A novel synthesis of 2-arylbenzimidazoles in molecular sieves-MeOH system and their antitubercular activity

Article abstract of DOI:10.1016/j.bmc.2018.07.049

Arylbenzimidazoles have been synthesized as antimycobacterial agents. An efficient synthesis has been developed for 2-arylbenzimidazoles from o-phenylenediamines and aromatic aldehydes in molecular sieves-methanol system. The methodology is straightforward to get 2-arylbenzimidazoles (3a–3z) in excellent yields with high chemoselectivity over 2-aryl-1-benzylbenzimidazoles (4a–4z). All these benzimidazole analogues were evaluated against M. tuberculosis in BACTEC radiometric assay. The compounds 4y and 4z exhibited potential antitubercular activity against M. tuberculosis H₃₇R_V, MIC at 16 μM and 24 μM respectively. The best compound of the series i.e. compound 4y was well tolerated by Swiss-albino mice in acute oral toxicity. Compound 4y possessing a diarylbenzimidazole core, can further be optimized for better activity.

Full text of DOI:10.1016/j.bmc.2018.07.049

Accepted Manuscript
A novel synthesis of 2-arylbenzimidazoles in molecular sieves-MeOH system
and their antitubercular activity
Amit K. Chaturvedi, Amit Kumar Verma, J.P. Thakur, Sudeep Roy, Shashi
Bhushan Tripathi, Balagani Sathish Kumar, Sadiya Khwaja, Naresh K. Sachan,
Ashok Sharma, Debabrata Chanda, Karuna Shanker, Dharmendra Saikia,
Arvind S. Negi
PII:
S0968-0896(18)31189-1
https://doi.org/10.1016/j.bmc.2018.07.049
BMC 14486
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 June 2018
27 July 2018
28 July 2018
Please cite this article as: Chaturvedi, A.K., Verma, A.K., Thakur, J.P., Roy, S., Bhushan Tripathi, S., Kumar, B.S.,
Khwaja, S., Sachan, N.K., Sharma, A., Chanda, D., Shanker, K., Saikia, D., Negi, A.S., A novel synthesis of 2-
arylbenzimidazoles in molecular sieves-MeOH system and their antitubercular activity, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.049
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A novel synthesis of 2-arylbenzimidazoles in molecular sieves-MeOH system and their
antitubercular activity
Amit K. Chaturvedi#a, Amit Kumar Verma#a, J.P. Thakura, Sudeep Royb, Shashi Bhushan Tripathia, Balagani
Sathish Kumara, Sadiya Khwajaa, Naresh K. Sachana, Ashok Sharmaa, Debabrata Chandaa, Karuna Shankera
Dharmendra Saikia*a, Arvind S. Negi*a
aCSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Kukrail Picnic Spot Road, Lucknow-
226 015, U.P., India.
bDepartment of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology
Technická 12, 61200 Brno, Czech Republic.
#Equal authorship
Abstract: Arylbenzimidazoles have been synthesized as antimycobacterial agents. An efficient synthesis has been
developed for 2-arylbenzimidazoles from o-phenylenediamines and aromatic aldehydes in molecular sieves-
methanol system. The methodology is straightforward to get 2-arylbenzimidazoles (3a-3z) in excellent yields with
high chemoselectivity over 2-aryl-1-benzylbenzimidazoles (4a-4z). All these benzimidazole analogues were
evaluated against M. tuberculosis in BACTEC radiometric assay compounds 4y and 4z exhibited potential
antitubercular activity against M. tuberculosis H37RV, MIC at 16 µM and 24 µM respectively. The best compound
of the series i.e. compound 4y was well tolerated by Swiss-albino mice in acute oral toxicity. Compound 4y
possessing a diarylbenzimidazole core, can further be optimized for better activity.
Keywords: arylbenzimidazoles; chemoselectivity; molecular sieves; antitubercular; DNA gyrase; acute oral toxicity
1. Introduction
Tuberculosis (TB) is a contagious air-borne bacterial disease caused by the infection from
Mycobacterium tuberculosis (Mtb) affecting mainly lungs. It preferably affects men over
women and mostly adults in their productive years. Globally, in year 2016, 10.4 million people
fell ill with TB and 1.7 million people died of it. Almost 64% of global cases are in India, China
and Russia.1 India has the largest infected population of TB patients in the world with an
estimated number of 2.8 million. Every year, 0.39 million new patients are added with 0.13
million as MDR and a mortality of 0.48 million.2 Over the period of time, the pathogen has
become resistant to two most powerful first line anti-TB drugs i.e. rifampicin and isoniazid and
also to second line anti-TB drug group fluoroquinolones. To tackle this situation, ‘directly
observed therapy-short course’ (DOTS) and DOTS-plus programs have been started. The
emergence of ‘total drug resistance’ (TDR) has exaggerated the situation. Due to present
scenario of drug resistance, there may be a critical situation in future. Hence, there is a need to
explore for new anti-TB molecules.
Benzimidazole is a biodynamic pharmacophore present in several natural products and drug
candidates, exhibiting various biological activities3 such as antibacterial,4 antimalarial,5
anticancer6 and anti-HIV7 etc. This moiety has also been good ligands for some of the crucial
receptors like DNA topoisomerase I,8 tubulin polymerase9 and estrogen receptors10 etc. There
are many drugs in clinics possessing benzimidazole basic core like albendazole and
mebendazole as antiparasitics, omeprazole as antiulcer, telmisartan and candesartan as
antihypertensives, bilastine as antihistamine, bendamustine as anticancer and droperidol as
antipsychotics (Figure 1).11
..........................................................................................
*Corresponding authors, E-mail::arvindcimap@rediffmail.com (AS Negi); d.saikia@cimap.res.in (Dharmendra Saikia);
CIMAP Communication No. CIMAP/PUB/2017/46
1

Being an important pharmacophore several synthetic strategies have been developed
previously.12,13 Among them the most common method for the synthesis of substituted 2-
arylbenzimidazoles involves through the direct condensation of substituted o-
phenylenediamines with a variety of substituted aromatic aldehydes in presence of a catalyst.
Various kinds of acidic reagents and catalysts have been used to promote this reaction,12a-s such
as I2-AcOH, p-TSA, BF3-Et2O, FeCl3, Zn2+-K10 (Clayzic), TiCl4, PhI(OAc)2, Ru and Ir
complexes, ZrO2, (NH4)2Ce(NO3)6-H2O2, Amberlite IR-120, Dowex, ZrOCl2 on
Montmorillonite-H2O, PbO2, CuI-proline, MgCl2.6H2O, VOSO4 and Ionic liquids etc. A good
number of nanoparticles has also been used in this synthesis like Cu nano-particles on
13e
13f
charcoal,13a Cu on SiO2,13b In2O3,13c Ce(NO3)3,13d MnO2,
and TiO2 etc. However, these
methods are associated with several drawbacks such as low reaction yields, inconvenient
procedures, use of expensive and excess of reagents, longer reaction times, tedious work-up
procedures, co-occurrence of several side reactions and impractical to scale-up for industrial
application. Therefore, there is continued interest in search for a better reagent for the synthesis
of substituted 2-arylbenzimidazoles in terms of operational simplicity, economic viability, with
greater selectivity and in particular industrial applicability.
In the present communication, we have synthesized 2-arylbenzimidazoles through an
efficient and chemoselective new methodology (Scheme 1). In a few cases 2-aryl-1-
benzylbenzimidazoles were also synthesized. All these benzimidazoles (3a-z and 4d, 4e, 4g,
4m, 4w-4z) were evaluated against Mycobacterium tuberculosis H37RV by BACTEC-460
radiometric assay. Two of the 2-aryl-1-benzylbenzimidazoles, 4y and 4z exhibited potent
antitubercular activity. We explored their targets through molecular docking studies and further
validated by gyrase supercoiling inhibition assay. The best analogue was also evaluated for in-
vivo toxicity in Swiss-albino mice.
O
H
N
O
H
N
O
H3CO
O
S
O
H
N
O
N
NH
NH
N
N
S
N
OCH3
Albendazole
Omeprazole
Mebendazole
OEt
N
N
COOH
CH3
N
N
OH
N
O
N
N
Bilastine
Telmisartan
N
N
HN
N
O
NH
CH3
N
N
O
COOH
O
N
N
HO
N
N
Cl
Cl
OEt
N
F
Candesartan
Bendamustine
Droperidol
Fig. 1. Various marketed drugs based on benzimidazole core
Scheme 1. Syntheses of 2-arylbenzimidazoles in molecular sieves-methanol system
R2
O
Molecular sieves 3Ao
MeOH, reflux
NH2
NH2
H
N
R2
H
R2
N
+
R2
+
R1
N
R1
N
R1
2
1.5h
3a-3y
4a-4y
1: R1=H/OCH3/CH3/Cl/NO2
2
30-89%
<8%

2. Results and discussion
2.1. Chemical synthesis
o-Phenylenediamine (1e) and 3,4,5-trimethoxybenzaldehyde (2e) on refluxing in methanol
for 1.5h in presence of molecular sieves (3Å) gave 2-arylbenzimidazole (3e) in 76% yield and
1,2-diarylbenzimidazole (4e) in 8%. However, when reaction time (1.5-8h) and molar ratio of
benzaldehydes (1: 2) were enhanced the yield of 1,2-diarylbenzimidazoles was up to 34%
(Table 1).
Table 1. Optimization of reaction time
OCH3
H3CO
O
OCH3
OCH3
H3CO
H
N
Molecular sieves (3/4/5Ao)
H3CO
H3CO
NH2
NH2
H
OCH3
OCH3
OCH3
+
+
N
Various solvents/reflux
1.5hs
N
OCH3
OCH3
N
3e
4e
S. Molecular
Substrates
Reaction Yield* Yield*
no.
sieves
(3Ao)
1g
molar ratios
time (h)
(%)
3e
(%)
4e
0
(diamine:aldehydes)
1.
2.
3.
4.
1.2:1
1.2:1
1.2:1
1.2:1
1.0
1.5
4
57
1g
1g
76
58
3
18
34
1g
8
62
* isolated yields
There was only 16% yield of 2-arylbenzimidazole (3e) in absence of molecular sieves.
Molecular sieves of 3Å pore size were much suitable to effectively trap water molecules
generated during condensation of 1 and 2 as compared to other two i.e. 4Å and 5Å pore size
molecular sieves (Table 2).
Table 2. Effect of pore size of molecular sieves on the synthesis of benzimidazoles.
OCH3
H3CO
O
OCH3
OCH3 +
H3CO
H
N
Molecular sieves (3/4/5Ao)
H3CO
H3CO
NH2
NH2
H
OCH3
OCH3
OCH3
+
N
Various solvents/reflux
1.5hs
N
OCH3
OCH3
N
3e
4e
S.
no.
Solvent Molecular Temp.
Reaction
time (h)
Yield
(%)
3e
Yield
(%)
4e
sieves
oC
1.
2.
3.
4.
MeOH ---
65
65
65
65
1.5
1.5
1.5
1.5
16
76
62
59
Traces
Traces
Traces
Traces
MeOH 3Å, 1g
MeOH 4Å, 1g
MeOH 5Å, 1g
*traces means <5%
Among the various organic solvents used in the reaction dichloromethane, chloroform and
toluene were least preferred (low yields) while, acetonitrile, tetrahydrofuran, dioxane and
methanol afforded better yields. But, methanol and THF were the most preferred due to high
chemoselectivity (Table 3).
3

Table 3. Effect of solvents on the synthesis of benzimidazoles.
S.
no.
Solvent
Molecular
sieves
Temp.
oC
Yield*
(%)
3e
Yield
(%)
4e
1.
2.
3.
4.
5.
6.
7.
8.
Dichloromethane
Chloroform
Acetonitrile
Toluene
Tetrahydrofuran
Methanol
1g
1g
1g
1g
1g
1g
1g
1g
40
62
82
110
66
65
78
98
46
49
57
47
62
76
52
64
06
traces
16
traces
traces
03
Ethanol
Dioxane
12
traces
The scope of the reaction was explored through diverse aldehydes and also
phenylenediamines (Table 4, 3a-y, entry 1-26). Thus, various kinds of substituted o-
phenylenediamines (1) were refluxed with a variety of substituted aromatic aldehydes (2) in
molecular sieve-MeOH system to get corresponding substituted 2-arylbenzimidazoles (3) in
moderate to good yields (30-89%). A minor amount (≤8%) of substituted 1-arylmethyl-2-
arylbenzimidazole (4) was also observed during the course of this synthetic protocol. Reaction
goes well with both electron-releasing and electron-withdrawing substitutions (Table 4, entry
6, 11 & 21) at aromatic aldehyde. Reaction goes well not only with aromatic aldehydes but also
with aliphatic aldehyde (Table 4, entry 20) and cinnamaldehyde (Table 4, entry 19) in good
yields. An electron withdrawing group at phenylenediamine ring reduced yield of the product
drastically (3r, 30%, Table 4, entry 18). In all these cases the yield of 1,2-diarylbenzimidazoles
was always less than 8%. The products were purified through column chromatography over
silica gel (60-120 mesh) using chloroform-acetone as eluant. The 2-arylbenzimidazoles were
confirmed through the various spectroscopic means (Supplementary information). Purity of
compounds 3x, 4y and 4z was found to be 94-97% by Reversed Phase HPLC. The yields of the
products i.e. 2-arylbenzimidazole (3e) and 1,2-diarylbenzimidazole (4e) were determined on
RP-HPLC (Shimadzu) using C-18 (Phenomenex, 4.6×26, 5 µm) column and mobile phase:-
Buffer (5.0mM CH3COONH4 + 1.0% AcOH) : CH3CN = 30:70, v/v at 1.0 mL/min, λmax at 254
nm in Photodiode array (PDA). Column purified products 3e and 4e were used as standard
compounds. Retension time (tR) of 3e and 4e were 3.44 min and 4.54 min respectively. The
yields of the products were found to be 76% (3e) and 3% (4e) shown in Table 4 (entry 6).
[Table 4, presently placed at last]
2.2. Biological evaluation
The antimycobacterial assay was done radiometrically as per previously reported method.
Rifampicin, streptomycin and isoniazid (Sigma Chemicals, USA), standard drugs for
tuberculosis were used as positive controls. As shown in Table 5, most of the compounds
showed activity against M. tuberculosis with MIC in 16-400 µM range. Two of the analogues
i.e. compound 4y and 4z exhibited potential antitubercular activity with MICs at 16 µM and 24
µM respectively. Compound 4m showed moderate activity (MIC, 67 µM). The standard drugs
rifampicin, streptomycin, and isoniazid had MICs 3 µM, 4 µM, and 1 µM respectively. The log
4

P value of most of the benzimidazole analogues was between 2-5, which is within the level of
druggability (3-5). However, compound 4y and 4z exhibited comparatively higher lipophilicity
than the others. Higher lipophilicity of the molecule helps in interacting with Mycobacterium
cell wall in the penetration of cell wall. Generally, a moderate to high level of lipophilicity is
preferred in the compounds to exhibit better antitubercular activity. Over 60% of its cell wall is
lipid due to which it poses difficulty to permeability to drug molecules, which ultimately
retards the transport of polar compounds through outer lipid layer of Mycobacterium and thus,
the pathogen is resistant to many antibiotics, acidic and alkaline compounds, resistant to
osmotic lysis via complement deposition, resistant to lethal oxidations and survival inside of
macrophages.
There were two types of products i.e. 2-arylbenzimidazoles (3a-3z) (Series 1) and 1-benzyl-
2-arylbenzimidazoles (4d, 4e, 4g, 4m, 4w-4z) (Series 2). In general, 1-benzyl-2-
arylbenzimidazoles possessed better antitubercular activity as compared to 2-
arylbenzimidazoles. Only one compound i.e. 3x (MIC=103 µM) belongs to 2-
arylbenzimidazoles (Series 1). In both the series, methoxy substitution at 5-position of
benzimidazole ring enhanced the activity (Activity 3x > 3g and 4z > 4m). In series 2 (i.e. 1-
benzyl-2-arylbenzimidazoles) 3,4-methylenedioxy substitution at 1-benzyl and 2-aryl (4m and
4z) exhibited potent antitubercular activity. This indicates that 3,4-methylenedioxy substitution
at 1-benzyl and 2-aryl along with 5-methoxy substitution in benzimidazole core is the most
desireable for better antitubercular activity.
Table 5. Minimum inhibitory concentration (MIC) of arylbenzimidazoles and standard drugs against M.
tuberculosis H37RV
S. Comp Structure
No d no.
Log
P
2.47
MIC*
µM
424
S.
No
6.
Compd no.
Structure
Log
P
4.45
MIC*
µM
16
H
N
OCH3
1.
3a
4y
OH
H3CO
N
N
N
H3CO
H
O
O
N
2.
2.74
2.9
406
103
7.
8.
4.26
3.72
24
3
3b
4z
N
H3CO
O
N
N
O
H3CO
CH3 CH3
H
N
HO
3.
Rifampicin
3x
O
O
OH
F
O
CH3
CH3
CH3
N
NH
H3CO
H3CO
OH OH
H3
C
N
N
O
OH
N
CH3
O
CH3
O
F
O
NH-NH2
4.
5.
5.51
2.64
312
67
9.
Isoniazid
0.64
8.01
1
4
4g
N
N
F
N
O
O
NH
H2N
HO
10.
Streptomycin
4m
NH
HN
H2N
OH
OH
O
N
N
N
H
O
O
O
CHO
H
HO
O
HOH2C
HO
O
NHMe
HO
F
6.
5.02
285
--
---
----
----
----
4x
N
N
F
H3CO
* MIC>500µM was considered as inactive
5

2.3. Molecular docking studies
Molecular docking results showed that compounds 3x (-7.0581), 4y (-10.2858), 4m (-8.3134),
and 4z (-10.5108) docked well with DNA gyrase. The study confirmed significant binding
affinity of compounds 4y and 4z to DNA gyrase (Table 6). The compounds 4z (-7.9873) and
4y (-8.0771) showed good interaction with the receptor thymidine monophosphate kinase
(TMK).The interaction results with the receptor Filamenting temperature-sensitive mutant Z
(FtsZ) was average with all the compounds. The stability of the compounds is attributed to
their hydrogen bond capability with the polar (including Basic) amino acids. The binding
involves the participation of common chain within the active sites between the compounds 3x,
4y, 4m, and 4z along with the positive controls. This includes ASN46, ASP73, ILE78,
GLY102, LYS103, LEU115, HIS116, GLY117, VAL118, GLY119, VAL120, SER121 and
LYS337 in case of DNA gyrase and GLY18, GLY19, ASN41, GLY105, THR106, GLY107
and ARG140 in case of FtsZ and Arg160 for 4m, 4y, and 4z and ASP9, LYS13, ARG14,
TYR39, PHE70, ARG74, ARG95 and ASN100 for 3x in case of Thymidine Monophosphate
Kinase. The contact atoms of ligand with the receptors have also been studied. The
comparative docking scores of all the compounds against different set of receptors are provided
in Fig. 2. Interaction diagrams of 4m and 4y with DNA gyrase are provided in Figures 3A and
3B.The overall finding suggests that all the synthesized molecules have shown good binding
affinity against DNA Gyrase B target protein. The molecule 4m have shown promising results
in biological evaluation which is well supported by Gyrase supercoiling inhibition assay.
DNA gyrase is an ATP-dependent enzyme that acts by creating a transient double-stranded
DNA break. It is unique in catalyzing the negative supercoiling of DNA and is essential for
efficient DNA replication, transcription, and recombination.DNA gyrase, the only Type II
topoisomerase in M. tuberculosis, is a validated, and pharmaceutically effective target for drug
discovery against this important pathogen.20
Fig.2: Comparative docking scores of 3x, 4m, 4y, and 4z with different receptors for anti-tubercular
property.
6

Table 6. Dock Scores of compounds with DNA gyrase
Molecule
S-Score
rmsd_refine E_Conf
E_Place
E_Score E_Refine E_Score
1
1
2
Ciprofloxacin
Colchicine
Ethambutol
Isoniazid
Pyrazinamide
Rifampicin
Streptomycin
4m
-7.6668
-9.1702
-7.8062
-5.2634
-4.8893
-11.4428
-9.9292
-8.3134
-10.5108
-10.2858
-7.0581
2.7102
1.8841
1.4366
0.9412
1.8731
3.8587
1.7627
1.5921
2.0278
1.5145
2.8008
82.1373
31.0714
68.3195
23.4909
51.2641
131.3949 -29.9244
118.9568 -88.8039
60.9514
46.1585
28.5883
31.9128
-93.3845
-45.3507
-61.3733
-55.0511
-50.3059
-12.8931 -41.1527
-10.0016 -9.5949
-11.1416 -23.9365
-7.6669
-9.1702
-7.0862
-5.2634
-4.8893
-9.3914
-8.1905
-6.9557
-16.2313
-13.9651
-50.7988 -11.4428
-12.4607 -43.0728
-13.6629 -45.0172
-14.8197 -45.1741 -10.5108
-11.5209 -40.3549 -10.2858
-9.9292
-8.3134
-69.2996
-81.7589
-98.7376
-57.9648
4z
4y
3x
-10.5723 -28.4175
-7.0581
Fig. 3A. Molecular Docking interaction diagram of compound 4m with DNA gyrase
Fig. 3B. Molecular Docking interaction diagram of compound 4y with DNA gyrase
2.4. Gyrase supercoiling inhibition assay
The molecular docking results were validated through gyrase supercoiling inhibition assay as
shown in Fig. 4. Benzimidazoles 3x, 4m, 4y, and 4z exhibited moderate activity
(4m>4z>4y>3x) against DNA gyrase enzyme. However, compound 4m exhibited better
activity as compared to rest three. Compounds 3x, 4y, and 4z showed poor inhibitory activity
against DNA gyrase. This assay is considered as an accurate predictor of target enzyme
inhibitory activity as the measurement of the inhibition of DNA gyrase supercoiling.21
7

Fig.4: Gyrase supercoiling inhibition by benzimidazoles 3x, 4m, 4y and 4z, ciprofloxacin was taken as
positive control.
2.5. Safety studies
In acute oral toxicity of the potent compound 4y, there were no observational changes in the
morbidity throughout the experimental period of seven days up to the dose level of 300 mg/kg
body weight. However, mortality (50%) and loss in body weight were observed at the dose
level of 1000 mg/kg body weight and the animals were depressed and inactive compared to
control. Blood and serum samples upon analysis showed non-significant changes in all the
parameters studied like total haemoglobin level, RBC count, WBC count, SGPT, SGOT,
cholesterol, triglycerides, albumin, serum protein, tissue protein, and reduced GSH content
(Table 7). However, ALKP activity and MDA level showed significant changes in group of
animals treated with compound 4y at 1000 mg/kg body weight as compare to control group and
other treatment groups. Animals on gross pathological study did not show change in any of the
organs studied including their absolute and relative weights (Figure 5) except that increase in
relative organ weight of liver at 1000 mg/kg body weight as compare to control group.
Therefore, the experiment showed that 4y is well tolerated by the Swiss albino mice up to the
dose level of 300 mg/kg body weight as a single acute oral dose. However, in-view of mortality
and toxicity of 4y at 1000 mg/kg, detailed sub-acute and chronic experiments need to be
carried out to look for mechanism of toxicity and organ toxicity on repeated exposure of the
lead compound 4y for its future development.14
8

Table 7. Effect of 4y as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on- body weight,
haemogram and serum biochemical parameters in Swiss albino mice (Mean±SE; n=6; a, b, c, d, significant
compared to control, 5 mg/kg, 50 mg/kg, 300 mg/kg respectively. P<0.05).
Dose of 4y at mg/kg body weight as a single oral dose
Parameters
Control
5 mg/kg
50 mg/kg
300 mg/kg
1000 mg/kg
22.86±0.75ab
28.25±0.54
28.47±0.73
27.61±0.85
25.47±0.74
Body weight (g)
RBC Count
(millions/mm3)
WBC Count
7.61±0.54
7.47±1.61
16.08±1.38
6.92±0.20
7.72±0.24
15.26±0.82
7.44±0.32
6.33±0.81
15.98±1.32
6.87±0.60
5.67±1.01
12.26±0.60
7.33±0.64
6.67±0.91
11.72±0.78
(thousands/mm3)
SGPT (U/L)
SGOT (U/L)
ALKP (U/L)
15.11±1.25
219.20±5.90
11.96±0.84
172.10±8.91
11.50±1.05
171.70±6.22
11.11±1.25
169.24±20.46
11.83±0.72
118.06±9.45ab
12.29±0.53
10.81±0.38
91.48±7.52
11.45±0.82
85.13±3.17
11.51±0.81
97.14±6.88
9.68±0.72
Haemoglobin (g/dL)
Cholesterol (mg/dL)
89.73±10.76
115.43±11.24
45.21±2.95
3.53±0.35
48.61±6.61
3.81±0.21
41.98±1.70
3.58±0.15
43.04±3.17
3.79±0.23
57.01±12.37
2.89±0.22
Triglycerides (mg/dL)
Albumin (mg/dL)
0.10±0.004
0.06±0.004
0.24±0.05
0.09±0.002
0.06±0.002
0.26±0.04
0.09±0.004
0.007±0.002
0.32±0.07
0.09±0.006
0.05±0.006
0.39±0.06
0.09±0.005
0.05±0.003
0.71±0.0.10abcd
Serum protein (mg/mL)
Tissue Protein(mg/mL)
MDA (nmole/ mg of
tissue protein )
GSH (µmole/mg of
tissue protein)
17.64±1.03
15.60±0.80
17.10±1.45
19.37±1.17
19.34±2.64
Fig. 5. Effect of compound 4y as a single acute oral dose at 5, 50, 300 and 1000mg/kg on absolute and relative
organ weights in Swiss albino mice. (mean±SE; n=6, Non significant changes were found compared to control).
Benzimidazoles are multifunctional nucleus possessing a wide range of pharmaceutical
properties.15 We previously reported (2013)16 a solid phase synthesis of 2-arylbenzimidazoles
on silica gel surface (100-200 mesh) without the use of any catalyst but less selectivity. The
present report is synthesis of 2-arylbenzimidazoles in solution phase with high
chemoselectivity and their antimycobacterial activity.
9

The development of resistance against most of the first line and second line clinical
antimycobacterial drugs by M. tuberculosis is a threat and needs immediate remedy to
counteract it. Recently, teixobactin has been isolated from a bacteria Eleftheria terrae having
excellent activity against gram positive bacteria including M. tuberculosis. It acts as a cell wall
synthesis inhibitor after binding to lipid II.17 Compound 4m exhibited moderate activity against
DNA gyrase as compared to ciprofloxacin while, other three (3x, 4y and 4z) were poorly
active. The antimycobacterial activities of benzimidazoles have got a special attention.19
Various anti-TB agents have been reported in the recent past.20 Earlier, benzimidazoles have
been reported as DNA gyrase inhibitors and FtsZ inhibitors.21 DNA gyrase, also known as
DNA topoisomerase II, catalyses the ATP dependent negative supercoiling of double stranded
closed circular DNA. Being a specific enzyme of M. tuberculosis, it is a well validated target
for antibacterial agents. It is found that the inhibitors of DNA gyrase are also active against
non-replicating Mycobacteria.22 However, M. tuberculosis has acquired resistance against
many DNA gyrase inhibitors.23 FtsZ, a protein tubulin named after ‘Filamenting temperature-
sensitive mutant Z’ has its vital role in bacterial cell division. It plays an important role in
bacterial cytokinesis.24 Previously, several antibacterials have been developed on
benzimidazole core. Albendazole and thiabendazole are potential FtsZ inhibitors previously
reported against Mycobacterium possessing benzimidazole core.20 There are several other anti-
TB leads acting as FtsZ inhibitors. Prof. Slayden’s group found that benzimidazole core based
FtsZ inhibitors led to the absence of septum formation.20
3. Conclusion
In summary, we have demonstrated a novel synthesis of 2-arylbenzimidazoles in molecular
sieve-MeOH system. Further, 1-benzyl-2-arylbenzimidazoles are potential antitubercular
agents. The potential antitubercular candidates 4y, 4z and 4m showed moderate affinity with
DNA gyrase as target. Most potent antitubercular compound 4y was tolerable up to 300 mg/kg
dose in safety studies. The potential leads may further be optimized for better antitubercular
activity.
4. Experimental
4.1. General
Melting points were determined in open glass capillaries using automated melting point
apparatus, Stanford Research System, USA and are uncorrected. Reactions were monitored on
Merck TLC plates UV254nm and 2% ceric sulphate in 10% aqueous sulphuric acid solution
spray was used for development. Column chromatography was carried out on silica gel (60-120
mesh) and hexane, chloroform-acetone were used as eluants. NMR spectra were recorded on
Bruker AVANCE 300/500MHz spectrometer with TMS as an internal standard (δ=0.00 ppm).
Solvents were dried as per standard methods. Electrospray mass were recorded on APC3000
LC-MS-MS (Applied Biosystem) and High Resolution Mass (HRMS) on Agilent 6520Q-TOF
after dissolving the compounds in methanol. Scanning Electron Microscope (SEM) Imaging
experiments were done on Zeiss Leica model SEMLEO430, UK after coating the sample with
Polakon Sputter coater with gold-palladium target at Birbal Sahni Institute of Paleobotany
10

(BSIP), Lucknow, India. Purity of compounds 3x, 4m, 4y and 4z was found to be 95-97% by
RP-HPLC.
4.2. Chemical synthesis
4.2.1. Typical experimental procedure for the synthesis of 2-arylbenzimidazoles:
o-Phenylenediamine (1, 260 mg, 2.4 mmol) and 3,4,5-trimethoxybenzaldehyde (2, 392 mg, 2
mmol) were dissolved in methanol (15 mL). To this reaction mixture, 1g molecular sieves 3Ao
were added and reaction mixture was refluxed for 1.5 h. On completion, it was filtered and
solvent was evaporated. The residue was purified through column chromatography over silica
gel (60-120 mesh) and eluted with hexane-chloroform. Compound 1,2-diarylbenzimidazole
(4e) was eluted at 70% chloroform-hexane polarity followed by 2-arylbenzimidazole (3e) at
2% acetone-chloroform.
4.3. Biological evaluation
4.3.1. Antitubercular assay
It was performed on BACTEC 460 radiometrically as per reported method [Supplementary
information S1]
4.3.2. In vivo acute oral toxicity
Acute oral toxicity of compound 4y was performed in accordance with the Organization for
Economic Co-operation and Development (OECD) test guideline No 423 (1987)
[Supplementary information S2]
4.3.3. Molecular docking studies
The docking studies of 3x, 4y and 4z were performed by MOE2014.09 software using DNA
gyrase_1EI1, Thymidine Monophosphate Kinase_1GSI and FtsZ_1RQ2 as receptors
(Supplementary information, S3).
4.3.4. Gyrase supercoiling inhibition assay
Gyrase supercoiling inhibition assay was carried out by the M. tuberculosis gyrase microplate
assay kit (Inspiralis, Innovation Centre, Norwich Research Park, Norwich, UK) as per
manufacturer’s protocol (Supplementary information, S4).
Acknowledgements
Authors are thankful to Director CIMAP for constant encouragement and support. Council of
Scientific and Industrial Research (CSIR) is duly acknowledged for financial support. Authors
also thank to Director, Senior Research Fellowships to two of the authors (AKV and SK) is
duly acknowledged. CIMAP-Chemical Central Facility is duly acknowledged for the analysis
of High Resolution Mass spectra.
References
1.
2.
Tuberculosis: WHO Factsheet, October 2017.
TB India, 2017, Revised National TB control Programme, Annual Status Report, DGHS,
MoHFW, Govt of India
3.
Yadav G, Ganguly S. Eur. J. Med. Chem. 2015; 97:419-443.
11

4.
5.
6.
7.
Chang CS, Liu JF, Lin HJ, Lin CD, Tang CH, Lu DY, Sing YT, Chen LY, Kao MC, Kuo SC,
Lai CH. Eur. J. Med. Chem. 2012; 48: 244-254.
Ndakala AJ, Gessner RK, Gitari PW, October N, White KL, Hudson A, Fakorede F, Shackleford
DM, Kaiser M, Yeates C, Charman SA, Chibale K. J. Med. Chem. 2011; 54: 4581-4589.
Omar MA, Shaker YM, Galal SA, Ali MM, Kerwin SM, Li J, Tokuda H, Ramadan RA, Diwani
HIE. Bioorg. Med. Chem. 2012; 20: 6989-7001.
Morninstar ML, Roth T, Farnsworth DW, Smith MK, Watson K, Buckheit RB, Das K, Zhang W,
Arnold E, Julias JG, Hughes SH, Michejda CJ. J. Med. Chem. 2007; 50: 4003-4015.
Kim JS, Gatto B, Yu C, Liu A, Liu LF, LaVoie EJ. J. Med. Chem. 1996; 39: 992-998.
Kamal A, Reddy MK, Shaik TB, Rajender, Srikanth YVV, Reddy VS, Kumar GB, Kalivendi SV.
Eur. J. Med. Chem. 2012; 50: 9-17.
8.
9.
10. Dettmann S, Szymanowitz K, Wellner A, Schiedel A, Muller CE, Gust R. Bioorg. Med. Chem.
2010; 18: 4905-4916.
11. Gaba M, Singh S, Mohan C. Eur. J. Med. Chem. 2014; 76: 494-505.
12. (a) Lin C, Lai PT, Liao SKS, Hung WT, Yang WB, Fang JM. J. Org. Chem. 2008; 73: 3848-3853;
b) Xiangming H, Huiqiang M, Yulu W. Arkivoc 2007; xiii: 150-154; (c). Tandon VK, Kumar M.
Tetrahedron Lett. 2006; 45: 4185-4187; (d). Singh MP, Sasmal S, Lu W, Chatterjee MN.
Synthesis 2000; 1380-1390; (e). Dhakshinamoorthy A, Kanagaraj K, Pitchumani K. Tetrahedron
Lett. 2011; 52: 69-73; (f). Nagawade RR, Shinde DB. Ind. J. Chem., Section B 2007; 46: 349-
351; (g). Du LH, Wang YG. Synthesis 2007; 675-678; (h) Blacker AJ, Farah MM, Hall MI,
Marsden SP, Saidi O, Williams JM. J. Org. Lett. 2009; 11: 2039-2042; (i). Behbahani FK, Ziaei
P, Fakhroueian Z, Doragi N. Monatsh. Chem. 2011; 42: 901-908; (j) Bahrami K, Khodaei MM,
Naali F. J. Org. Chem. 2008; 73: 6835-6837; k) Pasha MA, Nizam A. J. Saudi Chem. Soc. 2012;
16: 237-240; (l). Mukhopadhyay C, Datta A, Butcher RJ, Paul BK, Guchhait N, Singha R.
Arkivoc 2009; xiii: 1-22; (m). Rostamizadeh S, Amani AM, Aryan Ghaieni RHR. Monatsh.
Chem. 2009; 140: 547-549; (n). Patil VD, Patil J, Rege P, Dere G. Synth. Commun. 2011; 41: 58-
62; (o). Dio X, Wang Y, Jiang Y, Ma, D. J. Org. Chem. 2009; 74: 7974-7977; (p) Ghosh P,
Subba R. Tetrahedron Lett. 2015; 56: 2691-2694; (q) Digwal CS, Yadav U, Sakla AP, Ramya
PVS, Aaghaz S, Kamal A. Tetrahedron Lett. 2016; 57: 4012-4016; (r) Alvim HGO, deOliveira
HCB, Bataglion GA, Eberlin MN, Ramos LM, Silva WA. RSC Adv. 2016; 5: 69418-69422; (s)
Chakraborty A, Majumdar S, Maiti DK. Tetrahedron Lett. 2016; 57: 3298-3302.
13. (a) Sharghi H, Khalifeh R, Mansouri SG, Aberi M, Eskandari MM. Cat. Lett. 2011; 141: 1845-
1850; (b) Inamdar SM, More VK, Mandal SK. Tetrahedron Lett. 2013; 54: 579-583; (c) Santra
S, Majee A, Hajra A. Tetrahedron Lett. 2012; 53: 1974-1977; (d) Shelkar R, Sarode S, Nagarkar
J. Tetrahedron Lett. 2013; 54: 6986-6990; (e) Naeimi H, Babaei Z. Lett. Org. Chem. 2015; 12:
311-318; (f) Bahrami K, Khodaei MM, Naali F. J. Exp. NanoSci. 2016; 11: 148-160.
14. Ghosh MN. In Book: Fundamentals of Experimental Pharmacology (1st Edition), Scientific Book
Agency, Kolkata, 1986; pp.156.
15. (a) Akhtar W, Khan MF, Shaquiquzzaman M, Rizvi MA, Mehdi SH, Akhter M, Alam MM. Eur.
J. Med. Chem. 2017; 126: 705-753.
16. Chaturvedi AK, Negi AS, Khare P. RSC Advances 2013; 3: 4500-4504.
17.
Ling LL, Schneider T, Peoples AJ, Spoering AL, Engles I, Conlon BP, et al. Nature 2017; 517:
455-459 .
18. Keri RS, Rajappa CK, Patil SA, Nagaraja BM. Pharmacological Reports 2016; 68:1254-1265.
19. Klimesova V, Pour M, Stachel J, Waisser K, Kaustova J. Eur J Med Chem 2002; 37: 409-418.
12

20. (a) Hameed S, Raichukar A, Madhayapeddi P, Menasinakai S, Sharma S, Kaur P, Nandishaiah R,
Panduga V, Reddy J, Sambandamurthy V, Sriram D. ACS Med. Chem. Lett. 2014; 5: 820-825.
(b) Kumar K, Avasthi D, Lee SY, Zanardi I, Ruzsicska B, Knudson S, Tonge PJ, Slayden RA,
Ojima I. J. Med. Chem. 2011; 54: 374-381.
(c) Awasthi D, Kumar K, Slayden RA, Ojima I. J. Med. Chem. 2013; 56: 9756-9770.
(d) Knudson SE, Kumar K, Ojima I, Slayden RA. Tuberculosis 2014; 94: 271-293.
21. Barrett JF, BarnsteinJI, Krause HM, Hillard JJ, Ohemeng KA. Anal. Biochem. 1993, 214: 313-317.
22. Mdluli K, Ma Z. Infect. Disord. Drug Targets 2007; 7: 159-168.
23. Ginsberg AM, Spigelman M. Nature Med. 2007; 13: 290-294.
24. Li X, Ma S. Eur. J. Med. Chem. 2015; 95: 1-15.
....................................
13

Table 4. Synthesis of 2-arylbenzimidazoles (3) in molecular sieves-MeOH system
o-phenylene
diamine (1)
Product (3a-3y)
(2-arylbenzimidazole)
%Yield
(3a-3y)
Product (4a-4y)
(Diarylbenzimidazole)
%Yield
(4a-4y)
S. No.
aldehyde
(2)
H
N
NH2
NH2
CHO
1.
67
-
-
3a
4a
OH
N
HO
CHO
H
N
NH2
2.
3.
79
62
-
-
-
-
3b
3c
4b
4c
OCH3
N
H3CO
NH2
NH2
CHO
H
N
OH
HO
N
NH2
NH2
OCH3
OCH3
OCH3
H3CO
CHO
OCH3
H
4.
5.
73
76
7
8
3d
3e
4d
4e
N
OCH3
H3CO
NH2
N
OCH3
OCH3
OCH3
N
N
H3CO
H3CO
CHO
OCH3
H
OCH3
NH2
NH2
H3CO
H3CO
N
OCH3
N
OCH3
OCH3
OCH3
OCH3
OCH3
N
N
-
CHO
H
N
NH2
NH2
6.
7.
53
89
-
-
3f
4f
NO2
O2N
N
F
H
N
NH2
NH2
CHO
6
3g
4g
F
N
F
N
F
N
H
CHO
NH2
N
8.
59
-
-
3h
4h
N
N
NH2
NH2
N
Cl
Cl
H
N
9.
64
61
-
-
-
-
3i
3j
4i
4j
CHO
NH2
NH2
N
Cl
Cl
H
10.
N
CHO
Cl
NH2
N
Cl
H
CHO
NH2
NH2
11.
12.
67
59
-
-
-
-
3k
3l
4k
4l
N
CF3
N
F3C
CHO
CHO
H
NH2
NH2
N
N
N
N
O
NH2
NH2
O
O
O
H
O
13.
14.
71
67
8
-
3m
3n
4m
4n
N
O
N
O
N
N
O
CHO
CHO
OH
OCH3
NH2
NH2
H
N
-
H3CO
N
HO
NH2
NH2
NH2
H
N
15.
16.
74
83
-
-
-
-
3o
3p
4o
4p
OAc
AcO
N
OCH3
OCH3
H3CO
CHO
H
N
H3C
NH2
H3CO
N
H3C
OCH3
OCH3
NH2
NH2
H3CO
H3CO
CHO
CHO
H
17.
18.
19.
62
30
51
-
-
-
-
-
-
3q
3r
3s
4q
4r
4s
N
Cl
N
Cl
NH2
NH2
OCH3
OCH3
H3CO
H3CO
H
N
O2N
N
O2N
NH2
CHO
H
N
NH2
N
NH2
NH2
H
CH3-CH2-CHO
20.
21.
58
64
-
-
-
-
3t
4t
CH3
N
N
H
N
NH2
NH2
CHO
3u
4u
NO2
H3CO
N
O2N
H3CO
14

H3CO
HO
CHO
CHO
OCH3
OH
NH2
NH2
H
N
22.
23.
55
57
-
-
3v
4v
H3CO
H3CO
N
H3CO
OCH3
NH2
NH2
OCH3
OCH3
H
N
H3CO
5
3w
4w
H3CO
N
H3CO
OCH3
OCH3
OCH3
N
N
H3CO
F
H
NH2
NH2
CHO
24.
25.
26.
46
42
70
6
8
6
3x
3y
3z
4x
4y
4z
N
F
N
H3CO
F
H3CO
N
N
F
H3CO
H
N
OCH3
CHO
NH2
NH2
OCH3
N
H3CO
H3CO
H3CO
N
N
H3CO
H3CO
-
CHO
O
O
NH2
NH2
O
O
O
H
N
O
H3CO
N
H3CO
O
N
N
O
H3CO
Highlights
* An efficient and chemoselective new synthetic protocol for arylbenzimidazoles
* Three of the diarylbenzimidazoles antitubercular activity against M. tuberculosis H37RV
(MIC=16, 24 and 67 µM)
* In molecular docking studies, compounds showed moderate affinity to DNA gyrase of M.
tuberculosis.
* Compound 4m showed moderate gyrase supercoiling inhibition activity.
* Best analogue, 4y was non-toxic in in-vivo acute oral toxicity.
15