ 8.56 (1H,
bs, H-4), 7.99 (1H, dd, J = 8.6, 1.4 Hz, H-6), 7.54 (2H, dd,
J = 8.8, 5.2 Hz, H-2b/6b), 7.29 (2H, dd, J = 8.9, 4.8 Hz,
H-2a/6a), 7.23 (2H, m, H-3a/5a), 7.19 (1H, d, J = 8.6 Hz,
Grinding (mechanochemical) method
A mixture of methyl 3-amino-4-(4-fluoroaniline) benzoate
4 (1 mmol), various aldehydes (1 mmol), and iodine
(10 mol %) were ground together using a mortar and pestle

Med Chem Res
Table 1 Comparison of the
yields and duration of the final
step in the benzimidazole
synthesis using three different
methods
No. R1
Microwave
Grinding
Conventional
Time (min) Yielda (%) Time (min) Yielda (%) Time (h) Yielda (%)
5a Ph
3
3
3
3
5
8
6
6
5
5
5
4
5
5
6
5
4
8
3
3
6
6
96
93
89
85
90
85
88
90
85
90
90
90
95
97
92
90
85
85
95
95
90
90
10
10
10
10
10
45
20
25
20
10
20
20
20
25
25
30
35
40
30
30
25
25
55
53
50
48
45
20
30
25
10
20
35
40
56
50
50
30
20
20
50
50
30
30
10
8
90
85
75
74
70
60
72
60
65
70
87
85
80
75
73
60
60
76
75
75
70
70
5b 4-F Ph
5c 4-Cl Ph
5d 4-Br Ph
5e 4-CF3 Ph
10
10
8
5f
4-NO2 Ph
18
12
16
18
16
12
16
12
12
12
16
16
16
15
15
18
18
5g 4-CH3 Ph
5h 4-OCH3 Ph
5i
5j
4-NH2 Ph
4-OH Ph
5k 4-N(CH3)2 Ph
5l 4-S(CH3) Ph
5m 2-Naphthyl
5n α-(E)-prop-1-en-1-yl Ph
5o 2-(6-Chloroquinolinyl)
5p 2-OH-4,6-(OCH3)2 Ph
5q 3,4-(OH)2 Ph
5r 2,3,4-(OH)3 Ph
5s 2-Thiophenyl
5t
2-Furanyl
5u n-Butyl
5v n-Heptyl
a
Isolated yield after column chromatography
H-7), 7.02 (2H, t, J = 8.8 Hz, H-3b/5b), 3.95 (3H, s, H-11);
13C NMR (CDCl3, 100 MHz) δ 167.3 (C-10), 163.7 (d,
JCF = 250.4 Hz, C-4b), 162.5 (d, JCF = 249.2 Hz, C-4a),
153.0 (C-2), 142.1 (C-8), 140.2 (C-9), 132.2 (d, J = 3.2 Hz,
C-1a), 131.5 (d, J = 8.6 Hz, 2C, C-2b/6b), 129.2 (d, J = 8.7
Hz, 2C, C-2a/6a), 125.6 (C-5), 125.2 (C-6), 122.1 (C-4),
117.3 (d, J = 23.0 Hz, 2C, C-3a/5a), 115.8 (d, J = 21.8 Hz,
C-3b/5b), 110.0 (C-7), 52.2 (C-11); 19F NMR (CDCl3,
376.5 MHz) δ −110.71, −109.46; HRESIMS m/z (pos):
365.1111 C21H15F2N2O2 (calcd. 365.1102). * C-1b could
not be detected.
(C-9), 136.5 (C-4b), 132.1 (d, J = 3.0 Hz, C-1a), 130.7 (2C,
C-2b/6b), 129.1 (d, J = 8.7 Hz, 2C, C-2a/6a), 128.9 (C-3b),
127.4 (C-1b), 125.7 (C-5), 125.3 (C-6), 122.2 (C-4), 117.3
(d, J = 22.8 Hz, 2C, C-3a/5a), 110.1 (C-7), 52.2 (C-11); 19F
NMR (CDCl3, 376.5 MHz) δ −110.35; HRESIMS m/z
(pos): 381.0818 C21H15ClFN2O2 (calcd. 381.0806).
Methyl 2-(4-bromophenyl)-1-(4-fluorophenyl)-1H-benzo[d]
imidazole-5-carboxylate (5d) Cream colored solid (82%
yield, 97% purity); mp 203–205 °C; UV λmax (MeOH) nm
(log ε) 253 (3.28), 305 (2.92); IR υmax 3072 (CH), 1707
1
(C=O), 1357 (C–F), 1153 (C–O), 523 (C–Br) cm−1; H
Methyl 2-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-benzo[d]
imidazole-5-carboxylate (5c) Yellow solid (86% yield,
96% purity); mp 185–187 °C; UV λmax (MeOH) nm (log ε)
251 (3.23), 304 (2.85); IR υmax 3059 (CH), 1708 (C=O),
1357 (C–F), 1181 (C–O), 729 (C–Cl ) cm−1; 1H NMR
(CDCl3, 400 MHz) δ 8.58 (1H, bs, H-4), 8.01 (1H, d,
J = 8.5 Hz, H-6), 7.51 (2H, d, J = 8.4 Hz, H-2b/6b), 7.31
(4H, d, J = 8.5 Hz, H-2a/6a, H-3b/5b), 7.25 (2H, d, J = 6.44
Hz, H-3a/5a), 7.20 (1H, d, J = 8.8 Hz, H-7), 3.95 (3H, s,
H-11); 13C NMR (CDCl3, 100 MHz) δ 167.3 (C-10), 162.5
(d, JCF = 249.2 Hz, C-4a), 152.8 (C-2), 142.0 (C-8), 140.1
NMR (CDCl3, 400 MHz) δ 8.57 (1H, s, H-4), 8.00 (1H, dd,
J = 8.6, 1.4 Hz, H-6), 7.47 (2H, d, J = 8.6 Hz, H-3b/5b),
7.42 (2H, d, J = 8.6 Hz, H-2b/6b), 7.29 (2H, dd, J = 9.0, 4.8
Hz, H-2a/6a), 7.23 (2H, t, J = 10.8 Hz, H-3a/5a), 7.19 (1H,
d, J = 8.6 Hz, H-7), 3.95 (3H, s, H-11); 13C NMR (CDCl3,
100 MHz) δ 167.3 (C-10), 162.6 (d, JCF = 249.2 Hz, C-4a),
152.8 (C-2), 142.1 (C-8), 140.2 (C-9), 132.2 (d, J = 3.3 Hz,
C-1a), 131.9 (2C, C-3b/5b), 130.9 (2C, C-2b/6b), 129.1 (d,
J = 8.7 Hz, 2C, C-2a/6a), 127.9 (C-5), 125.7 (C-1b), 125.4
(C-6), 124.9 (C-4b), 122.2 (C-4), 117.4 (d, J = 22.8 Hz, 2C,
C-3a/5a), 110.1 (C-7), 52.2 (C-11); 19F NMR (CDCl3,

Med Chem Res
376.5 MHz) δ −110.53; HRESIMS m/z (pos): 425.0310
C21H15BrFN2O2 (calcd. 425.0301).
132.4 (d, J = 3.6 Hz, C-1a), 129.4 (2C, C-3b/5b), 129.3 (2C,
C-2b/6b), 129.2 (d, J = 8.7 Hz, 2C, C-2a/6a), 125.7 (C-1b),
125.5 (C-5), 125.1 (C-6), 121.8 (C-4), 117.2 (d, J = 22.8 Hz,
2C, C-3a/5a), 110.0 (C-7), 52.2 (C-11), 21.4 (C-7b); 19F
NMR (CDCl3, 376.5 MHz) δ −111.18; HRESIMS m/z
(pos): 361.1354 C22H18FN2O2 (calcd. 361.1352).
Methyl 1-(4-fluorophenyl)-2-(4-(trifluoromethyl)phenyl)-
1H-benzo[d]imidazole-5-carboxylate (5e) White solid
(84% yield, 98% purity); mp 171–173 °C; UV λmax
(MeOH) nm (log ε) 250 (2.83), 304 (2.5); IR υmax 3081
(CH), 1712 (C=O), 1358 (C–F), 1196 (C–O) cm−1; 1H
NMR (CDCl3, 400 MHz) δ 8.59 (1H, bs, H-4), 8.01 (1H,
dd, J = 8.6, 1.4 Hz, H-6), 7.68 (2H, d, J = 8.3 Hz, H-3b/5b),
7.58 (2H, d, J = 8.3 Hz, H-2b/6b), 7.30 (2H, dd, J = 8.9, 4.8
Hz, H-2a/6a), 7.24 (2H, t, J = 10.5 Hz, H-3a/5a), 7.21 (1H,
d, J = 8.6 Hz, H-7), 3.95 (3H, s, H-11); 13C NMR (CDCl3,
100 MHz) δ 167.2 (C-10), 162.6 (d, JCF = 249.4 Hz, C-4a),
152.3 (C-2), 142.3 (C-8), 140.3 (C-9), 132.6 (C-1b), 132.1
(d, J = 3.16 Hz, C-1a), 131.8 (q, JCF = 260.0 Hz, C-7b),
129.7 (2C, C-3b/5b), 129.1 (d, J = 8.7 Hz, 2C, C-2a/6a),
125.8 (C-5), 125.6 (C-6), 125.5 (q, J = 3.9 Hz, 2C, C-2b/
6b), 122.5 (C-4), 117.5 (d, J = 22.8 Hz, 2C, C-3a/5a), 110.2
(C-7), 52.2 (C-11); 19F NMR (CDCl3, 376.5 MHz)
δ −62.98; HRESIMS m/z (pos): 415.1078 C22H15F4N2O2
(calcd. 415.1070).
Methyl 1-(4-fluorophenyl)-2-(4-methoxyphenyl)-1H-benzo
[d]imidazole-5-carboxylate (5h) White solid (78% yield,
98% purity); mp 138–140 °C; UV λmax (MeOH) nm (log ε)
225 (2.90), 258 (3.00), 307 (2.75); IR υmax 3080 (CH), 1710
1
(C=O), 1385 (C–F), 1180 (C–O) cm−1; H NMR (CDCl3,
400 MHz) δ 8.54 (1H, d, J = 1.5 Hz, H-4), 7.96 (1H, dd, J =
8.5, 1.5 Hz, H-6), 7.48 (2H, d, J = 8.9 Hz, H-2b/6b), 7.29
(2H, dd, J = 8.7, 4.8 Hz, H-2a/6a), 7.21 (2H, t, J = 8.7 Hz,
H-3a/5a), 7.16 (1H, d, J = 8.5 Hz, H-7), 6.83 (2H, d, J = 8.9
Hz, H-3b/5b), 3.94 (3H, s, H-11), 3.79 (3H, s, H-7b); 13C
NMR (CDCl3, 100 MHz) δ 167.6 (C-10), 162.5 (d, JCF
=
248.6 Hz, C-4a), 161.0 (C-4b), 154.0 (C-2), 142.3 (C-8),
140.3 (C-9), 132.6 (d, J = 3.3 Hz, C-1a), 131.0 (2C, C-2b/
6b), 129.2 (d, J = 8.7 Hz, 2C, C-2a/6a), 125.3 (C-5), 124.8
(C-6), 121.8 (C-4), 121.3 (C-1b), 117.2 (d, J = 22.7 Hz, 2C,
C-3a/5a), 114.1 (2C, C-3b/5b), 109.9 (C-7), 55.3 (C-7b),
52.1 (C-11); 19F NMR (CDCl3, 376.5 MHz) δ −111.23;
HRESIMS m/z (pos): 377.1310 C22H18FN2O3 (calcd.
377.1301).
Methyl 1-(4-fluorophenyl)-2-(4-nitrophenyl)-1H-benzo[d]
imidazole-5-carboxylate (5f) Yellow solid (70% yield;
98% purity); mp 214–216 °C; UV λmax (MeOH) nm (log ε)
229 (2.84), 320 (2.47); IR υmax 3078 (CH), 1714 (C=O),
1510 (N–O), 1380 (C–F), 1204 (C–O) cm−1; 1H NMR
(CDCl3, 400 MHz) δ 8.60 (1H, bs, H-4), 8.18 (2H, d, J =
8.1 Hz, H-3b/5b), 8.04 (1H, d, J = 8.4 Hz, H-6), 7.76 (2H, d,
J = 8.1 Hz, H-2b/6b), 7.32 (2H, m, H-2a/6a), 7.27 (2H, m,
H-3a/5a), 7.23 (1H, d, J = 8.4 Hz, H-7), 3.96 (3H, s, H-11);
13C NMR (CDCl3, 100 MHz) δ 167.1 (C-10), 162.9 (d,
JCF = 250.2 Hz, C-4a), 151.2 (C-4b), 148.4 (C-2), 142.0 (C-
8), 140.3 (C-9), 134.9 (C-1b), 131.8 (d, J = 3.1 Hz, C-1a),
130.3 (2C, C-2b/6b), 129.2 (d, J = 8.7 Hz, 2C, C-2a/6a),
126.2 (C-5), 126.1 (C-6), 123.7 (2C, C-3b/5b), 122.6 (C-4),
117.7 (d, J = 22.8 Hz, 2C, C-3a/5a), 110.4 (C-7), 52.3 (C-
11); 19F NMR (CDCl3, 376.5 MHz) δ −109.63; HRESIMS
m/z (pos): 392.1055 C21H15FN3O4 (calcd. 392.1047).
Methyl 2-(4-aminophenyl)-1-(4-fluorophenyl)-1H-benzo[d]
imidazole-5-carboxylate (5i) Orange solid (75% yield,
97% purity); mp 225–227 °C; UV λmax (MeOH) nm (log ε)
233 (3.51), 327 (3.41); IR υmax 3473 (NH), 3077 (CH),
1710 (C=O), 1382 (C–F), 1197 (C–O) cm−1; 1H NMR
(DMSO-d6, 400 MHz) δ 8.26 (1H, d, J = 1.3 Hz, H-4), 7.83
(1H, dd, J = 8.5, 1.3 Hz, H-6), 7.52 (2H, dd, J = 8.7, 5.0 Hz,
H-2a/6a), 7.44 (2H, t, J = 8.7 Hz, H-3a/5a), 7.21 (2H, d, J =
8.6 Hz, H-2b/6b), 7.17 (1H, d, J = 8.5 Hz, H-7), 6.49 (2H, d,
J = 8.6 Hz, H-3b/5b), 5.61 (2H, s, H-7b), 3.88 (3H, s, H-11);
13C NMR (DMSO-d6, 100 MHz) δ 166.7 (C-10), 161.8 (d,
JCF = 244.9 Hz, C-4a), 155.1 (C-2), 150.6 (C-4b), 142.4 (C-
8), 140.5 (C-9), 132.9 (d, J = 3.0 Hz, C-1a), 130.4 (2C, C-
2b/6b), 129.9 (d, J = 8.9 Hz, 2C, C-2a/6a), 123.9 (C-5),
123.6 (C-6), 120.0 (C-4), 117.0 (d, J = 22.9 Hz, 2C, C-3a/
5a), 115.4 (C-1b), 113.1 (2C, C-3b/5b), 110.0 (C-7), 52.0
(C-11); 19F NMR (DMSO-d6, 376.5 MHz) δ −112.32;
HRMS (m/z): 362.1307 C21H17FN3O2 (calcd. 362.1305).
Methyl
1-(4-fluorophenyl)-2-p-tolyl-1H-benzo[d]imida-
zole-5-carboxylate (5g) White solid (80% yield, 96%
purity); mp 156–158 °C; UV λmax (MeOH) nm (log ε) 214
(3.21), 250 (3.36), 304 (2.93); IR υmax 3074 (CH), 1711
1
(C=O), 1381 (C–F), 1185 (C–O) cm−1; H NMR (CDCl3,
400 MHz) δ 8.57 (1H, bs, H-4), 7.98 (1H, dd, J = 8.5, 1.3
Hz, H-6), 7.43 (2H, d, J = 8.1 Hz, H-2b/6b), 7.29 (2H, dd,
J = 8.7, 4.8 Hz, H-2a/6a), 7.21 (2H, t, J = 8.7 Hz, H-3a/5a),
7.18 (1H, d, J = 8.5 Hz, H-7), 7.13 (2H, d, J = 8.1 Hz, H-3b/
5b), 3.94 (3H, s, H-11), 2.34 (3H, s, H-7b); 13C NMR
(CDCl3, 100 MHz) δ 167.3 (C-10), 162.5 (d, JCF = 248.7
Hz, C-4a), 154.0 (C-2), 140.6 (C-8), 140.1 (2C, C-4/4b),
Methyl 1-(4-fluorophenyl)-2-(4-hydroxyphenyl)-1H-benzo
[d]imidazole-5-carboxylate (5j) White solid (80% yield,
96% purity); mp 252–254 °C; UV λmax (MeOH) nm (log ε)
226 (2.88), 259 (2.92), 308 (2.74); IR υmax 3058 (CH), 1710
1
(C=O), 1384 (C-F), 1196 (C-O) cm−1; H NMR (MeOH-
d4, 400 MHz) δ 8.41 (1H, d, J = 1.4 Hz, H-4), 7.97 (1H,
dd, J = 8.6, 1.4 Hz, H-6), 7.42 (2H, dd, J = 8.7, 4.9 Hz,

Med Chem Res
H-2a/6a), 7.38 (2H, d, J = 8.7 Hz, H-2b/6b), 7.33 (2H, t, J =
8.7 Hz, H-3a/5a), 7.27 (1H, d, J = 8.6 Hz, H-7), 6.76 (2H, d,
J = 8.7 Hz, H-3b/5b), 3.94 (3H, s, H-11); 13C NMR
Methyl 1-(4-fluorophenyl)-2-(naphthalen-2-yl)-1H-benzo
[d]imidazole-5-carboxylate (5m) Yellowish solid (85%
yield, 99% purity); mp 169–171 °C; UV λmax (MeOH) nm
(log ε) 210 (3.33), 252 (3.62), 311 (3.10); IR υmax 3064
(CH), 1701 (C=O), 1362 (C–F), 1156 (C–O) cm−1; 1H
NMR (CDCl3, 400 MHz) δ 8.61 (1H, bs, H-4), 8.09 (1H, s,
H-2b), 8.01 (1H, dd, J = 8.6, 1.2 Hz, H-6), 7.80 (1H, d, J =
8.2 Hz, H-9b), 7.75 (2H, t, J = 8.8 Hz, H-4b/7b), 7.58 (1H,
dd, J = 8.2, 1.6 Hz, H-10b), 7.51 (1H, t, J = 7.0 Hz, H-5b*),
7.47 (1H, t, J = 7.0 Hz, H-6b*), 7.33 (2H, dd, J = 8.2, 4.7
Hz, H-2a/6a), 7.22 (1H, d, J = 8.6 Hz, H-7), 7.20 (2H, t, J =
8.2 Hz, H-3a/5a), 3.96 (3H, s, H-11); 13C NMR (CDCl3,
100 MHz) δ 167.4 (C-10), 162.5 (d, JCF = 248.8 Hz, C-4a),
154.0 (C-2), 142.4 (C-8), 140.3 (C-9), 133.7 (C-3b), 132.8
(C-1b), 132.6 (d, J = 3.2 Hz, C-1a), 130.0 (C-2b), 129.2 (d,
J = 8.7 Hz, 2C, C-2a/6a), 128.7 (C-7b), 128.2 (C-4b), 127.5
(C-9b#), 127.7 (C-6b#), 126.7 (C-5b), 126.4 (C-5), 125.8
(C-10b), 125.5 (C-8b), 125.2 (C-6), 122.2 (C-4), 117.2 (d,
J = 22.8 Hz, 2C, C-3a/5a), 110.0 (C-7), 52.2 (C-11); 19F
NMR (CDCl3, 376.5 MHz) δ −110.01; HRESIMS m/z
(MeOH– d4, 100 MHz) δ 168.8 (C-10), 164.1 (d, JCF
=
246.7 Hz, C-4a), 161.1 (C-4b), 156.6 (C-2), 143.0 (C-8),
141.5 (C-9), 133.8 (d, J = 3.4 Hz, C-1a), 132.4 (2C, C-2b/
6b), 130.9 (d, J = 9.0 Hz, 2C, C-2a/6a), 126.5 (C-5), 125.9
(C-6), 121.7 (C-4), 120.8 (C-1b), 118.1 (d, J = 23.2 Hz, 2C,
C-3a/5a), 116.5 (2C, C-3b/5b), 111.5 (C-7), 52.7 (C-11);
19F NMR (MeOH- d4, 376.5 MHz) δ −113.54; HRESIMS
m/z (pos): 385.0970 C21H15FN2O3Na (calcd. 385.0964).
Methyl 2-(4-(dimethylamino)phenyl)-1-(4-fluorophenyl)-
1H-benzo[d]imidazole-5-carboxylate (5k) Brown solid
(90% yield, 87% purity); mp 184–186 °C; UV λmax
(MeOH) nm (log ε) 205 (3.18), 235 (3.30), 337 (3.23); IR
υmax 3049 (CH), 1707 (C=O), 1363 (C–F), 1196 (C–O)
1
cm−1; H NMR (CDCl3, 400 MHz) δ 8.54 (1H, bs, H-4),
7.94 (1H, dd, J = 8.5, 1.4 Hz, H-6), 7.45 (2H, d, J = 8.9 Hz,
H-2b/6b), 7.34 (2H, dd, J = 8.8, 4.8 Hz, H-2a/6a), 7.24
(2H, t, J = 8.8 Hz, H-3a/5a), 7.13 (1H, d, J = 8.5 Hz, H-7),
6.59 (2H, d, J = 8.9 Hz, H-3b/5b), 3.95 (3H, s, H-11), 2.98
(6H, s, H-7b/8b); 13C NMR (CDCl3, 100 MHz) δ 167.5
(C-10), 162.3 (d, JCF = 248.0 Hz, C-4a), 154.8 (C-4b),
151.1 (C-2), 142.4 (C-8), 140.4 (C-9), 133.1 (d, J = 3.2 Hz,
C-1a), 130.5 (2C, C-2b/6b), 129.3 (d, J = 8.7 Hz, 2C, C-2a/
6a), 124.9 (C-1b), 124.3 (C-6), 121.3 (C-4), 117.0 (d, J =
22.8 Hz, 2C, C-3a/5a), 115.7 (C-5), 111.4 (C-3b/5b), 109.4
(C-7), 52.0 (C-11), 40.0 (2C, C-7b/8b); 19F NMR (CDCl3,
376.5 MHz) δ −111.47; HRESIMS m/z (pos): 390.1620
C23H21FN3O2 (calcd. 390.1618).
#
(pos): 397.1349 C25H18FN2O2 (calcd. 397.1352). * and
assignments may be interchanged.
(E)-Methyl 1-(4-fluorophenyl)-2-styryl-1H-benzo[d]imida-
zole-5-carboxylate (5n) White solid (87% yield, 97%
purity); mp 210–212 °C; UV λmax (MeOH) nm (log ε) 227
(3.21), 268 (3.04), 332 (3.06); IR υmax 3078 (CH), 1704
(C=O), 1635 (C=C), 1392 (C-F), 1154 (C-O) cm−1; 1H
NMR (CDCl3, 400 MHz) δ 8.52 (1H, bs, H-4), 8.05 (1H, d,
J = 16.0 Hz, H-1b), 7.95 (1H, dd, J = 8.5, 1.0 Hz, H-6), 7.46
(2H, m, H-4b/8b), 7.43 (2H, m, H-2a/6a), 7.31–7.35 (5H,
m, H-5b, H-6b, H-7b, H-3a/5a), 7.13 (1H, d, J = 8.5 Hz, H-
7), 6.74 (1H, d, J = 16.0 Hz, H-2b), 3.94 (3H, s, H-11); 13C
NMR (CDCl3, 100 MHz) δ 167.4 (C-10), 162.8 (d, JCF
=
Methyl 1-(4-fluorophenyl)-2-(4-(methylthio)phenyl)-1H-
benzo[d]imidazole-5-carboxylate (5l) Yellow solid (90%
yield, 90% purity); mp 182–184 °C; UV λmax (MeOH) nm
(log ε) 207 (3.16), 238 (3.21), 277 (2.96), 316 (3.14); IR
υmax 3059 (CH), 1697 (C=O), 1377 (C–F), 1155 (C–O)
249.1 Hz, C-4a), 152.5 (C-2), 142.3 (C-8), 139.6 (C-9),
138.9 (C-1b), 135.5 (C-3b), 130.9 (d, J = 3.1 Hz, C-1a),
129.5 (2C, C-5b/7b), 129.4 (2C, d, J = 8.8 Hz, C-2a/6a),
128.8 (C-6b), 127.4 (2C, C-4b/8b), 125.6 (C-5), 124.9 (C-
6), 121.5 (C-4), 117.3 (d, J = 23.9 Hz, 2C, C-3a/5a), 112.8
(C-2b), 109.6 (C-7), 52.2 (C-11); 19F NMR (CDCl3, 376.5
1
cm−1; H NMR (CDCl3, 400 MHz) δ 8.55 (1H, d, J = 1.4
Hz, H-4), 7.97 (1H, dd, J = 8.5, 1.4 Hz, H-6), 7.46 (2H, d,
J = 8.5 Hz, H-2b/6b), 7.30 (2H, dd, J = 7.2, 4.8 Hz, H-2a/
6a), 7.22 (2H, t, J = 7.2 Hz, H-3a/5a), 7.19 (1H, d, J = 7.3
Hz, H-7), 7.14 (2H, d, J = 8.5 Hz, H-3b/5b), 3.94 (3H, s, H-
11), 2.46 (3H, s, H-7b); 13C NMR (CDCl3, 100 MHz) δ
167.3 (C-10), 162.5 (d, JCF = 249.1 Hz, C-4a), 153.5 (C-2),
142.2 (2C, C-9/4b), 140.1 (C-9), 132.3 (d, J = 3.2 Hz, C-
1a), 129.6 (2C, C-2b/6b), 129.2 (d, J = 8.7 Hz, 2C, C-2a/
6a), 125.6 (C-1b), 125.5 (2C, C-3b/5b), 125.2 (C-6), 124.8
(C-5), 121.8 (C-4), 117.3 (d, J = 22.8 Hz, 2C, C-3a/5a),
109.9 (C-7), 52.2 (C-11), 14.9 (C-7b); 19F NMR (CDCl3,
376.5 MHz) δ −110.83; HRESIMS m/z (pos): 415.0885
C22H17FN2O2SNa (calcd. 415.0892).
MHz) δ −110.43; HRESIMS m/z (pos): 373.1355
C23H18FN2O2 (calcd. 373.1352).
Methyl 2-(6-chloroquinolin-2-yl)-1-(4-fluorophenyl)-1H-
benzo[d]imidazole-5-carboxylate (5o) Yellowish solid
(85% yield, 98% purity); Purity 98%; mp 228–230 °C; UV
λmax (MeOH) nm (log ε) 257 (3.43), 321 (2.94), 336 (2.98);
IR υmax 3068 (CH), 1702 (C=O), 1386 (C–F), 1190 (C–O),
750 (C–Cl) cm−1; 1H NMR (CDCl3, 400 MHz) δ 8.64 (1H,
bs, H-4), 8.47 (1H, d, J = 8.6 Hz, H-9b), 8.21 (1H, d, J = 8.6
Hz, H-10b), 8.03 (1H, dd, J = 8.6, 1.3 Hz, H-6), 7.72 (1H, d,
J = 9.2 Hz, H-4b), 7.46 (2H, m, H-5b/7b), 7.38 (2H, dd, J =
9.2, 4.8 Hz, H-2a/6a), 7.24 (2H, t, J = 9.2 Hz, H-3a/5a), 7.19

Med Chem Res
(1H, d, J = 8.6 Hz, H-7), 3.96 (3H, s, H-11); 13C NMR
(CDCl3, 100 MHz) δ 167.3 (C-10), 162.5 (d, JCF = 247.3
Hz, C-4a), 151.2 (C-2), 149.2 (C-1b), 147.3 (C-8b), 141.9
(C-8), 141.2 (C-9), 136.5 (C-10b), 135.8 (C-6b), 133.7 (d,
J = 3.1 Hz, C-1a), 129.6 (d, J = 8.7 Hz, 2C, C-2a/6a), 128.8
(C-7b*), 128.6 (C-5b*), 128.5 (C-4b), 126.2 (C-3b), 126.0
(C-6), 125.8 (C-5), 122.7 (C-4), 121.6 (C-9b), 116.3 (d, J =
22.8 Hz, 2C, C-3a/5a), 110.7 (C-5), 52.2 (C-11); 19F NMR
(CDCl3, 376.5 MHz) δ −112.10; HRESIMS m/z (pos):
432.0931 C24H16ClFN3O2 (calcd. 432.0915). *assignments
may be interchanged.
Methyl 1-(4-fluorophenyl)-2-(2,3,4-trihydroxyphenyl)-1H-
benzo[d]imidazole-5-carboxylate (5r) Green solid (85%
yield, 99% purity); mp 214–216 °C; UV λmax (MeOH) nm
(log ε) 230 (3.27), 322 (3.0); IR υmax 3483 (O-H), 3069
(CH), 1709 (C=O), 1345 (C–F), 1152 (C–O) cm−1; 1H
NMR (MeOH– d4, 400 MHz) δ 8.34 (1H, d, J = 1.4 Hz, H-
4), 7.92 (1H, dd, J = 8.5, 1.4 Hz, H-6), 7.46 (2H, dd, J = 8.7,
4.9 Hz, H-2a/6a), 7.36 (2H, t, J = 8.7 Hz, H-3a/5a), 7.12
(1H, d, J = 8.5 Hz, H-7), 6.35 (1H, d, J = 8.9 Hz, H-6b),
6.15 (1H, d, J = 8.9 Hz, H-5b), 3.93 (3H, s, H-11); 13C
NMR (MeOH– d4, 100 MHz) δ 168.9 (C-10), 164.4 (d, JCF
= 247.2 Hz, C-4a), 155.2 (C-2), 149.5 (C-4b), 149.4 (C-2b),
141.5 (C-8), 141.0 (C-9), 134.6 (C-3b), 134.2 (d, J = 3.3 Hz,
C-1a), 131.1 (d, J = 8.9 Hz, 2C, C-2a/6a), 126.5 (C-5),
125.8 (C-6), 121.0 (C-4), 120.4 (C-6b), 118.3 (d, J = 23.3
Hz, 2C, C-3a/5a), 111.0 (C-7), 107.8 (C-5b), 106.5 (C-1b),
52.7 (C-11); 19F NMR (MeOH– d4, 376.5 MHz) δ −113.12;
HRESIMS m/z (pos): 417.0874 C21H15FN2O5Na (calcd.
417.0863).
Methyl
1-(4-fluorophenyl)-2-(2-hydroxy-4,6-dimethoxy-
phenyl)-1H-benzo[d]imidazole-5-carboxylate (5p) White
solid (65% yield, 99% purity); mp 68–70 °C; UV λmax
(MeOH) nm (log ε) 229 (2.86), 267 (2.33), 297 (3.93); IR
υmax 3398 (O–H), 3074 (CH), 1712 (C=O), 1359 (C–F),
1153 (C–O) cm−1; 1H NMR (CDCl3, 400 MHz) δ 8.51 (1H,
bs, H-4), 8.00 (1H, dd, J = 8.5, 1.0 Hz, H-6), 7.31 (1H, d,
J = 8.5 Hz, H-7), 7.26 (2H, dd, J = 8.6, 4.9 Hz, H-2a/6a),
7.16 (2H, t, J = 8.6 Hz, H-3a/5a), 6.32 (1H, d, J = 1.9 Hz, H-
3b), 5.77 (1H, d, J = 1.9 Hz, H-5b), 3.94 (3H, s, H-11), 3.77
(3H, s, H-8b), 3.15 (3H, s, H-9b); 13C NMR (CDCl3, 100
MHz) δ 167.2 (C-10), 163.1 (C-4b), 162.2 (d, JCF = 247.1
Hz, C-4a), 160.1 (C-2b), 157.6 (C-6b), 151.8 (C-2), 140.4
(C-8), 138.5 (C-9), 133.8 (d, J = 2.8 Hz, C-1a), 126.9 (d,
J = 8.5 Hz, 2C, C-2a/6a), 125.5 (C-5), 125.0 (C-6), 120.7
(C-4), 116.3 (d, J = 22.8 Hz, 2C, C-3a/5a), 110.0 (C-7), 96.2
(C-1b), 94.0 (C-3b), 90.8 (C-5b), 55.4 (C-8b), 54.3 (C-9b),
52.2 (C-11); 19F NMR (CDCl3, 376.5 MHz) δ −112.41;
HRESIMS m/z (pos): 445.1168 C23H19FN2O5 Na (calcd.
445.1176).
Methyl 1-(4-fluorophenyl)-2-(thiophen-2-yl)-1H-benzo[d]
imidazole-5-carboxylate (5s) White solid (74% yield,
90% purity); mp 156–158 °C; UV λmax (MeOH) nm (log ε)
219 (3.26), 254 (3.28), 322 (3.18); IR υmax 3073 (CH), 1711
1
(C=O), 1386 (C-F), 1192 (C–O) cm−1; H NMR (CDCl3,
400 MHz) δ 8.53 (1H, bs, H-4), 7.95 (1H, dd, J = 8.5, 1.0
Hz, H-6), 7.41 (2H, dd, J = 8.2, 4.8 Hz, H-2a/6a), 7.39 (1H,
d, J = 4.4 Hz, H-3b), 7.30 (2H, t, J = 8.2 Hz, H-3a/5a), 7.03
(1H, d, J = 8.5 Hz, H-7), 7.02 (1H, bs, H-5b), 6.95 (1H, t,
J = 4.4 Hz, H-4b), 3.93 (3H, s, H-11); 13C NMR (CDCl3,
100 MHz) δ 167.3 (C-10), 163.2 (d, JCF = 249.6 Hz, C-4a),
149.0 (C-2), 142.2 (C-8), 142.1 (C-1b), 140.6 (C-9), 131.7
(d, J = 3.4 Hz, C-1a), 130.2 (d, J = 8.8 Hz, 2C, C-2a/6a),
129.4 (C-3b), 129.2 (C-5b), 127.7 (C-4b), 125.5 (C-5),
125.1 (C-6), 121.7 (C-4), 117.5 (d, J = 22.8 Hz, 2C, C-3a/
5a), 109.7 (C-7), 52.1 (C-11); 19F NMR (CDCl3, 376.5
Methyl
2-(3,4-dihydroxyphenyl)-1-(4-fluorophenyl)-1H-
benzo[d]imidazole-5-carboxylate (5q) Cream solid (65%
yield, 97% purity); mp 230–232 °C; UV λmax (MeOH) nm
(log ε) 226 (3.17), 264 (2.91), 313 (2.85); IR υmax 3501 (O-
H), 3062 (CH), 1712 (C=O), 1377 (C–F), 1177 (C–O)
MHz)
δ −109.62; HRESIMS m/z (pos): 353.0761
C19H14FN2O2S (calcd. 353.0760).
1
cm−1; H NMR (MeOH– d4, 400 MHz) δ 8.40 (1H, d, J =
Methyl 1-(4-fluorophenyl)-2-(furan-2-yl)-1H-benzo[d]imi-
dazole-5-carboxylate (5t) Dark brown solid (76% yield,
98% purity); mp 139–141 °C; UV λmax (MeOH) nm (log ε)
258 (3.15), 313 (3.11), 326 (3.01); IR υmax 3069 (CH), 1711
1.5 Hz, H-4), 7.97 (1H, dd, J = 8.5, 1.5 Hz, H-6), 7.42 (2H,
dd, J = 8.7, 4.9 Hz, H-2a/6a), 7.33 (2H, t, J = 8.7 Hz, H-3a/
5a), 7.26 (1H, d, J = 8.5 Hz, H-7), 7.01 (1H, d, J = 2.1 Hz,
H-2b), 6.86 (1H, dd, J = 8.2, 2.2 Hz, H-6b), 6.7 (1H, d, J =
8.2 Hz, H-5b), 3.95 (3H, s, H-11); 13C NMR (MeOH– d4,
100 MHz) δ 168.8 (C-10), 164.1 (d, JCF = 246.8 Hz, C-4a),
156.9 (C-2), 149.2 (C-3b), 146.6 (C-4b), 143.0 (C-8), 141.5
(C-9), 133.8 (d, J = 2.8 Hz, C-1a), 130.9 (d, J = 8.9 Hz, 2C,
C-2a/6a), 126.5 (C-5), 125.9 (C-6), 123.1 (C-6b), 121.6 (C-
4), 121.2 (C-1b), 118.1 (2C, d, J = 23.2 Hz, C-3a/5a), 117.7
(C-2b), 116.3 (C-5b), 111.5 (C-7), 52.7 (C-11); 19F NMR
(MeOH- d4, 376.5 MHz) δ −113.67; HRESIMS m/z (pos):
379.1094 C21H16FN2O4 (calcd. 379.1110).
1
(C=O), 1359 (C–F), 1195 (C–O) cm−1; H NMR (CDCl3,
400 MHz) δ 8.53 (1H, d, J = 1.4 Hz, H-4), 7.96 (1H, dd, J =
8.5, 1.4 Hz, H-6), 7.48 (1H, bs, H-3b), 7.40 (2H, dd, J = 8.5,
4.7 Hz, H-2a/6a), 7.29 (2H, td, J = 8.5, 2.2 Hz, H-3a/5a),
7.06 (1H, d, J = 8.5 Hz, H-7), 6.39–6.40 (2H, m, H-4b/5b),
3.93 (3H, s, H-11); 13C NMR (CDCl3, 100 MHz) δ 167.3
(C-10), 163.0 (d, JCF = 249.2 Hz, C-4a), 145.7 (C-2), 144.9
(C-3b), 143.6 (C-8), 140.0 (C-9), 131.7 (d, J = 3.2 Hz, C-
1a), 129.8 (d, J = 8.8 Hz, 2C, C-2a/6a), 125.7 (C-5), 125.4
(C-6), 122.0 (C-4), 117.2 (d, J = 22.8 Hz, 2C, C-3a/5a),

Med Chem Res
113.6 (C-5b), 111.8 (C-4b), 109.8 (C-7), 52.1 (C-11); 19F
NMR (CDCl3, 376.5 MHz) δ −110.00; HRESIMS m/z
(pos): 337.0994 C19H14FN2O3 (calcd. 337.0988).
collected at intervals of 0.5° in a 6° range with the exposure
time of 10 s per frame. The final cell constants were cal-
culated from a set of 4762 strong reflections from the actual
data collection. Data collection method involved ω scans of
width 0.5°. Data reduction was carried out using the pro-
gram System Administrator’s Integrated Network Tool+.
The structure was solved by direct methods using SHELXS
and refined. Non-H atoms were first refined isotropically
and then by anisotropic refinement with full-matrix least-
squares calculations based on F2 using SHELXS. All H
atoms were positioned geometrically and allowed to ride on
their respective parent atoms. All H atoms were refined
isotropically. The absorption correction was based on fitting
a function to the empirical transmission surface as sampled
by multiple equivalent measurements. The final least-
squares refinement of 265 parameters against 4762 data
resulted in residuals R (based on F2 for I ≥ 2σ) and wR
(based on F2 for all data) of 0.0465 and 0.1048, respec-
tively. The final difference Fourier map was featureless. The
programs Olex-2 and Ortep-3 were used within the WinGX
software package to prepare artwork representation (Spek
2003; Farrugia 2012). Crystallographic data (excluding
structure factors) for the structure in this paper has been
deposited with the Cambridge Crystallographic Data Cen-
tre, CCDC, 12 Union Road, Cambridge CB21EZ, UK.
Copies of the data can be obtained free of charge on quoting
the depository number CCDC 1454700 (fax: +44-1223-
336-033; E-mail: deposit@ccdc.cam.ac.uk, http://www.
ccdc.cam.ac.uk).
Methyl 1-(4-fluorophenyl)-2-propyl-1H-benzo[d]imidazole-
5-carboxylate (5u) Cream solid (70% yield, 100% purity);
mp 102–104 °C; UV λmax (MeOH) nm (log ε) 227 (3.16),
267 (2.40); IR υmax 3083 (CH), 1715 (C=O), 1360 (C–F),
1166 (C–O) cm−1; 1H NMR (CDCl3, 400 MHz) δ 8.49 (1H,
d, J = 1.4 Hz, H-4), 7.94 (1H, dd, J = 8.5, 1.4 Hz, H-6), 7.34
(2H, dd, J = 8.2, 5.2 Hz, H-2a/6a), 7.29 (2H, t, J = 8.2 Hz,
H-3a/5a), 7.05 (1H, d, J = 8.5 Hz, H-7), 3.92 (3H, s, H-11),
2.76 (2H, t, J = 7.5 Hz, H-1b), 1.76–1.86 (2H, sestet, H-2b),
0.94 (3H, t, J = 7.5 Hz, H-3b); 13C NMR (CDCl3, 100 MHz)
δ 167.4 (C-10), 162.8 (d, JCF = 249.3 Hz, C-4a), 156.9 (C-
2), 141.7 (C-8), 139.5 (C-9), 131.2 (d, J = 2.8 Hz, C-1a),
129.2 (d, J = 8.8 Hz, 2C, C-2a/6a), 125.1 (C-5), 124.8 (C-6),
121.2 (C-4), 117.3 (d, J = 22.8 Hz, 2C, C-3a/5a), 109.6 (C-
7), 52.1 (C-11), 29.4 (C-1b), 21.1 (C-2b), 13.9 (C-3b); 19F
NMR (CDCl3, 376.5 MHz) δ −110.53; HRESIMS m/z
(pos): 335.1161 C18H17FN2O2Na (calcd. 335.1172).
Methyl 1-(4-fluorophenyl)-2-hexyl-1H-benzo[d]imidazole-
5-carboxylate (5v) Brown solid (70% yield, 61% purity);
mp 66–68 °C; UV λmax (MeOH) nm (log ε) 227 (3.53), 264
(2.81); IR υmax 3075 (CH), 1708 (C=O), 1399 (C–F), 1156
1
(C–O) cm−1; H NMR (CDCl3, 400 MHz) δ 8.48 (1H, bs,
H-4), 7.94 (1H, dd, J = 8.5, 1.2 Hz, H-6), 7.36 (2H, dd, J =
8.6, 5.0 Hz, H-2a/6a), 7.30 (2H, t, J = 8.6 Hz, H-3a/5a), 7.06
(1H, d, J = 8.5 Hz, H-7), 3.94 (3H, s, H-11), 2.76 (2H, t, J =
7.5 Hz, H-1b), 1.77 (2H, quintet, J = 7.5 Hz, H-2b),
1.25–1.35 (2H, m, H-3b), 1.23–1.26 (2H, m, H-5b),
0.88–0.90 (2H, m, H-4b), 0.84 (3H, t, J = 6.8 Hz, H-6b);
13C NMR (CDCl3, 100 MHz) δ 167.6 (C-10), 162.9 (d,
JCF = 249.0 Hz, C-4a), 157.2 (C-2), 141.7 (C-8), 139.6 (C-
9), 131.5 (d, J = 3.1 Hz, C-1a), 129.2 (d, J = 8.8 Hz, 2C, C-
2a/6a), 124.8 (C-5), 124.5 (C-6), 121.3 (C-4), 117.2 (d,
J = 22.8 Hz, 2C, C-3a/5a), 109.4 (C-7), 52.0 (C-11), 31.3
(C-4b), 28.9 (C-3b), 27.6 (C-1b), 27.6 (C-2b), 22.4 (C-5b),
13.9 (C-6b); 19F NMR (CDCl3, 376.5 MHz) δ −110.80;
HRESIMS m/z (pos): 355.1816 C21H24FN2O2 (calcd.
355.1822).
In vitro antimicrobial studies
The synthesized compounds were tested for their anti-
bacterial and antifungal activity in vitro against the Gram
+ve bacterial strains: Staphylococcus aureus (ATCC 25923)
and methicillin resistant S. aureus (MRSA) (ATCC BAA-
1683) and Gram -ve strains (Escherichia coli (ATCC
25922), Klebsiella pneumonia (ATCC 31488), and Pseu-
domonas aeruginosa (ATCC 27853)) using ciprofloxacin
and ampicillin as standards. For the antifungal assay Can-
dida albicans (ATCC 10231) was used with tioconazole as
the reference drug. A disc diffusion assay was used as an
initial screening test to select potentially active compounds
using a concentration of 10 mg mL−1. Minimum bactericidal
concentrations (MBCs) were only carried out on com-
pounds that showed a zone of inhibition between 8–35 mm
(5b, 5e, 5h, 5k–l, and 5o–t).
Single crystal X-ray diffraction analysis
Crystals suitable for X-ray diffraction were obtained by
slow evaporation in a combination of ethyl acetate and n-
hexane at room temperature. The crystal evaluation and data
collection were performed on a Bruker Smart APEX II
diffractometer with Mo Kα radiation (λ = 0.71073 Å). The
diffractometer to crystal distance was set at 4 cm. The initial
cell matrix was obtained from three series of scans at dif-
ferent starting angles. Each series consisted of 12 frames
The microbial cultures were grown overnight at 37 °C in
nutrient broth (UKZN Biolab, South Africa), adjusted to a
0.5 McFarland standard using distilled water and lawn
inoculated onto Mueller-Hinton agar (MHA) plates.
A volume of 10 μL of each sample (23.20–32.04 µM in 1
mL DMSO) was inoculated onto antibiotic assay discs

Med Chem Res
(6 mm diameter) and placed on the MHA plates which were
incubated overnight at 37 °C for 24 h. After the incubation
period, zones of inhibition were measured in mm. Com-
pounds showing an inhibition zone of >9 mm were selected
to determine their MBC values using the broth dilution
assay with ampicillin and ciprofloxacin as the controls
following the method in Andrews (2001).
For the broth dilution method the microbial cultures
(adjusted to 0.5 McFarland) were prepared as described
previously for the disc diffusion method. The test com-
pounds were dissolved in DMSO (10 mg mL−1) and subject
to a 50% serial dilution in 1 mL Eppendorf tubes with
Mueller-Hinton broth, inoculated with bacterial cultures
(20 μL) and then incubated at 37 °C for 18 h. The total
volume in each Eppendorf was 200 μL. A volume of 10 µL
of each dilution was spotted on MHA plates and incubated
at 37 °C for 18 h to determine the MBC (µM). Ampicillin,
ciprofloxacin and tioconazole served as the standard drugs
for the antimicrobial and antifungal studies, respectively.
All experiments were performed in duplicate.
representative compounds (RCs) (5h and 5r) were gener-
ated at physiological pH using the “Prepare Ligands”
module and subsequently minimized. The partial atomic
charges on each atom were developed using the CHARMm
force field. A binding sphere of diameter 7.6 Å was devel-
oped around the active site residues. The automated docking
was performed using the CDocker algorithm (Wu et al.
2003) by generating new conformations of the ligands using
the molecular dynamics method. The best pose of each RC
was chosen based on the scoring function (-CDOCKER
energy), and subjected further to binding energy
calculations.
Results and discussion
Chemistry
Three fundamental methodologies were used in this study:
microwave, conventional and mechanochemical synthesis.
A series of 22 novel benzimidazole molecules (5a–5v) were
synthesized employing these synthetic methods with dif-
ferent substituents at C-2 on the benzimidazole skeleton.
The different substituents were all aldehyde derived
(Table 1). These contained para halogenated phenyl groups,
phenyl groups with para substituted electron donating and
withdrawing groups as well as other heterocyclic ring
moieties such as quinoline, furan, naphthalene and thio-
phene, and the alkyl chains butane and heptane. In addition
to the different substituents present on the aromatic ring
attached to C-2, an ester moiety was present at C-5 and a
4-fluorophenyl moiety at N-1 (Scheme 1).
The synthesis started with the esterification of 4-fluoro-3-
nitrobenzoic acid (1), since the subsequent nucleophilic step
is easier to perform with esters rather than acids due to the
acidic nature of benzoic acids. The conversion to the ester
(2) occurred in 95% yield. The fluoro substituent on the
ester was then substituted with 4-fluoroanilne producing 3
in a 90% yield. Prior to the aldehyde being added, the nitro
group of the methyl benzoate (3) was reduced (92% yield)
with zinc and ammonium formate forming the o-diamino
phenyl precursor (4) to the benzimidazoles (Scheme 1).
In the last step of the synthesis, different aldehydes were
added to the precursor 4 using three methods: (i) without the
use of solvents or catalysts in a microwave at 110 °C, (ii)
grinding equimolar amounts of 4 and the aldehydes in the
presence of iodine as a catalyst at room temperature and (iii)
refluxing the amino benzoate (4) and various aldehydes
with ethanol in the presence of Na2S2O5 to produce 22
benzimidazoles (5a–v). The probable mechanism of the
final step is imine formation of the aldehyde with the pri-
mary amino group followed by nucleophilic attack by the
secondary amine to the imine carbon. Proton transfer from
In vitro antioxidant studies
The scavenging activity (antioxidant capacity) of the syn-
thesized fluorinated benzimidazole compounds (5a–5v) on
the stable radical, DPPH was evaluated according to a
method by Murthy et al. (2012) with some modifications. A
volume of 150 μL of methanolic solution of the test com-
pounds at different concentrations (1000, 500, 200, 50, 20
and 10 μg mL−1) was mixed with 2850 μL of the methanolic
solution of DPPH (0.1 mM). An equal amount of MeOH
and DPPH without sample served as a control. After 30 min
of reaction at room temperature in the dark, the absorbance
was measured at 517 nm against methanol as a blank using
a UV spectrophotometer as mentioned above. The percen-
tage free radical scavenging activity was calculated
according to the following equation:
À
Á
ꢀ
ꢁ
AcontrolꢀAsample
% Scavenging activity ¼
´ 100
Ablank
Where “Acontrol” is the absorbance of the control reaction
(containing all reagents except the test compound) and
“Asample” is the absorbance of the reagents with a particular
test compound.
Molecular docking methodology
The “Prepare Protein” module was used to protonate the
amino acid residues of the X-ray structure of topoisomerase
II DNA-gyrase (PDB ID: 2XCT, resolution 3.35 Å). The
native ligand and water molecules were removed from the
protein, while the manganese ion (Mn2+) present in the
active site was retained. Different conformations of the

Med Chem Res
Scheme 1 Synthesis of fluorinated benzimidazoles 5a–5v; a MW 110 °C, 3–8 min; b grinding, I2 (10 mol%), 5–45 min; c Na2S2O5 (20 mol%),
EtOH, reflux, 8–18 hr
one nitrogen to the other followed by loss of H2 resulted in
the formation of the benzimidazoles 5a–5v (Scheme 2).
Their structures were confirmed by NMR spectroscopy (1D
and 2D NMR) and single crystal X-ray diffraction. Of the
three methods used for the synthesis, the best results were
achieved under microwave conditions at 110 °C for 3–8 min
with yields of between 85–96% (Table 1).
The aromatic protons of phenyl group H-2a/6a appeared
as a dd at δΗ 7.52 (J = 9.8, 5.0 Hz), coupling to both F and
H-3a/5a, the latter appearing as a triplet at δΗ 7.44, due to
the same J values (8.6 Hz) for both coupling to the F and to
H-2a/6a. The C-4a fluorinated carbon resonance occurred as
a doublet at δC 161.8 (J = 244.9 Hz). These assignments
were supported by HMBC correlations between C-4a and
H-2a/6a as well as H-3a/5a and C-1a, which occurred at
δ 132.9 (d, J = 3.0 Hz). C-2a/6a appeared as a doublet at δ
129.9 (J = 8.9 Hz) and C-3a/5a appeared as a doublet at δ
117.0 (J = 22.9 Hz).
Structural elucidation
Using 5i (methyl 2-(4-aminophenyl)-1-(4-fluorophenyl)-
1H-benzo[d]imidazole-5-carboxylate) as a representative
fluorinated benzimidazole, H-7, H-6 and H-4 on the ben-
zimidazole core skeleton occurs at δΗ 7.17 (d, J = 8.5 Hz),
7.83 (dd, J = 8.5, 1.5 Hz) and 8.26 (d, J = 1.3 Hz) respec-
tively. The ester methyl resonance (H-11) occurred as a
singlet at δΗ 3.89. The ester carbonyl resonance (C-10) was
identified from the HMBC correlation to H-11 and appeared
at δC 166.7. The two aromatic C-N singlet resonances, C-8
and C-9 were assigned to δC 142.4 and 140.5. C-8 was
distinguished from C-9 by a heteronuclear multiple bond
correlation (HMBC) to H-6. C-5 to which the ester group
was attached was assigned to δC 123.9 and showed an
HMBC correlation to H-7. C-2 of the imidazole core ske-
leton was present at δC 155.1.
For the phenyl group (B) attached to C-2, the aromatic
proton resonance of H-2b/6b appeared as a doublet at δΗ
7.21 (J = 8.6 Hz) and H-3b/5b appeared at δH 6.49 (d, J =
8.6 Hz). An HMBC correlation between the corresponding
C-3b/5b carbon resonance at δ 113.1 and the proton reso-
nance at δH 5.61 (NH2) confirmed the assignment of C-3b/
5b. The H-2b/6b resonance was confirmed by a HMBC
correlation with C-2. Selected HMBC correlations used in
the structural elucidation of 5i are provided in Fig. 1.
Antimicrobial activity
The trioxygenated derivatives 5p and 5r showed broad
spectrum activity against all strains of bacteria (S. aureus,
E. coli, K. pneumonia and P. aeruginosa) with the

Med Chem Res
Scheme 2 Proposed mechanism for the final step of the benzimidazole synthesis
trioxygenated molecules from the 22 compounds that was
synthesized. Even the dihydroxy substituted compound 5q
showed poor antimicrobial activity in comparison to 5p and
5r. It is likely that this trioxygenation has something to do
with its broad spectrum activity. However, the test com-
pounds were not as active as ciprofloxacin, whose MBC
was at least one order of magnitude better than the test
compounds, including 5r and 5p.
The benzimidazole 5a with an unsubstituted 2-phenyl
group was relatively unreactive and when the halogens, F
(5b), Cl (5c), and Br (5d) were placed at the para position
of the 2-substituted phenyl group, only the fluorinated
compound showed some form of antibacterial activity,
showing the best activity against K. pneumonia at 107.3
µM. This activity was enhanced when the fluorine was
replaced with a trifluoromethyl group (5e), which was
active against MRSA with an MBC of 70.7 μM and against
K. pneumonia (24.3 μM) and C. albicans (94.3 μM).
Compounds 5f-5o, 5q, 5s, and 5t with various substituents
at the para position of the 2-substituted phenyl group
(Table 1) were all inactive as antimicrobial agents.
Fig. 1 Selected HMBC correlations of compound 5i (H→C)
exception of MRSA. Compound 5p had MBC values of
115.7, 92.5 115.7 and 14.5 µM and 5r MBC values of 74.3,
49.6, 37.2 and 25.6 µM against S. aureus, E. coli, P. aer-
uginosa and K. pneumoniae, respectively. The activity of
both these compounds were 10 fold greater than ampicillin
in the same assay. Compound 5r was 2b,3b,4b-trisub-
stituted with hydroxyl groups at each of the positions on the
aromatic B ring and 5p was 2b,4b,6b-trisubstituted with a
hydroxy group at the 2b-position and methoxy groups at the
4b and 6b positions. Consequently, these were the only two
Antioxidant activity
For antioxidant activity, the DPPH radical scavenging
activity assay was used. The DPPH radical scavenging

Med Chem Res
Fig. 2 The orientation of 5r (a)
and 5h (b) in the active site of
topoisomerase II DNA-gyrase.
Ligands are shown as sticks
(lemon color) and amino acids
of the protein as lines. Green
dotted lines represent classical
H-bonds, yellow dotted lines,
non-classical H-bonds and red
dotted lines hydrophobic
interactions (color figure online)
assay measures the ability to scavenge free radicals in situ
(Murthy et al. 2012). The trioxygenated compounds 5r
(IC50 of 306.71 μM) and 5p (IC50 of 386.55 μM) with
hydroxy and methoxy groups showed antioxidant activity
comparable to ascorbic acid (264.50 μM). Two other com-
pounds, 5q (IC50 of 1129.0 μM) with dihydroxy moieties
and 5j with a monohydroxy group (IC50 of 1326.93 μM)
was approximately four times worse than ascorbic acid. All
the other compounds were five times or more, worse than
ascorbic acid. Thus, the only compounds that could be
considered as having antioxidant potential were 5r and 5p.
Other methods to test for antioxidant activity was not car-
ried out as this was only a preliminary antioxidant test for
these compounds.
DNA-gyrase. 3D-coordinates of the enzyme co-crystallized
with its inhibitor, ciprofloxacin (pdb id: 2XCT (Bax et al.
2010)), were downloaded from the RCSB protein data bank.
A binding sphere covering the active site was generated and
docking of the RCs was performed using the CDOCKER
module (Wu et al. 2003) implemented in Discovery Studio
ver 4.0 (Accelrys) along with ciprofloxacin.
The binding affinity of the docked compounds was
assessed by computing their binding energies (BEs) [Ecom-
plex-(Eprotein+Eligand)]. The more negative the BE, the
stronger the interaction with the enzyme. Compound 5r
exhibited stronger interaction (BE = −124.2 kcal mol−1)
with the enzyme in comparison to 5h (BE = −24.3 kcal
mol−1). This consequently supports the experimental results
as 5r showed the highest MBC in the experimental assay
performed. The BE of 5r had a difference of approximately
30 kcal mol−1 from the computed BE of ciprofloxacin
(−156 kcal mol−1).
Molecular docking
Several drugs including ciprofloxacin and lomefloxacin
exhibit antibacterial action by inhibiting bacterial type II
topoisomerases (topoisomerase IV and DNA-gyrase) (Mit-
scher 2005). In order to support the antibacterial results and
gain some insight into the binding characteristics of the
compounds with bacterial proteins, two representative
compounds (RCs), 5r (most potent) and 5h (least potent)
were docked into the empty active site of topoisomerase II
The best docked pose of 5r and 5h in the active site of
topoisomerase II DNA-gyrase (Fig. 2) showed that both the
RCs interacted with similar amino acid residues (Arg458,
Lys460 and Glu477). This was also observed for cipro-
floxacin (Fig. 3). Compound 5r (Fig. 2a), the most active
antibacterial agent, formed a classical hydrogen bond with
Ser 1084 (1.98 Å) and four non-classical hydrogen bonds

Med Chem Res
Fig. 3 The orientation of
ciprofloxacin in the active site of
topoisomerase II DNA-gyrase.
The ligand is shown in sticks
(lemon) and amino acids of the
protein are depicted in line
format. Classical H-bonds
(green dotted), non-classical
H-bonds (yellow dotted) and co-
ordinate bonds (golden dotted)
(color figure online)
Table 2 Antimicrobial activity
of the synthesized compounds,
MBCs in μM
No.
Gram-positive
bacteria
Gram-negative bacteria
Fungus
S.aureus
MRSA
E.coli
P.aeruginosa
K.pneumoniae
C.albicans
5b
268.19
141.49
2492.54
803.02
398.18
1087.38
115.65
206.61
74.32
–
536.42
70.73
1661.69
401.51
1992.48
906.13
555.21
439.06
446.03
–
321.83
282.98
103.85
200.76
149.43
407.76
92.53
536.42
565.98
623.12
250.93
199.24
362.46
115.65
–
107.28
24.32
830.85
301.11
149.43
724.92
14.45
–
214.56
94.33
5e
5h
1246.27
803.02
398.18
362.46
369.85
3305.99
421.24
443.81
1859.62
–
5k
5l
5o
5p
5q
232.45
49.56
5r
37.15
–
25.55
–
5s
1109.50
348.66
447.18
1.84
5t
–
–
–
–
Ampicilin
Ciprofloxacin
Tioconazole
55.89
1.84
894.36
7.36
1788.73
1.84
447.18
3.68
–
–
–
–
–
–
100.84
Table 3 Antioxidant activity of the synthesized compounds by the
DPPH assay (IC50 in μM)
Table 4 Hydrogen bond interactions for 5k
D-H…A
d(D-H) d(H…A) d(D…A)
<(DHA)
No.
IC50 (μM)
C(5b)-H(5b)…O(2)#1 0.95
C(8b)-H(8bi)…O(2)#1 0.98
2.49
2.63
2.59
2.46
2.52
3.4101(14) 162.3
5j
1326.93
386.55
1129.01
306.71
264.50
3.3278(15) 128.3
3.1002(13) 113.6
3.2737(14) 143.3
3.4611(14) 171.9
5p
C(7)-H(7)…F(1)#2
C(2a)-H(2a)…N(3)#3
C(6a)-H(6a)…N(3)#4
0.95
0.95
0.95
5q
5r
Ascorbic Acid
with Arg458 (2.61 Å), Lys460 (2.45 Å) and Glu477 (2.71
Å, 2.76 Å). The least active compound (5h) displayed three
hydrogen bonds with Glu477 (2.61 Å) and Lys460 (3.03 Å,
3.05 Å) in addition to hydrophobic interactions with Arg458
(Fig. 2b). The fewer and relatively longer hydrogen bond
distances in 5h could have led to its weaker binding with
the enzyme and in turn lower activity. Ciprofloxacin (the
native ligand) fitted nicely into the active site of the enzyme
through five hydrogen bonds with Glu477 (2.78 Å, 2.80 Å),
Lys460 (2.34 Å, 2.40 Å) and Arg458 (2.14 Å) amino resi-
dues and formed another two co-ordinate bonds with Mn2+
(Fig. 3). However, both compounds failed to show any
interaction with Mn2+, which may be responsible for them
not being as active as ciprofloxacin. (Tables 2 and 3)

Med Chem Res
Fig. 4 Ortep diagram for 5k
the National Research Foundation (NRF), South Africa, and the South
African Research Chairs Initiative of the Department of Science and
Technology.
Crystal structure
The crystal structure of the parent compound, methyl 2-(4-
(dimethylamino)phenyl)-1-(4-fluorophenyl)-1H-benzo[d]
imidazole-5-carboxylate (5k) was obtained and the refine-
ment data are contained in the Supplementary file (Table
S1). Compound 5k crystallized in the monoclinic P 21/c
space group with two molecules in the asymmetric unit. The
ester group and N-dimethyl group point away from each
other and all groups are in the plane except the 4-fluoro
phenyl group, which is perpendicular to the benzimidazole
moiety on N-1. Weak hydrogen bond interactions (C–H…
O, C–H…F and C–H…N) were observed in the crystal
structure (Table 4). Further selected bond lengths were
described in the supplementary file (Table S2). An ortep
diagram of 5k is given in Fig. 4.
Compliance with ethical standards
Conflict of interest The authors declare no competing financial
interests.
References
Abdullah JM, Sulaiman MM, Mohammed SJ (2012) Microwave-
assisted one-pot synthesis of 2-aryl (1H) benzimidazoles without
catalyst. J Educ Sci 25(1):58–63
Al-Mohammed NN, Alias Y, Abdullah Z, Shakir RM, Taha EM,
Hamid AA (2013) Synthesis and antibacterial evaluation of some
novel imidazole and benzimidazole sulfonamides. Molecules
18:11978–11995
Andrews JM (2001) Determination of minimum inhibitory con-
centrations. J Antimicrob Chemother 48(S1):5–16
Conclusion
Arora S (2011) Study of some benzimidazole compounds as anti-
bacterial and antifungal agents. J Pharm Sci Res 3(7):1310–1314
Azizian J, Torabi P, Noei J (2016) Synthesis of benzimidazoles and
benzoxazoles using TiCl3OTf in ethanol at room temperature.
Tetrahedron Lett 57(2):185–18
Banerjee M, Chatterjee A, Kumar V, Bhutia ZT, Khandare DG, Majik
MS, Roy BG (2014) A simple and efficient mechanochemical
route for the synthesis of 2-aryl benzothiazoles and substituted
benzimidazoles. RSC Adv 4:39606–39611
We have developed an efficient, cost effective and faster
route for 2-substituted fluorinated benzimidazoles via a
solvent and catalyst free microwave method. Compounds
5p and 5r with trioxygenated substitution showed better
activity than ampicillin against the microbial strains used in
the assays. These two compounds could be good leads for
antimicrobial agents and will lead to further trioxygenated
substituted benzimidazoles being synthesized in order to
improve on the antibacterial activity and possibly find an
excellent candidate to be developed into an antiobiotic.
Barot KP, Nikolova S, Ivanov I, Ghate MD (2013) Novel research
strategies of benzimidazole derivatives: a review. Mini-Rev Med
Chem 13(10):1–27
Bax BD, Chan PF, Eggleston DS, Fosberry A, Gentry DR, Gorrec F,
Giordano I, Hann MM, Hennessy A, Hibbs M, Huang J, Jones E,
Jones J, Brown KK, Lewis CJ, May EW, Saunders MR, Singh O,
Spitzfaden CE, Shen C, Shillings A, Theobald AJ, Wohlkonig A,
Pearson ND, Gwynn MN (2010) Type IIA topoisomerase inhi-
bition by a new class of antibacterial agents. Nature 466:935–943
Biron KK (2006) Antiviral drugs for cytomegalovirus diseases. Anti-
viral Res 71:154–163
Acknowledgements The authors gratefully acknowledge the Centre
for High Performance Computing (CHPC), an initiative supported by
the Department of Science and Technology of South Africa for the
Discovery Studio facility. This research was supported by grants from

Med Chem Res
Borhade AV, Tope DR, Patil DR (2012) An efficient synthesis of
benzimidazole by cyclization–oxidation processes using Fe/MgO
as a heterogeneous recyclable catalyst. J Chem Pharm Res 4(5):
2501–2506
Carvalho LCR, Ribeiro D, Seixas RSGR, Silva AMS, Nave M,
Martins AC, Erhardt S, Fernandes E, Cabrita EJ, Marques MMB
(2015) Synthesis and evaluation of new benzimidazole based
COX inhibitors: a naproxen-like interaction detected by STD-
NMR. RSC Adv 5:49098–49109
Chen C, Chen C, Li B, Tao J, Peng J (2012) Aqueous synthesis of 1-
H-2-substituted benzimidazoles via transition-metal-free intra-
molecular amination of aryl iodides. Molecules 17:12506–12520
Das R, Mehta D, Bhardawaj H (2012) An overview on microwave
mediated synthesis. Int J Res Dev Pharm Life Sci 1(2):32–39
Eren B, Bekdemir Y (2014) Simple, mild, and highly efficient
synthesis of 2-substituted benzimidazoles and bis-
benzimidazoles. Quim Nova 37(4):643–647
(trifluoromethyl)-benzimidazole derivatives. Bioorg Med Chem
Lett 11:187–190
Panda SS, Malik R, Jain SC (2012) Synthetic approaches to 2-aryl-
benzimidazoles: A review. Curr Org Chem 16(16):1905–1919
Park S, Jung J, Cho EJ (2014) Visible-light-promoted synthesis of
benzimidazoles. Eur J Org Chem 2014:4148–4154.
Patil AA, Kamble SB, Rashinkar GS, Salunkhe RS (2014) Ultrasound
promoted synthesis of 1,2-disubstituted benzimidazoles using
aqueous hydrotropic solution. Chem Sci Rev Lett 3(10):214–220
Prajapti SK, Nagarsenkar A, Guggilapu SD, Babu BN (2015) B
(C6F5)3 as versatile catalyst: an efficient and mild protocol for the
one-pot synthesis of functionalized piperidines and 2-substituted
benzimidazole derivatives. Tetrahedron Lett 56:6795–6799
Rajasekhar KK, Ananth VS, Nithiyananthan TS, Hareesh G, Kumar
PN, Reddy RSP (2010) Comparative study of conventional and
microwave induced synthesis of selected heterocyclic molecules.
Int J ChemTech Res 2(1):592–597
Farrugia LJ (2012) WinGX and ORTEP for windows: an update. J
Appl Cryst 45:849–854
Rao SS, Reddy CVR, Dubey PK (2014) An ultrasound mediated green
synthesis of benzimidazolylthiounsaturatednitriles using water as
a green solvent. Org Chem Int:5. doi:10.1155/2014/403803
Rithe SR, Jagtap RS, Ubarhande SS (2015) One pot synthesis of
substituted benzimidazole derivatives and their charcterization. J
Chem 8(2):213–217
Saberi A (2015) Efficient synthesis of Benzimidazoles using zeolite,
alumina and silica gel under microwave irradiation. Iranian J Sci
Technol 39(A1):7–10
Saleh V, Khazaei A, Abizadeh H, Saednia S (2015) Synthesis, char-
acterization, and application of poly(N,N’-dibromo-N-ethyl-
naphthyl-2,7-disulfonamide) as an efficient reagent for the synthesis
of 2-arylbenzimidazole and 2-aryl-1-arylmethyl-1H-1,3-benzimi-
dazole derivatives. Org Chem Int:9. doi:10.1155/2015/635630
Singla M, Ranjan R, Mahiya K, Mohapatra SC, Ahmad S (2015)
Nitric oxide inhibition, antioxidant, and antitumour activities of
novel copper(II) bis-benzimidazole diamide nanocoordination
complexes. New J Chem 39:4316–4327
Funel JA, Brodbeck S, Guggisberg Y, Litjens R, Seidel T, Struijk M,
Abele S (2014) Diastereospecific enolate addition and atom-
efficient benzimidazole synthesis for the production of L/T calcium
channel blocker ACT-280778. Org Process Res Dev 18:1674–1685
Gawande MB, Shelke SN, Zboril R, Varma RS (2014) Microwave-
assisted chemistry: synthetic applications for rapid assembly of
nanomaterials and organics. Acc Chem Res 47:1338–1348
Gurvinder S, Maninderjit K, Mohan C (2013) Benzimidazoles: The
latest information on biological activities. Int Res J Pharm 4(1):
82–87
Jacob J (2012) Microwave assisted reactions in organic chemistry: A
review of recent advances. Int J Chem 4(6):29–43
Jain ZJ, Kankate RS, Chaudhari BN, Kakad RD (2013) Action of
benzimidazolo-piperazinyl derivatives on dopamine receptors.
Med Chem Res 22:520–530
Kalyankar TM, Pekamwar SS, Wadher SJ, Tiprale PS, Shinde GH
(2012) Review on benzimidazole derivative. Int J Chem Pharm
Sci 3(4):1–10
Spek AL (2003) Single-crystal structure validation with the program
PLATON. J Appl Cryst 36:7–13
Kamal A, Narasimha Rao MP, Swapna P, Srinivasulu V, Bagul C,
Shaik AB, Mullagiri K, Kovvuri J, Reddy VS, Vidyasagar K,
Nagesh N (2014) Synthesis of β-carboline–benzimidazole con-
jugates using lanthanum nitrate as a catalyst and their biological
evaluation. Org Biomol Chem 12:2370–2387
Kattimani PP, Kamble RR, Meti GY (2015) Expedient synthesis of
benzimidazoles using amides. RSC Adv 5:29447–29455
Keri RS, Hiremathad A, Budagumpi S, Nagaraja BM (2015) Com-
prehensive review in current developments of benzimidazole-
based medicinal chemistry. Chem Biol Drug Des 86(1):19–65
Khanna L, Panda SS, Khanna P (2012) Synthetic routes to symmetric
bisbenzimidazoles: A review. Mini-Rev Org Chem 9(4):381–396
Kuş C, Ayhan-Kılcıgil G, Tunçbilek M, Altanlar N, Çoban T, Can-Eke
B, Iscan M (2009) Synthesis, antimicrobial and antioxidant
activities of some benzimidazole derivatives. Lett Drug Des
Discovery 6(5):1–6
Mitscher LA (2005) Bacterial topoisomerase inhibitors: quinolone and
pyridone antibacterial agents. Chem Rev 105:559–592
Murthy PS, Manjunatha MR, Sulochannama G, Naidu MM (2012)
Extraction, characterization and bioactivity of coffee anthocya-
nins. Eur J Biol Sci 4(1):13–19
Narasimhan B, Sharma D, Kumar P (2010) Benzimidazole: a medic-
inally important heterocyclic moiety. Med Chem Res 21(3):
269–283
Tateyama K, Wada K, Miura H, Hosokawa S, Abe R, Inoue M (2016)
Dehydrogenative synthesis of benzimidazoles under mild condi-
tions with supported iridium catalysts. Catal Sci Technol
6:1677–1684
Tomic M, Kundakovic M, Butorovic B, Jana B, Andric D, Rogli G,
Ignjatovic D, Rajacic SK (2004) Pharmacological evaluation of
selected arylpiperazines with atypical antipsychotic potential.
Bioorg Med Chem Lett 14:4263–4266
Vyas KV, Ghate M (2010) Substituted benzimidazole derivatives as
angiotensin II–AT1 receptor antagonist: a review. Mini-Rev Med
Chem 10(14):1366–1384
Wu G, Robertson DH, Brooks CL, Vieth M (2003) Detailed analysis
of grid-based molecular docking: A case study of CDOCKER-A
CHARMm-based MD docking algorithm. J Comput Chem
24(13):549–62
Xiangming H, Huiqiang M, Yulu W (2007) p-TsOH catalyzed synth-
esis of 2-arylsubstituted benzimidazoles. ARKIVOC 13:150–154
Yoon YK, Ali MA, Wei AC, Choon TS, Ismail R (2015) Synthesis
and evaluation of antimycobacterial activity of new benzimida-
zole aminoesters. Eur J Med Chem 93:614–624
Yoon YK, Ali MA, Wei AC, Choon TS, Osman H, Parang K, Shirazi
AN (2014) Synthesis and evaluation of novel benzimidazole
derivatives as sirtuin inhibitors with antitumor activities. Bioorg
Med Chem 22:703–710
Navarrete-Vázquez G, Cedillo R, Hernández-Campos A, Yépez L,
Hernández-Luis F, Valdez J, Morales R, Cortés R, Hernández M,
Castillo R (2001) Synthesis and antiparasitic activity of 2-
Zhang ZH, Yin L, Wang YM (2007) An expeditious synthesis of
benzimidazole derivatives catalyzed by lewis acids. Catal Com-
mun 8:1126–1131