Antituberculosis agents bearing the 1,2-disubstituted benzimidazole scaffold

Article abstract of DOI:10.1007/s00044-017-1784-2

Abstract: The emergence of drug-resistant strains in recent years has fueled the epidemic of tuberculosis. This necessitates the development of new chemical scaffolds to curb resistant tuberculosis for effective control of this disease. In this study, we have designed and synthesized two series of benzimidazole derivatives. Their antimycobacterial activities were initially evaluated using Mycobacterium tuberculosis H₃₇R_V strains. The most potent analog (6h) was further assessed using various drug-resistant M. tuberculosis strains. This report described the importance of benzimidazoles as new antitmycobacterial agents targeting both the M. tuberculosis H₃₇R_V as well as the drug-resistant-tuberculosis strains. The trifluoromethyl group which was essential for antimycobacterial activity was also highlighted. Graphical Abstract: Two series of benzimidazole derivatives and their antimycobacterial activities were evaluated using M. tuberculosis H₃₇R_V (MTB-H₃₇R_V) strains. Compound 6h was identified as the most potent among all synthesized compounds. The most potent analog was further assessed using various drug-resistant MTB strains. In addition, the trifluoromethyl was identified as an important substitution in giving good antimycobacterial effect. [InlineMediaObject not available: see fulltext.]

Full text of DOI:10.1007/s00044-017-1784-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1784-2
ORIGINAL RESEARCH
Antituberculosis agents bearing the 1,2-disubstituted
benzimidazole scaffold
1,2
Chee Wei Ang1 Mohamed Ashraf Ali3,4 Hasnah Osman5
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●
●
●
Keng Yoon Yeong
Soo Choon Tan1
Received: 16 May 2016 / Accepted: 5 January 2017
© Springer Science+Business Media New York 2017
Abstract The emergence of drug-resistant strains in recent
years has fueled the epidemic of tuberculosis. This neces-
sitates the development of new chemical scaffolds to curb
resistant tuberculosis for effective control of this disease.
In this study, we have designed and synthesized two series
of benzimidazole derivatives. Their antimycobacterial
activities were initially evaluated using Mycobacterium
tuberculosis H37RV strains. The most potent analog (6h)
was further assessed using various drug-resistant M.
tuberculosis strains. This report described the importance of
benzimidazoles as new antitmycobacterial agents targeting
both the M. tuberculosis H37RV as well as the drug-
resistant-tuberculosis strains. The trifluoromethyl group
which was essential for antimycobacterial activity was also
highlighted.
Graphical Abstract Two series of benzimidazole deriva-
tives and their antimycobacterial activities were evaluated
using M. tuberculosis H37RV (MTB-H37RV) strains.
Compound 6h was identified as the most potent among all
synthesized compounds. The most potent analog was
further assessed using various drug-resistant MTB strains.
In addition, the trifluoromethyl was identified as an
important substitution in giving good antimycobacterial
effect.
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1784-2) contains supplementary material,
which is available to authorized users.
* Keng Yoon Yeong
yeong.kengyoon@monash.edu
kyyeong@gmail.com
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Keywords Tuberculosis Benzimidazoles
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Antimycobacterial Drug-resistance Trifluoromethyl
1
Institute for Research in Molecular Medicine, Universiti Sains
Malaysia, Minden, 11800 Penang, Malaysia
2
School of Science, Monash University Malaysia Campus, Jalan
Lagoon Selatan, Bandar Sunway, 47500 Selangor, Malaysia
Introduction
3
New Drug Discovery Research, Department of Medicinal
Chemistry, Alwar Pharmacy College, Alwar, Rajasthan 301030,
India
Tuberculosis (TB) remains a global health problem and the
second leading cause of death from an infectious disease
worldwide after AIDS (Dutt and Stead 1999). The intro-
duction of first-line drugs such as streptomycin, p-amino-
salicylic acid, and isoniazid for TB treatment some 50 years
4
New Drug Discovery Research, Department of Medicinal
Chemistry, Sunrise University, Alwar, Rajasthan 301030, India
5
School of Chemical Sciences, Universiti Sains Malaysia, Minden,
11800 Penang, Malaysia

Med Chem Res
ago has witnessed a remarkable decline in TB cases.
The active TB is currently treated with a four first-line
drug regimen comprising mainly isoniazid (INH),
rifampicin (RMP), pyrazinamide, and ethambutol for a
period of at least 6 months (Snider and Roper 1992;
Bass et al. 1994). However, the infectious disease
has been undergoing resurgence since the last two
decades as Mycobacterium tuberculosis (MTB), the bacteria
which cause TB, gain resistance to the above-mentioned
drugs.
By definition, multidrug-resistant TB (MDR-TB) strains
lose their susceptibility to two of the first-line drugs used in
therapy, while extensively drug-resistant TB strains are in
addition, resistant to at least one fluoroquinolone and
one of the injectable anti-TB drugs, such as kanamycin
(Dorman and Chaisson 2007). Treatment of MDR-TB
requires longer time and is much more expensive compared
to TB. The problem is compounded with the recent
isolation of strains resistant to all of the standard first- and
second-line drugs used in TB therapy, giving rise to the
likely threat of a virtually incurable infection (Velayati
et al. 2009).
Materials and methods
Chemistry
All chemicals were supplied by Sigma-Aldrich (USA) and
Merck Chemicals (Germany). Thin layer chromatography
(TLC) using silica gel G were performed in the solvent
system chloroform–methanol (9:1). The spots were located
under short (254 nm)/long (365 nm) UV light. Elemental
analyses were performed on Perkin Elmer 2400 Series II
CHN Elemental Analyzer and were within 0.4% of
1
the calculated values. H and 13C NMR were performed
on Bruker Avance 300/500 (1H: 300/500 MHz, 13C:
75/125 MHz) spectrometer in CDCl3 using tetra-
methylsilane as internal standard. Mass spectra were
recorded on Varian 320-MS TQ liquid chromatography/
mass spectrometry using electrospray ionization (ESI). X-
ray structural analysis was carried out using Bruker
SMART APEXII charge-coupled device area-detector
diffractometer.
Synthesis of ethyl-4-fluoro-3-nitrobenzoate (1)
Recently, there have been reports on utilizing benzimi-
dazole derivatives to counter TB from work done by our
group (Yoon et al. 2013, 2015) as well as by others (Pieroni
et al. 2011; Kumar et al. 2011; Gong et al. 2014). Benzi-
midazoles, such as albendazole and thiabendazole were
shown to inhibit the activity of MTB, albeit at relatively
high concentration (MIC between 60 and 160 µM) (Slayden
et al. 2000). Benzimidazoles were previously demonstrated
to also inhibit the GTPase activity of MTB FtsZ and septum
formation (Margalit et al. 2004; Slayden et al. 2006), further
strengthening the notion of using benzimidazoles as anti-
tubercular agent. Moreover, benzimidazoles share structural
similarity with indoles, which are found to be present in
mycobacterial InhA inhibitors such as Genz-10850 (Kuo
et al. 2003). However, these reported inhibitors suffer
mostly from its poor cellular activity, which may be due to
low membrane permeability or activation of efflux pump
although they have very potent in vitro (InhA) inhibitory
activity (Kuo et al. 2003).
Therefore, developing a benzimidazole-based com-
pound, which has good membrane permeability as anti-
mycobacterial agent, is an interesting proposition. Our
previous studies have underlined the antimycobacterial
potential of benzimidazole derivatives in cell-based assay
using M. tuberculosis H37RV (MTB-H37RV) strains.
In the present work, we continued to expand our library of
compounds in the search of new antitubercular agents,
by reporting the synthesis and activity study of two new
series of benzimidazole derivatives. Key findings such as
the importance of trifluoromethyl group were also
discussed.
4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in
ethanol (50 mL) and concentrated H2SO4 (2 mL) for 8 h.
After completion of reaction (as evident from TLC), the
solvent was evaporated under reduced pressure. The aqu-
eous layer was extracted with ethyl acetate (25 mL × 3).
The organic layer was dried over Na2SO4 and concentrated
under reduced pressure to yield 1 as cream-colored powder
(75%).
Synthesis of 4-(2-substituted amino)-3-nitro-ethylbenzoate (2)
Ethyl-4-fluoro-3-nitrobenzoate, 1 (0.5 g, 2.34 mmol), amine
[for 5: N-(3-aminopropyl)imidazole; for 6: aniline] (2.58
mmol) and N,N-diisopropylethylamine, DIPEA (0.49 mL,
2.78 mmol) were mixed in dichloromethane (10 mL). The
reaction mixture was stirred overnight at room temperature.
After completion of reaction (as evident from TLC), the
reaction mixture was washed with water (10 mL × 2) fol-
lowed by 10% Na2CO3 solution (10 mL). The organic layer
was dried over Na2SO4 and concentrated under reduced
pressure to afford 2 as yellow solid (80–90%).
Synthesis of ethyl-3-amino-4-(2-substituted amino)
benzoate (3)
4-(2-Substituted amino)-3-nitro-ethylbenzoate, 2 (1 mmol),
ammonium formate (3 mmol) and Pd/C (50 mg) were mixed
in ethanol (10 mL). The reaction mixture was refluxed until
completion (solution turned colorless). The reaction mixture
was then filtered through Celite 545. The filtrate was

Med Chem Res
evaporated under reduced pressure. It was resuspended in
ethyl acetate (20 mL) and washed with water (10 mL × 2),
dried over Na2SO4 and evaporated to dryness to yield 3
(55–65%).
131.18, 137.76, 151.74, 153.40, 157.67, and 167.89. ESI-
MS: m/z 391.2 [M+H]+. Anal. calcd for C22H22N4O3:
C, 67.68%; H, 5.68%; N, 14.35%. Found: C, 67.66%;
H, 5.62%; N, 14.50%.
General procedure for the preparation of sodium bisulfite
addcuts of 4-substituted benzaldehyde (4a–h)
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-methoxyphenyl)-
1
1H-benzo[d]imidazole-5-carboxylate (5c) Yield: 82%; H
1
NMR (500 MHz, CDCl3): δ H NMR: 1.44 (3H, t, J = 7.0
Appropriate benzaldehyde (10 mmol) was dissolved in
ethanol (20 mL). Sodium metabisulfite (15 mmol) in 5 mL
water was added in portion over 5 min. The reaction
mixture was stirred at room temperature for 1 h and sub-
sequently stirred at 4 °C overnight. The precipitate formed
was filtered and dried to afford sodium bisulfite adducts
(55–90%).
Hz), 2.10–2.30 (2H, m), 3.88 (2H, t, J = 6.5 Hz), 3.91 (3H,
s), 4.27 (2H, t, J = 7.5 Hz), 4.43 (2H, q, J = 7.0 Hz), 6.79
(1H, s), 7.07 (1H, s), 7.28 (1H, d, J = 8.5 Hz), 7.39 (1H, s),
7.77 (2H, d, J = 8 Hz), 7.80 (2H, d, J = 8 Hz), 8.09 (1H, dd,
J = 1.5 Hz, 8.5 Hz), 8.55 (1H, s). 13C NMR: 14.62, 28.94,
41.01, 43.93, 59.24, 61.67, 108.15, 109.29, 120.12, 121.80,
130.93, 137.22, 150.04, 151.47, 151.62, and 167.68.
ESI-MS: m/z 405.2 [M+H]+. Anal. calcd for C23H24N4O3:
C, 68.30%; H, 5.98%; N, 13.85%. Found: C, 68.16%; H,
5.93%; N, 13.95%.
General procedure for the preparation of 2-substituted
benzimidazole derivatives (5a–h and 6a–h)
Ethyl-3-amino-4-(2-substituted amino)benzoate, 3 (1 mmol)
and various sodium bisulfite adducts, 4 (1.5 mmol) were
dissolved in DMF (5 mL). The reaction mixture was stirred
at 90 °C under N2 atmosphere for 24 h. After completion of
reaction (evident by TLC), the reaction mixture was diluted
in ethyl acetate (25 mL) and washed with water (10 mL ×
3). The organic layer was collected, dried over Na2SO4 and
evaporated under reduced pressure to afford compounds the
final compounds in 52–92% yields.
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-morpholinophe-
nyl)-1H-benzo[d]imidazole-5-carboxylate
(5d) Yield:
77%; 1H NMR (300 MHz, CDCl3): δ 1H NMR: 1.44 (3H, t,
J = 7.2 Hz), 2.10–2.30 (2H, m), 3.29 (4H, t, J = 4.5 Hz),
3.88 (2H, t, J = 6.6 Hz), 3.92 (4H, t, J = 4.5 Hz), 4.31 (2H,
t, J = 6.6 Hz), 4.43 (2H, q, J = 7.2 Hz), 6.75 (1H, s), 7.08
(1H, s), 7.32 (1H, d, J = 8.4 Hz), 7.58 (2H, d, J = 8.4 Hz),
7.59 (1H, s), 7.66 (2H, d, J = 8.4 Hz), 8.08 (1H, dd, J = 1.5
Hz, 8.4 Hz), 8.56 (1H, s). 13C NMR: 14.39, 30.79, 41.77,
43.89, 60.86, 63.40, 108.90, 112.01, 116.32, 118.53,
121.79, 123.95, 125.01, 129.86, 130.03, 136.97, 138.55,
142.90, 151.54, 155.98, and 167.11. ESI-MS: m/z 460.3 [M
+H]+. Anal. calcd for C26H29N5O3: C, 67.95%; H, 6.36%;
N, 15.24%. Found: C, 68.12%; H, 6.22%; N, 15.28%.
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-phenyl-1H-benzo
[d]imidazole-5-carboxylate (5a) Yield: 84%; 1H NMR
(300 MHz, CDCl3): δ 1.45 (3H, t, J = 7.2 Hz), 2.20–2.30
(2H, m), 3.88 (2H, t, J = 6.6 Hz), 4.31 (2H, t, J = 6.6 Hz),
4.44 (2H, q, J = 7.2 Hz), 6.75 (1H, s), 7.08 (1H, s), 7.30
(1H, d, J = 8.4 Hz), 7.32 (1H, d, J = 8.4 Hz), 7.58 (2H, d,
J = 8.4 Hz), 7.59 (1H, s), 7.66 (2H, d, J = 8.4 Hz), 8.08
(1H, dd, J = 1.5 Hz, 8.4 Hz), 8.57 (1H, s). 13C NMR
(75 MHz, CDCl3): 14.67, 61.29, 110.14, 122.56, 124.70,
125.41, 126.09, 128.68, 129.22, 129.83, 130.54, 139.05,
143.10, 156.19, and 167.44. Electrospray ionization-mass
spectroscopy (ESI-MS): m/z 375.2 [M+H]+. Anal. calcd for
C22H22N4O2: C, 70.57%; H, 5.92%; N, 14.96%. Found: C,
70.61%; H, 5.97%; N, 14.93%.
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-chlorophenyl)-
1H-benzo[d]imidazole-5-carboxylate (5e) Yield: 75%;
1
1H NMR (300 MHz, CDCl3): δ H NMR: 1.44 (3H, t, J =
6.9 Hz), 2.20–2.40 (2H, m), 4.09 (2H, t, J = 6.6 Hz), 4.33
(2H, t, J = 6.6 Hz), 4.43 (2H, q, J = 6.9 Hz), 6.98 (1H, s),
7.07 (1H, s), 7.50–7.70 (6H, m), 8.08 (1H, dd, J = 1.5 Hz,
8.7 Hz), 8.40 (1H, s). 13C NMR: 14.43, 28.99, 41.00, 43.03,
61.05, 109.96, 123.41, 124.66, 125.92, 126.13, 127.45,
131.54, 137.44, 137.97, 142.72, 151.37, 154.59, and
167.86. ESI-MS: m/z 410.1 [M+H]+. Anal. calcd for
C22H21N4O2Cl: C, 64.62%; H, 5.18%; N, 13.70%. Found:
C, 64.67%; H, 5.22%; N, 13.80%.
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-hydroxyphenyl)-
1H-benzo[d]imidazole-5-carboxylate (5b) Yield: 76%; 1H
1
NMR (300 MHz, CDCl3): δ H NMR: 1.45 (3H, t, J = 7.0
Hz), 2.20–2.30 (2H, m), 3.88 (2H, t, J = 6.5 Hz), 4.30 (2H,
t, J = 7.5 Hz), 4.43 (2H, q, J = 7.0 Hz), 6.79 (1H, s), 7.07
(1H, s), 7.28 (1H, d, J = 8.5 Hz), 7.39 (1H, s), 7.77 (2H, d,
J = 8 Hz), 7.80 (2H, d, J = 8 Hz), 8.09 (1H, dd, J = 1.5 Hz,
8.5 Hz), 8.56 (1H, s). 13C NMR: 14.62, 29.05, 42.01, 43.83,
62.02, 107.83, 109.87, 110.24, 111.06, 123.07, 126.59,
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-bromophenyl)-
1
1H-benzo[d]imidazole-5-carboxylate (5f) Yield: 85%; H
1
NMR (300 MHz, CDCl3): δ H NMR: 1.45 (3H, t, J = 7.2
Hz), 2.20–2.40 (2H, m), 3.92 (2H, t, J = 6.6 Hz), 4.27 (2H,
t, J = 6.6 Hz), 4.43 (2H, q, J = 7.2 Hz), 6.79 (1H, s), 7.12
(1H, s), 7.28 (1H, d, J = 8.4 Hz), 7.38 (1H, s), 7.52 (2H, d,

Med Chem Res
J = 8.4 Hz), 7.68 (2H, d, J = 8.4 Hz), 8.09 (1H, dd, J = 1.5,
8.4 Hz), 8.56 (1H, s). 13C NMR: 14.55, 29.01, 41.24, 43.94,
61.03, 108.10, 122.65, 124.78, 125.40, 126.19, 128.77,
129.21, 129.82, 130.22, 130.56, 137.05, 139.11, 143.13,
150.12, 156.25, and 167.18. ESI-MS: m/z 453.2 [M+H]+.
Anal. calcd for C22H21N4O2Br: C, 58.29%; H, 4.67%; N,
12.36%. Found: C, 58.22%; H, 4.77%; N, 12.40%.
7.35–7.60 (5H, m), 7.47 (2H, d, J = 9 Hz), 7.99 (1H, dd,
J = 1.5, 9 Hz), 8.56 (1H, s). 13C NMR (125 MHz, CDCl3):
14.26, 61.05, 118.95, 120.87, 122.54, 124.79, 126.95,
127.26, 128.08, 129.17, 130.34, 132.96, 136.15, 140.28,
143.03, 158.69, and 167.00. ESI-MS: m/z 359.1 [M+H]+.
Anal. calcd for C22H18N2O3: C, 73.73%; H, 5.06%;
N, 7.82%. Found: C, 73.56%; H, 5.16%; N, 7.88%.
Ethyl
1-(3-(1H-imidazol-1-yl)propyl)-2-(4-(trifluoro-
Ethyl
2-(4-methoxyphenyl)-1-phenyl-1H-benzo[d]imida
methoxy)phenyl)-1H-benzo[d]imidazole-5-carboxylate
(5g) Yield: 92%; 1H NMR (500 MHz, CDCl3): δ 1H
NMR: 1.43 (3H, t, J = 7.0 Hz), 2.10–2.30 (2H, m), 3.90
(2H, t, J = 6.5 Hz), 4.27 (2H, t, J = 7.5 Hz), 4.43 (2H, q,
J = 7.0 Hz), 6.79 (1H, s), 7.07 (1H, s), 7.28 (1H, d, J = 8.5
Hz), 7.39 (1H, s), 7.77 (2H, d, J = 8 Hz), 7.80 (2H, d, J = 8
Hz), 8.09 (1H, dd, J = 1.5, 8.5 Hz), 8.56 (1H, s). 13C NMR:
14.40, 29.15, 40.92, 43.73, 61.74, 110.15, 121.18, 122.80,
124.99, 127.88, 129.26, 130.76, 131.59, 137.85, 139.06,
143.04, 150.78, 151.44, 154.83, and 166.98. ESI-MS:
m/z 459.2 [M+H]+. Anal. calcd for C23H21N4O3F3:
C, 60.26%; H, 4.62%; N, 12.43%. Found: C, 60.32%;
H, 4.62%; N, 12.34%.
-zole-5-carboxylate (6c) Yield: 80%; 1H NMR (500
MHz, CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 3.83 (3H, s), 4.43
(2H, q, J = 7.1 Hz), 6.85 (2H, d, J = 9 Hz), 7.24 (1H, d, J
= 9 Hz), 7.35 (2H, d, J = 9 Hz), 7.50–7.97 (5H, m), 7.98
(1H, dd, J = 1.5 Hz, 9 Hz), 8.59 (1H, s). 13C NMR (125
MHz, CDCl3): 14.30, 59.05, 61.25, 116.07, 119.88, 121.31,
122.79, 123.79, 124.00, 129.18, 129.28, 130.31, 133.05,
138.77, 139.41, 143.08, 159.32, and 168.19. ESI-MS: m/z
373.1 [M+H]+. Anal. calcd for C23H20N2O3: C, 74.18%;
H, 5.41%; N, 7.52%. Found: C, 74.09%; H, 5.35%;
N, 7.64%.
Ethyl 2-(4-morpholinophenyl)-1-phenyl-1H-benzo[d]imida-
zole-5-carboxylate (6d) Yield: 84%; H NMR (500 MHz,
1
Ethyl 1-(3-(1H-imidazol-1-yl)propyl)-2-(4-(trifluoromethyl)
phenyl)-1H-benzo[d]imidazole-5-carboxylate (5h) Yield:
90%; 1H NMR (500 MHz, CDCl3): δ 1H NMR: 1.43 (3H, t,
J = 7.0 Hz), 2.20–2.30 (2H, m), 3.92 (2H, t, J = 6.5 Hz),
4.27 (2H, t, J = 7.5 Hz), 4.43 (2H, q, 7.0 Hz), 6.76 (1H, s),
7.09 (1H, s), 7.28 (1H, d, J = 8.5 Hz), 7.39 (1H, s), 7.77
(2H, d, J = 8 Hz), 7.80 (2H, d, J = 8 Hz), 8.09 (1H, dd, J =
1.5, 8.5 Hz), 8.56 (1H, s). 13C NMR: 14.42, 30.89, 41.93,
43.81, 61.12, 109.30, 118.29, 122.78, 125.24, 125.85,
126.10, 126.21, 129.54, 130.43, 132.49, 133.32, 137.01,
138.37, 142.67, 153.33, and 166.79. ESI-MS: m/z 444.2
[M+H]+. Anal. calcd for C23H21N4O2F3: C, 62.44%;
H, 4.78%; N, 12.88%. Found: C, 62.44%; H, 4.78%;
N, 12.89%.
CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 3.22 (4H, t, J = 5.0 Hz),
3.85 (4H, t, J = 5.0 Hz), 4.44 (2H, q, J = 7.1 Hz), 6.80 (2H,
d, J = 9 Hz), 7.20 (1H, d, J = 9 Hz), 7.37 (2H, d, J = 9 Hz),
7.50–7.60 (5H, m), 7.99 (1H, dd, J = 1.5 Hz, 9 Hz), 8.60
(1H, s). 13C NMR (125 MHz, CDCl3): 14.40, 47.97, 52.13,
60.90, 66.67, 109.84, 114.19, 121.43, 124.16, 124.58,
125.49, 127.46, 128.95, 130.07, 130.64, 136.80, 140.23,
143.92, 151.95, and 167.07. ESI-MS: m/z 428.2 [M+H]+.
Anal. calcd for C26H25N3O3: C, 73.05%; H, 5.89%;
N, 9.83%. Found: C, 73.06%; H, 5.86%; N, 9.80%.
Ethyl 2-(4-chlorophenyl)-1-phenyl-1H-benzo[d]imidazole-
5-carboxylate (6e) Yield: 81%; 1H NMR (500 MHz,
CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 4.42 (2H, q, J = 7.1 Hz),
7.25 (1H, d, J = 9 Hz), 7.28 (2H, d, J = 9 Hz), 7.40–7.60
(5H, m), 7.54 (2H, d, J = 9 Hz), 8.00 (1H, dd, J = 1.5, 9
Hz), 8.52 (1H, s). 13C NMR (125 MHz, CDCl3): 14.40,
61.64, 110.28, 111.80, 116.26, 122.70, 122.97, 124.65,
125.67, 127.91, 129.20, 130.35, 131.18, 137.50, 140.76,
142.71, 152.68, and 167.48. ESI-MS: m/z 377.1 [M+H]+.
Anal. calcd for C22H17N2O2Cl: C, 70.12%; H, 4.55%;
N, 7.43%. Found: C, 70.10%; H, 4.52%; N, 7.54%.
Ethyl
1,2-diphenyl-1H-benzo[d]imidazole-5-carboxylate
(6a) Yield: 77%; 1H NMR (300 MHz, CDCl3): δ 1.43
(3H, t, J = 7.2 Hz), 4.42 (2H, q, J = 7.2 Hz), 7.20–7.60
(11H, m), 7.99 (1H, dd, J = 1.5, 8.4 Hz), 8.60 (1H, s). 13C
NMR (75 MHz, CDCl3): 14.51, 61.42, 110.34, 111.80,
111.99, 112.56, 113.14, 113.91 115.67, 118.82, 119.05,
130.24, 131.00, 136.56, 140.28, 142.44, 152.16, and
167.88. ESI-MS: m/z 343.1 [M+H]+. Anal. calcd for
C22H18N2O2: C, 77.17%; H, 5.30%; N, 8.18%. Found: C,
77.06%; H, 5.35%; N, 8.32%.
Ethyl 2-(4-bromophenyl)-1-phenyl-1H-benzo[d]imidazole-
5-carboxylate (6f) Yield: 79%; 1H NMR (500 MHz,
CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 4.44 (2H, q, J = 7.1 Hz),
7.24 (1H, d, J = 9 Hz), 7.32 (2H, d, J = 9 Hz), 7.40–7.60
(5H, m), 7.58 (2H, d, J = 9 Hz), 8.00 (1H, dd, J = 1.5, 9
Hz), 8.60 (1H, s). 13C NMR (125 MHz, CDCl3): 14.39,
61.65, 110.30, 111.82, 116.37, 122.75, 123.89, 124.65,
Ethyl
2-(4-hydroxyphenyl)-1-phenyl-1H-benzo[d]imida-
zole-5-carboxylate (6b) Yield: 77%; 1H NMR (500
MHz, CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 4.42 (2H, q,
J = 7.1 Hz), 7.19 (2H, d, J = 9 Hz), 7.28 (1H, d, J = 9 Hz),

Med Chem Res
125.67, 127.91, 129.20, 130.32, 131.08, 137.50, 140.76,
142.71, 152.69, and 167.50. ESI-MS: m/z 421.1 [M+H]+.
Anal. calcd for C22H17N2O2Br: C, 62.72%; H, 4.07%;
N, 6.65%. Found: C, 62.76%; H, 4.02%; N, 6.73%.
Multidrug-resistant M. tuberculosis MIC testing
The broth micro dilution assay format, following guidelines
established by the Clinical and Laboratory Standards
Institute, is routinely utilized for MIC testing. Briefly,
testing was conducted using 96-well, U-bottom micro plates
with an assay volume of 0.2 mL/well. First, the test media
was added to each well. Test compounds, dissolved in
appropriate solvent and diluted in test media, were then
added to appropriate wells at twice the intended starting
concentration and serially diluted two-fold across the plate.
The assay plates were subsequently incubated at 37 °C for
7 days. Following incubation, the plates were read visually
and individual wells scored for inhibition. In addition, an
absorbance reading was taken via a plate reader and the
results recorded. Testing was conducted in duplicate. The
MIC was reported as the lowest concentration of drug that
inhibits growth of the organism. The strains that were used
in this study were isoniazid-resistant (SRI 1367), rifampin-
resistant (SRI 1369) and ofloxacin-resistant (SRI 4000)
MTB.
Ethyl 1-phenyl-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]
imidazole-5-carboxylate (6g) Yield: 81%; 1H NMR
(500 MHz, CDCl3): δ 1.43 (3H, t, J = 7.1 Hz), 4.43 (2H, q,
J = 7.1 Hz), 7.20 (1H, d, J = 9 Hz), 7.28 (2H, d, J = 9 Hz),
7.40–7.60 (5H, m), 7.55 (2H, d, J = 9 Hz), 7.99 (1H, dd,
J = 1.5, 9 Hz), 8.54 (1H, s). 13C NMR (125 MHz, CDCl3):
14.34, 61.56, 111.03, 111.79, 116.30, 121.80, 123.06,
123.99, 124.66, 126.86, 127.25, 129.24, 131.51, 131.82,
136.50, 140.79, 142.70, 154.05, and 167.50. ESI-MS: m/z
427.1 [M+H]+. Anal. calcd for C23H17N2O3F3: C, 64.79%;
H, 4.02%; N, 6.57%. Found: C, 64.69%; H, 4.11%;
N, 6.54%.
Ethyl 2-(4-(trifluoromethyl)phenyl)-1-phenyl-1H-benzo[d]
imidazole-5-carboxylate (6h) Yield: 89%; 1H NMR
(500 MHz, CDCl3): δ 1.44 (3H, t, J = 7.1 Hz), 4.44 (2H, q,
J = 7.1 Hz), 7.27 (1H, d, J = 9 Hz), 7.33–7.60 (5H, m),
7.58 (2H, d, J = 9 Hz), 7.70 (2H, d, J = 9 Hz), 8.03 (1H, dd,
J = 1.5, 9 Hz), 8.63 (1H, s). 13C NMR (125 MHz, CDCl3):
14.38, 61.03, 110.32, 122.45, 122.67, 124.84, 125.33,
125.36, 125.41, 125.97, 127.29, 130.30, 132.89,
136.15, 140.28, 142.43, 152.26, and 166.89. ESI-MS:
m/z 411.1 [M+H]+. Anal. calcd for C24H23N3O2:
C, 67.31%; H, 4.18%; N, 6.83%. Found: C, 67.30%;
H, 4.12%; N, 6.88%.
Cytotoxicity assay
Vero cells were treated with 50 µM of interested compounds
and allowed to adhere for 72 h. Then, the proliferative
activity was determined by MTT assay (CellTiter 96 Non-
radioactive cell proliferation assay; Promega, Madison, WI)
to monitor the number of viable cells according to the
manufacturer’s instructions. Briefly, the dye solution was
added at 15 μL/well. After 1 h of incubation at 37 °C in a
humidified 5% CO2 atmosphere, 100 µL of the stop solution
was added into each well. Absorbance was recorded at 570
nm. All experiments were done in triplicate, and the pro-
liferation rate was calculated as the ratio of absorbance
under each experimental condition to that of the control.
Biological evaluation
Antimycobacterial activity against MTB-H37RV strains
All the synthesized compounds 5a–5h and 6a–6h were
tested for their antimycobacterial activity in vitro against
MTB-H37RV using a modified high throughput screen assay
adapted from Collins and Franzblau (1997). The end-point
detection was assessed by the Promega reagent BacTiter-
GloTM microbial cell viability (BTG) assay and was com-
pared with microdilution AlamarBlue broth assay. Com-
pounds screened in dose response were tested in ten two-
fold dilutions from 100 to 0.195 µM. Three standard drugs
(INH, cycloserine, and pyrimethamine) were used as
references for the assay. Data were analyzed using the IDBS
Activity Base software and the dose response result was
analyzed using a four parameter logistic fit to the data
(Excel Fit equation 205) with the maximum and minimum
locked at 100 and 0.
Results and discussion
Synthesis and characterization of compounds
The synthetic scheme is a four-step pathway (Fig. 1) leading
to the formation of a variety of benzimidazole derivatives as
described previously (Yoon et al. 2015). Esterification
of 4-fluoro-3-nitro benzoic acid in ethanol, with con-
centrated sulfuric acid as catalyst furnished the ethyl ester 1
(75%). The ethylbenzoate 1 was then treated with amine
[for R1 = 5: N-(3-aminopropyl)imidazole; for R1 = 6: ani-
line] and DIPEA in dry dichloromethane at room tem-
perature to afford amino compound 2. It was subsequently
reduced to intermediate 3 (60%) using ammonium formate
and 10% Pd/C. Intermediate 3 was then refluxed with the

Med Chem Res
Fig. 1 Protocol for synthesis of
final compounds 5a–h and 6a-h
Biological activity
The synthesized compounds 5a–h and 6a–h were tested for
their antimycobacterial activity against MTB-H37Rv in a
screening assay adapted using the Promega reagent BTG.
The BTG assay is a quantitative ATP assay for bacteria
using luciferase production as an end-point detection point.
Data was analyzed using the IDBS Activity Base software
and the dose response result was analyzed using a four
parameter logistic fit to the data (Excel Fit equation 205)
with the maximum and minimum locked at 100 and 0. From
these curves, IC90 and IC50 values were calculated.
As references, three standard drugs, namely cycloserine,
isoniazid, and pyrimethamine were also evaluated in the
assays.
Of the 16 benzimidazole analogs that have been syn-
thesized, five compounds showed MIC of ≤100 µM against
MTB-H37Rv strains (Table 1). Consistent with our previous
findings, the most potent compounds from both series
contained a trifluoromethyl group (5h and 6h), suggesting
the potential of this substituent in giving good anti-
mycobacterial effect against MTB-H37Rv. Although the
reason behind this has not been fully elucidated, the ability
of both 5h and 6h to permeate into the bacterial cell wall
could be one possible reason for their better inhibitory
Fig. 2 ORTEP diagram of compound 6h
bisulfite adduct of various aromatic aldehydes (4a–h) in
DMF overnight to afford benzimidazole derivatives 5a–h
and 6a–h in moderate to good yields (52–93%). Column
chromatography purification for the final compounds were
done in solvent system chloroform–methanol (9:1) using
Silica Gel 60 (0.063–0.200 mm). The structures of the
newly synthesized compounds were then characterized
using NMR, CHN, mass spectrometry, and X-ray analysis
where possible (Fig. 2) (Yoon et al. 2012).
activity. With
a
steric parameter (Es) of −2.4,
trifluoromethyl group (–CF3) is relatively highly lipophilic
(Elliott 1995; Welch and Eswarakrishnan 1991) and
permeates cell membrane easily, which may give the
trifluoromethylated compounds superior activity compared
to the rest of the tested compound. Thus, combining the

Med Chem Res
Table 1 Antimycobacterial activity of synthesized compounds against MTB-H37Rv strains
Compound
BTG
Alamar blue
Cytotoxicity
(µg/mL)
IC50
IC90
MIC
IC50
IC90
MIC
(µM)
(µM)
(µM)
(µM)
(µM)
(µM)
5a
>100
>100
90.11
>100
44.58
>100
34.77
11.53
53.87
>100
>100
36.05
>100
>100
56.71
8.96
>100
>100
99.72
>100
>100
>100
70.84
13.20
70.79
>100
>100
45.27
>100
>100
>100
12.46
26.38
0.20
NA
NA
NA
NA
NA
NA
100
50
>100
>100
94.43
>100
>100
>100
48.80
11.86
58.43
>100
>100
37.57
>100
>100
>100
11.27
24.76
0.19
>100
>100
>100
>100
>100
>100
84.98
13.74
77.61
>100
>100
47.00
>100
>100
>100
12.65
28.01
>5
NA
NA
NA
NA
NA
NA
100
50
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
5b
5c
5d
5e
5f
5g
5h
6a
100
NA
NA
50
100
NA
NA
50
6b
6c
6d
6e
NA
NA
NA
12.50
100
0.31
100
NA
NA
NA
50
6f
6g
6h
Cycloserin
Isoniazid
Pyrimethamine
23.55
0.13
100
NA
100
24.27
46.37
25.09
28.00
NA: Data not available due to poor or no activity
Fig. 3 Dose response curves of
compound 6h against
Mycobacterium tuberculosis
H37RV strains (MTB-H37RV)
using a BacTiter-Glo (BTG) and
b Alamar Blue method. Red
lines indicated the half maximal
inhibitory concentration (IC50);
outliers (in blue boxes) were
eliminated from curve fitting
(color figure online)
benzimidazole backbone with the –CF3 group may have
synergistic advantages.
effective against drug-resistant MTB as well. As the ben-
zimidazole scaffold is still considered novel in TB drug
discovery, the most potent compound found in the screen-
ing assay (6h) was subsequently tested in several drug-
resistant MTB strains. Interestingly, 6h was demonstrated to
possess antimycobacterial activities against several drug-
resistant MTBs such as isoniazid-resistance MTB (INH-R
MTB and strain SRI 1367), rifampin-resistance MTB
(RMP-R MTB and strain SRI 1369) and ofloxacin-
resistance MTB (OFX-R MTB and strain SRI 4000) with
MIC of 15.24, 30.50, and 60.00 µM, respectively (Table 2).
INH and RMP are two of the most important anti-
tuberculosis drugs in the market today and among drug-
The most potent analog, ethyl 2-(4-(trifluoromethyl)
phenyl)-1H-benzo[d]imidazole-5-carboxylate (6h) showed
IC50 at 8.96 µM (Fig. 3a), IC90 at 12.46 µM, and MIC at
12.50 µM using BTG method. By comparison, it gave IC50
of 11.27 µM (Fig. 3b), IC90 of 12.65 µM and MIC of 50.00
µM using the Alamar Blue method. It was found to be
more active than the standard drugs cycloserine and pyr-
imethamine, but was less potent compared to the first-line
drug isoniazid.
However, as many TB cases now are multidrug-resistant,
it is important to have new drugs in the pipeline that can be

Med Chem Res
Table 2 MIC values of the most potent analog 6h against resistant
strains of MTB
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Conclusion
In conclusion, we have described the synthesis and MTB-
H37Rv inhibitory activities of 16 benzimidazole derivatives.
In line with our previous work, the present work clearly
demonstrated that the trifluoromethyl group is essential for
potent MTB-H37Rv inhibitory activity. Although currently
none of the benzimiadzole-based compounds are as potent
as INH, compound such as 6h warrant further investiga-
tions as the benzimidazoles still have much potential to be
modified to be useful as second-line drug to counter some of
the MDR-TB cases. Moreover, they were found to be non-
cytotoxic toward mammalian cells. Further work on
trifluoromethyl-substituted benzimidazoles are ongoing in
our laboratory.
Acknowledgements The authors wish to express their gratitude and
appreciation to Pharmacogenetics and Novel Therapeutics Research
Cluster, Institute for Research in Molecular Medicine, Universiti Sains
Malaysia for supporting this work. This work was funded through
Research Grant No.RUC (1001/PSK/8620012). This work was also
supported by the National Institutes of Health and the National Insti-
tute of Allergy and Infectious Diseases (contract no.
HHSN272201100012I).
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interests.

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