Discovery of new leads against Mycobacterium tuberculosis using scaffold hopping and shape based similarity

Article abstract of DOI:10.1016/j.bmc.2017.07.034

BM212 [1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)-methyl-pyrrole] is a pyrrole derivative with strong inhibitory activity against drug resistant Mycobacterium tuberculosis and mycobacteria residing in macrophages. However, it was not pursued because o

Full text of DOI:10.1016/j.bmc.2017.07.034

Accepted Manuscript
Discovery of new leads against Mycobacterium tuberculosis using Scaffold
Hopping and Shape based Similarity
Ravindra D. Wavhale, Elvis A.F. Martis, Premlata K. Ambre, Baojie Wan, Scott
G Franzblau, Krishna R. Iyer, Kavita Raikuvar, Katarzyna Macegoniuk, Łukasz
Berlicki, Santosh R. Nandan, Evans C. Coutinho
PII:
S0968-0896(17)30612-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.034
BMC 13871
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2017
30 June 2017
17 July 2017
Please cite this article as: Wavhale, R.D., Martis, E.A.F., Ambre, P.K., Wan, B., Franzblau, S.G., Iyer, K.R.,
Raikuvar, K., Macegoniuk, K., Berlicki, L., Nandan, S.R., Coutinho, E.C., Discovery of new leads against
Mycobacterium tuberculosis using Scaffold Hopping and Shape based Similarity, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.034
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Discovery of new leads against Mycobacterium tuberculosis using Scaffold
Hopping and Shape based Similarity
Ravindra D. Wavhale , Elvis A. F. Martis , Premlata K. Ambre , Baojie Wan§, Scott G
Franzblau§, Krishna R. Iyer , Kavita Raikuvar , Katarzyna Macegoniuk˥, Łukasz Berlicki˥,
†
Santosh R. Nandan┴ and Evans C. Coutinho
Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Mumbai
400098, India.
┴Ambernath Organics, 307/314, Creative Industries Premises, Kalina, Mumbai 400098, India.
§Institute for Tuberculosis Research, University of Illinois, Chicago 60612, USA.
˥Department of Bioorganic Chemistry, Wroclaw University of Technology, Wroclaw 50-370,
Poland.
†For Correspondence: E-mail: evans.coutinho@bcp.edu.in
Tel No. +91-22-26670905. Fax No. +91-22-26670816
1

1
2
Graphical Abstract
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2

4
Abstract
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BM212 [1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)-methyl-pyrrole] is
a
pyrrole
derivative with strong inhibitory activity against drug resistant Mycobacterium tuberculosis
and mycobacteria residing in macrophages. However, it was not pursued because of its poor
pharmacokinetics and toxicity profile. Our goal was to design and synthesize new
antimycobacterial BM212 analogues with lower toxicity and better pharmacokinetic profile.
Using the scaffold hopping approach, three structurally diverse heterocycles - 2,3-
disubstituted imidazopyridines, 2,3-disubstituted benzimidazoles and 1,2,4-trisubstituted
imidazoles emerged as promising antitubercular agents. All compounds were synthesized
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13
14
15
16
17
18
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1
through easy and convenient methods and their structures confirmed by IR, H NMR, 13C
NMR and MS. In-vitro cytotoxicity studies on normal kidney monkey cell lines and HepG2
cell lines, as well as metabolic stability studies on rat liver microsomes for some of the most
active compounds, established that these compounds have negligible cytotoxicity and are
metabolically stable. Interestingly the benzimidazole compound (4a) is as potent as the
parent molecule BM212 (MIC 2.3 µg/ml vs 0.7-1.5 µg/ml), but is devoid of the toxicity
against HepG2 cell lines (IC50 203.10 µM vs 7.8 µM).
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Key-words: BM212, scaffold hopping, anti-tubercular activity, microplate alamar blue assay,
2,3-disubstituted benzimidazoles, 1,2,4-trisubstituted imidazoles, 2,3-disubstituted
imidazopyridines, Mycobacterium tuberculosis.
3

Cytotoxicity
Metabolic
stability
M. tuberculosis
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1 Introduction
The WHO report 2016 gives an estimate of 10.4 million new TB cases with 1.4
million TB deaths in 20151. In recent times, drug resistance has emerged as the greatest
problem. As a result, physicians are left with limited choice of drugs, increasing the risk of
therapy failures. Deidda et al. had identified BM212, a 1,5-diarylpyrrole derivative, with
promising activity against multidrug-resistant clinical isolates of Mycobacterium tuberculosis
and also against those residing within macrophages (MICs between 0.7 and 1.5 µg/ml)2.
These results offered a ray of hope for the emergence of a new anti-tubercular agent (Figure
1), however it was soon realized that BM212 suffers from poor bioavailability and severe
toxicity. In the pursuit of new derivatives of BM212 with improved pharmacokinetics and
toxicity profile3-7, optimization strategies were focused on modification of the 1,5-diphenyl
substituents and the side chain at the 3-position of the pyrrole ring. Several BM212
analogues like BM521, BM533, BM579 and Compound I were synthesized with good
biological profiles (MIC ranging from 0.2 to 0.12 µM) and comparatively better Protection
Indices (PI = CC50/MIC, ranging from 127.5 to 1937.5) than BM212 (Figure 1), but all suffer
from high HepG2 toxicity6 (Table 1).
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BM212
BM521
BM533
BM579
I
40
41
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Figure 1 BM212 and their analogs
4

clearance in
mouse
VERO cells
HepG2Cells
microsomes
IC50 (µM)
IC50 (µM)
T1/2 (Min)
BM212
BM521
BM533
BM579
I
5
-
7.8
24.01
>2423.77
5.90
>30
0.16
0.20
0.16
0.12
178.80
>302.26
15.20
50.15
8.2
<5
10
15.3
>30
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44
Table 1 In vitro cytotoxicity on VERO cells and HEPG2 cells and metabolic stability studies
of BM212 and analogs6.
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To further augment the safety and efficacy profile of BM212 and develop potent
antitubercular agents through exploration of the diversity in the chemical space of BM212,
we embarked on the scaffold hopping approach to seek a replacement for the central pyrrole
ring in BM212. This strategy identified imidazole, benzimidazole and imidazopyridine
scaffolds as promising replacement moieties. Furthermore, the Rapid Overlay of Chemical
Structures8-10 (ROCS) program was employed to search analogs of these three scaffolds with
similar 3D shape and volume outlines as BM212. The Tanimoto shape similarity coefficient
(TSSC) was used to quantify the shape similarity, and those molecules with TSSC greater
than 0.60 were advanced for further studies. This approach led to a library of twenty
compounds comprising 1,2,4-trisubstituted imidazoles, 2,3-disubstitued benzimidazoles and
2,3-disubstituted imidazopyridines.
These compounds were profiled for their
antimycobacterial activity and cytotoxicity potential.
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60
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Figure 2: BM212 and representative molecules belonging to the scaffolds benzimidazole (4),
imidazole (8), and imidazopyridine (12) that were designed using scaffold hopping and
ROCS.
ROCS Shape Tanimoto
BM212→
Conf. 1 Conf. 2 Conf. 3 Conf. 4 Conf. 5
Data set
Compound 4
Compound 8
Compound 12
0.73
0.73
0.65
0.71
0.71
0.65
0.71
0.7
0.72
0.68
0.66
0.71
0.66
0.66
0.63
62
63
64
Table 2: The ROCS TSSC of compounds 4, 8 and 12 with respect to different conformations
of BM212
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66
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2 Results and Discussion
2.1 Molecular Design and Shape based complementarity studies with BM212
The scaffold hopping approach11 was used to identify heterocycles that could replace the
original core (pyrrole ring). Figure 3 illustrates the shape of BM212 along with the
pharmacophoric features that are essential for its bioactivity, namely, 1) a central
hydrophobic core, 2) hydrogen bond acceptor and 3) two adjacent aromatic rings.
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71
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Figure 3 Representation of BM212 and pharmacophore features with its molecular shape.
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The scaffold hopping approach identified imidazole, benzimidazole and imidazopyridine as
three cores that could potentially replace the pyrrole ring. Derivatives of the identified cores
were sought such that the 3D shape and volume of the new molecules were like the shape and
volume of BM212. We surmised that by means of this strategy, the new derivatives would
retain the potency inherent to BM212 (MIC 0.7-1.5 µg/ml). The Tanimoto shape similarity
coefficient (TSSC) was used to quantify the shape similarity with BM212, and is expressed
quantitatively in the range 0 to 1.0. A value of 1.0 indicates complete similarity, while 0
indicates no similarity (Table 2). A database curated from literature was virtually screened
against the five lowest energy conformations of BM212.
The 2,3-disubstitued
benzimidazoles (e.g. compound 4) with TSSC between 0.70 to 0.73 is closest in shape to
BM212; next is the 1,2,4-trisubstituted imidazoles (e.g compound 8) with TSSC 0.66 to 0.73,
followed by the 2,3-disubstituted imidazopyridines (e.g. compound 12) with TSSC 0.63 to
0.66. In case of 2,3-disubstituted benzimidazoles (Figure 4a), the two phenyl rings of
benzimidazole superimpose well onto the two phenyl rings of BM212 and the benzimidazole
core overlays the pyrrole ring. A similar arrangement is seen for the 1,2,4-trisubstituted
imidazole (Figure 4b), the phenyl rings at the 1- and 2-positions overlap in a fashion similar
to 2,3-disubstituted benzimidazole; the phenyl ring at the 4-position points towards the
piperazine attached at the 3-position of BM212. With the 2,3-disubstituted imidazopyridines,
the 2-phenyl ring overlaps with the 2-phenyl ring of BM212 (Figure 4c), however the 3-
substituent (in this case the morpholine ring) as opposed to our expectations did not match
7

94
the piperazine moiety of BM212 but instead lies over the 3-phenyl ring of BM212.
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96
97
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Figure 4: Molecular superimposition of a- compound 4, b- compound 8, and c- compound
12 on BM212. Compounds 4, 8 and 12 are represented in grey color while the BM212 is
shown in green color.
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2.2 Chemistry
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112
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Synthesis of 2,3-disubstituted benzimidazoles12 (4a-4k), 1,2,4-trisubstituted imidazoles13 (8a-
8b) and 2,3-disubstituted imidazopyridines14,15 (12a-12g) are described in Schemes 1–3.
Scheme 1 shows a one pot synthetic route for 2,3-disubstituted benzimidazoles using o-
fluoro-nitrobenzene (1), a substituted aniline (2) and a substituted benzaldehyde (3).
Substituted 1,2,4-trisubstituted imidazoles were synthesized as shown in Scheme 2, in which
a substituted aniline (6), a substituted benzaldehyde (7), ammonium acetate and
phenacylbromide (5) are heated at 130oC without any solvent.
2,3-Disubstituted
imidazopyridines were synthesized in two steps (Scheme 3) as follows: in the first step, 2-
phenyimidazopyridines (11) were synthesized by cyclization of a substituted 2-aminopyridine
(9) and a substituted phenacylbromide (10). Then, in the second step, morpholine or
piperidine was attached via a methyl linker at the 3-position of the imidazopyridine by the
Mannich reaction.
2.2.1 Scheme 1 Synthesis of 2,3-disubstituted benzimidazoles
8

N
N
114
R1
NO2
F
(i)
R1CHO
3
R2NH2
2
1
R2
4a-4k
115
116
(i) DMSO, sodium dithionite, 130oC.
2.2.2 Scheme 2 Synthesis of 1,2,4-trisubstituted imidazoles
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118
(i) 130oC for 2 Hrs.
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2.2.3 Scheme 3 Synthesis of 2,3-disubstituted imidazo[1,2-a]pyridine
121
122
(i) ethanol, reflux (ii) acetic acid, morpholine/piperidine, ~ 36% formalin solution, room
temperature.
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2.3 Biological evaluation
2.3.1 Antitubercular activity
The antitubercular activity was measured on Mycobacterium tuberculosis H37Rv strain by
the microplate alamar blue assay (MABA) as per published protocols16. The initial
9

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concentration of the compounds was fixed at 50 µg/ml (Table 3). Alamar blue is a general
indicator used to check cellular growth and/or viability; basically, the blue non-fluorescent
oxidized form of the dye resazurin in the reducing environment of living cells turns into
resorufin which is fluorescent pink. An inhibitor of mycobacterial growth will prevent this
color change. Rifampicin and isoniazid were used as standards. Compounds that showed
95% inhibition at 50 µg/ml were further evaluated at lower concentrations to finally
determine the minimum inhibitory concentration (MIC) for 95% inhibition. The results show
that six compounds are active at less than 20 µg/ml against Mycobacterium tuberculosis
H37Rv. The most potent compound 4a has MIC of 2.3µg/ml.
The 2,3-disubstituted benzimidazoles were found to be the most promising among the three
classes of molecules with MICs of 2.3 and 8.0 µg/ml for 4a and 4b respectively. On closer
inspection, we find that for optimal antitubercular activity, the 4-methoxy group as R1 and the
4-chloro substitution as R2 on the benzimidazole ring are critical. A shift in the position of
the R1 methoxy group from the para to the meta position lowers the activity (4h, MIC >50
µg/ml), as also the addition of a methoxy group at the ortho position e.g. 4c and 4k with
MICs of 48.3 µg/ml and 20.3 µg/ml respectively. We also notice that the antitubercular
activity is very sensitive to the R2 substituent; moving the chloro substituent from the para to
the meta position leads to complete abolishment of activity, e.g. 4f (MIC >50 µg/ml, 0%
inhibition). Further, we observe that replacing the chloro substituent with other halogens like
bromine (4d, MIC >50 µg/ml, 92% inhibition) or fluorine (4g, MIC >50 µg/ml, 6%
inhibition) leads to decrease in the activity. Replacement of the chloro substituent, with 2,5-
dimethyl (4e, MIC >50 µg/ml, 80% inhibition) or 4-isopropyl (4i, MIC >50 µg/ml, 68%
inhibition) or 3-methoxy (4i, MIC > 50 µg/ml, 14% inhibition) is detrimental to the activity.
A drastic drop in the antitubercular activity, indicates that the chloro substituent at R2 is
essential.
In the 1,2,4-trisubstituted imidazole series, two molecules (8a and 8b), have MICs of 20.3
µg/ml (8a) and 17.8 µg/ml (8b). Both molecules contain at the 4th position of the 2-
substituted phenyl ring, a hydrogen bond donor/acceptor group which is in line with the
pharmacophore requirement for good antitubercular activity.
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In the 2,3-disubstituted imidazopyridine class, five of the seven compounds have MICs below
50 µg/ml; these are 12c with MIC of 17.0 µg/ml, 12a with MIC of 22.2 µg/ml, 12b MIC 23.9
µg/ml, 12f MIC 33.7 µg/ml and 12d MIC 40 µg/ml. This highlights the potential of 2,3-
disubstituted imidazopyridines to be developed as new antitubercular agents. Interestingly, a
minor change in the position of the methyl group from the 6th to the 7th position of the
imidazopyridine ring, removal/addition of the 4-fluoro group on the 2-phenyl ring and
replacement of the morpholine moiety with piperidine, show dramatic effects on the
antitubercular activity. The methyl group on the 6th position of the imidazopyridine ring and
the 4-fluoro on the 2-substituted phenyl ring impart the highest antitubercular activity as seen
in compound 12c with MIC of 17.0 µg/ml. A change in the position of the methyl group
from the 6th to the 7th position as in compound 12b results in a slight drop in the activity with
MIC of 23.9 µg/ml. Removal of the methyl group but retaining the 4-fluoro group, as in 12c
and 12a, preserve the antitubercular activity; however, removal of the 4-fluoro group while
retaining the 7-methyl group as in 12d, weakens the activity (MIC 40 µg/ml). Hence, we
reckon that both the methyl and fluoro groups on the imidazopyridine ring are imperative for
antitubercular activity, as is evinced by the MIC values of molecules 12g, 12e and 12f
174
M. tuberculosis H37Rv
Comp.
4a
R1
R2
R3
MIC (µg/mL)
4-OCH3
4-Cl
H
2.3
11

4-OCH3, 3-
OH
H
H
4b
8.0
4-OCH3
4-OH
O
H
H
H
H
8a
8b
20.3
17.8
22.2
23.9
17.0
40.0
>50
4-F
4-F
4-F
H
H
12a
O
7-CH3
6-CH3
7-CH3
H
12b
O
12c
O
12d
CH2
CH2
O
H
12e
H
7-CH3
H
12f
33.7
>50
H
12g
Isoniazid
Rifampicin
0.11
0.03
A larger library of 2,3-disubstituted benzimidazoles
2,4-di(OCH3)
4-OCH3
4-Cl
4-Br
4c
4d
4e
48.3
> 50 (92)
> 50 (80)
4-OCH3
2,5-
di(CH3)
4-OCH3
4-OCH3
3-OCH3
3-Cl
4-F
> 50 (0)
> 50 (6)
> 50 (67)
4f
4g
4h
4-Cl
12

4-OCH3
4-
> 50 (68)
4i
CH(CH3)2
4-OCH3
3-OCH3
4-Cl
> 50 (14)
20.3
4j
3,4-di(OCH3)
4k
Isoniazid
Rifampicin
0.06
0.50
175
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Table 3 Structures and in vitro antitubercular activity against M. tuberculosis H37Rv.
2.3.2 Assessment of selectivity – antitubercular vs antimicrobial/antifungal activity
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In order to access how selective are the compounds against tuberculosis, we decided to
evaluate the compounds for their activity on gram negative bacteria Escherichia coli,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, the gram
positive bacteria Staphylococcus aureus, and the fungi Candida albicans and Cryptococcus
neoformans (Table 4). Expect for compounds 4d, 4h and 4j with 50% inhibition of
Actinobacter baumanni at the relatively high dose of 32 µg/ml, others show poor antibacterial
and antifungal activity and is in sharp contrast to their antitubercular activity. This result
highlights the selectivity of these molecules against Mycobacterium tuberculosis over gram
positive bacteria, gram negative bacteria and fungi. The reason for this selectivity is
unknown at present.
13

Test
Percent inhibition at 32 µg/ml
comp.
Staphylococcus
aureus
Escherichia
coli
Klebsiella.
pneumoniae
(G -ve)
Acinetobacter
baumannii
(G -ve)
Pseudomonas
Candida
albicans
(Fungi)
Cryptococcus
neoformans
var. grubii
(Fungi)
aeruginosa
(G +ve)
(G -ve)
(G -ve)
Strain
Strain
Strain
Strain
Strain
Strain
ATCC 90028
Strain
ATCC43300
ATCC 25922
ATCC 700603
ATCC 19606
ATCC 27853
ATCC 208821
Inh. 1
-19.12
7.70
Inh.2
4.59
Inh. 1
-6.11
-8.29
-5.75
-5.33
-8.47
-3.59
-4.10
-8.63
-5.93
-3.74
-10.30
-5.08
-2.98
Inh.2
-6.64
Inh. 1
2.12
Inh.2
-20.26
-1.42
3.25
Inh. 1
-3.17
-9.27
-6.12
1.56
Inh.2
32.03
-0.58
1.56
Inh. 1
9.75
7.73
8.86
3.52
5.72
7.07
5.18
2.25
6.29
11.23
-3.23
2.98
11.03
Inh.2
1.34
Inh. 1
12.9
Inh.2
12.19 -12.27
6.64 -6.62
Inh. 1
Inh.2
-2.00
-9.81
-15.36
-9.29
-8.42
-12.59
-13.28
6.16
4a
4b
4c
4d
4e
4f
15.48
14.19
15.00
9.57
-6.80
5.26
1.32
11.26
3.53
10.95
12.30
10.01
15.99
12.65
-13.23
6.25
-7.22
9.39
-34.01
-28.18
19.67
24.46
12.82
13.04
15.74
9.80
2.68 -10.75
9.75 14.66
-12.42
-11.99
-6.55
7.06
-12.80
-17.93
-3.40
-5.95
-3.27
-7.58
-1.06
7.53
58.14
38.80
-12.48
-8.46
49.38
36.14
59.86
42.64
34.76
8.91
11.65
4.32
14.01
12.72
6.63
3.50
1.69
4.66
-7.27
-3.58
14.12
11.62
7.95
-7.86
-9.90
50.78
23.76
19.72
39.94
23.06
-5.84
7.76
4g
4h
4i
-7.46
1.62
1.69 -12.49
4.73 19.65
-15.77
-11.64
-5.29
-3.23
2.46
28.10
14.16
5.26
12.36
20.44
9.81
13.05
8.76
8.23
7.06
-8.36
4.02
11.02
-3.73
0.95
4j
7.42
8.57
4k
8a
12a
1.03
-15.61
-6.92
-3.37
-0.26
13.72
19.16
16.56
14.35
7.63
3.07
13.34
30.28
-3.93
-2.71
17.53
0.63
26.40 -18.35
5.39 1.63
-19.70
-11.02
22.29
-13.56
14

12b
12c
12d
12e
12g
20.12
25.15
15.85
14.77
28.57
21.75
26.23
23.49
19.41
26.09
-4.19
-2.48
-7.53
-2.22
-2.72
-12.41
-6.51
14.39
14.46
7.77
-5.28
-6.29
-1.06
-5.80
2.52
0.94
-1.99
-9.54
1.56
-16.15
1.09
9.71
9.45
26.98
-2.06
15.15
17.70
16.50
2.74
6.17
1.68
3.93
0.30
7.44
3.40
-1.19
1.85
-2.86
-0.09
-9.98
-4.43
3.04
-12.61
-12.91
-11.19
-5.58
10.45
6.13
10.63
11.45
9.04
0.89 -11.62
11.72
13.77
2.41
6.38
9.01
2.71
-2.71
189
190
Table 4. Antimicrobial and antifungal activity on selective strains
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199
200
201
202
203
204
205
206
2.3.3 Cytotoxicity and Metabolic stability studies
A selected set of compounds were investigated for their cytotoxicity potential on normal
kidney monkey cell lines17 and HepG2 cell lines (Table 5). In this study, the concentration
of a compound to inhibit 50% growth of the cells (GI50), as well as the concentration to kill
50% of cells (LC50) and finally the concentration to inhibit total growth of cells (TGI) were
measured. Compounds 4a, 4b, 4c, 4d and 4k of the 2,3-disubstituted benzimidazole series
were found to exhibit low cytotoxicity at concentrations below 10 µM against normal kidney
monkey cell lines. The IC50 against HepG2 cell lines for compounds 4a, 4d and 4k were
found to be 203.10 µM, 112.63 µM and 49.90 µM respectively. In the in-vitro metabolic
stability studies compounds 4a, 4d and 4k were found to be metabolic stable in rat liver
microsomes. There was no meaningful change found in the peak area ratio of compound to
internal standard for compound 4a, whereas 4d and 4k showed about 2% and 5% decrease
respectively after 60 min of incubation. Overall it appears that the three compounds are
stable to oxidative metabolism mediated by CYP450s and FMO. The positive control p-
nitrophenol showed significant conversion to p-nitrocatechol (about 17%) in 60 minutes.
207
Comp.
Cytotoxicity studies
Metabolic
stability
studies
M. tuberculosis H37Rv
MIC (µg/mL)
Normal monkey kidney cell line
VERO
HepG2
% Turnover by rat
liver microsomes
in 60 min.
Cell line
GI50
(µM)
29
LC50
TGI
IC50
(µM)
(µM)
(µM)
8.4×105 5×103 203.10
0
-
4a
4b
2.3
8.0
4.5×103
5.0
-
1×103
-
70
200
2
-
> 50 (92)
20.3
112.63
49.90
-
2
5
-
4d
8.2
5×103
4k
0.02
0.2×103
ADR
-
208
209
Table 5 In vitro toxicity measured on normal monkey kidney cells and HEPG2 cell lines.
GI50 = concentration of drug causing 50% inhibition of cell growth; LC50 = concentration of
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drug causing 50% cell kill; TGI = concentration of drug causing total inhibition of cell
growth; ADR = adriamycin, positive control.
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3 Materials and Methods
All solvents and reagents used for the synthesis were of general reagent grade. All reactions
were monitored by thin layer chromatography with Merck pre-coated silica plates (GF254).
Column chromatography was used to purify the final products. Melting points were recorded
using a Büchi capillary melting point apparatus and are uncorrected. Infrared spectra were
recorded (KBr disc method) on a Jasco FT-IR 5300 spectrophotometer. NMR spectra were
recorded on a Bruker Avance-II model with H frequency of 400 MHz and 13C resonance
1
frequency of 100 MHz. Chemical shifts are reported in parts per million (ppm) downfield
from tetramethylsilane (TMS) used as the internal standard. Mass spectra were recorded on
an Agilent Technologies 1260 Infinity LC equipped with an Agilent Technologies 6120
Quadrupole mass spectrometer. Purity was evaluated by reversed-phase HPLC (RP-HPLC)
on an Agilent Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm column with a flow rate of 1.5
mL/min. Compounds were eluted with gradient mixtures of A) 0.1% formic acid in water
and B) Methanol. Compound purity was confirmed by elution with linear gradients of 95%
A and 5% B for the initial 3.5 min, which was switched to 100% B for the 3.5 min to 7.5 min
interval and finally to 95% A and 5% B for the last 7.5 to 10 min period. HPLC data are
reported with percentage purity in parentheses following the retention time.
230
3.1 Computational details
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Shape based similarity studies were carried out with the ROCS module in the Open Eye
Scientific Software running on the Windows platform. ROCS can rapidly compare and rank
molecules based on the three-dimensional shape similarity. To begin, various conformations
of the reference molecule BM212 were generated using the confgen module in Schrödinger
suite 2014, employing the OPLS 2005 forcefield to define the atom types, bond length, bond
angles, torsions, improper and the intra-molecular non-bonded terms. The various
conformations of BM212 were used to screen molecules by shape as the yard stick. Those
closely related in shape and volume to BM212 were selected for structural optimization.
Shape based screening was carried out on a database curated from the literature that
comprised of different five membered, six membered and fused heterocyclic molecules. Of
the various conformations generated for BM212, the five lowest energy structures were used
as the query for shape-based screening. The TSSC was used to rank molecules which were
then selected based on their Tanimoto score, synthetic feasibility and drug like properties.
The last attribute i.e. drug likeness was assessed with the QikProp module in the Schrödinger
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suite (Schrödinger LLC, New York, 2014).
3.2 Biological evaluation
3.2.1 Microplate Alamar Blue Assay and cytotoxicity studies -
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All compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis
H37Rv by the Microplate Alamar Blue assay (MABA) according to the well-established
protocol, while cytotoxicity studies against Normal monkey kidney cells were carried out as
per the protocol published by Kanyawim Kirtikara et. Al17.
HepG2 Cytotoxicity Assay
Cell seeding was done by taking 100-200 µl of desired cell suspension (human
Hepatocarcinoma Cell Line HepG2) in a 96-well plate at required cell density (25,000-50,000
cells per well), without the test agent. Cells were then allowed to adhere to the culture plate
for about 24 hours. The plate was then incubated at 37oC in a 5% CO2 atmosphere for 24
hours. After 24 hours the growth medium was removed and freshly prepared compound
solutions were serially diluted (300µM, 100µM, 50µM, 10µM, 1µM 0.1µM). 100µl of each
dilution in a 96 –well plate, in triplicates, were seed. After the incubation period of 24 hours,
the plates were removed from the incubator and MTT reagent added to a final concentration
of 10% of the total volume. This volume was the same volume that was used while
determining the optimum cell density. The plate was wrapped in an aluminum foil to avoid
exposure to light and put back into the incubator for 2 to 4 hours. The culture medium was
then aspirated without disturbing the monolayer. Solubilization solution was then added
(100% DMSO) in an amount equal to the culture volume and the solution stirred gently in a
gyratory shaker to enhance the dissolution. The absorbance was read on a Spectostar Nano
ELISA plate reader at 570 nm and IC50 values were calculated.
268
3.2.2 Antimicrobial and antifungal assay
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276
Primary antimicrobial screening by whole cell growth inhibition was done in duplicate (n =
2) at a single concentration of 32 µg/mL. The study was carried out against five bacteria,
namely, Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603),
Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 27853) and
Staphylococcus aureus (ATCC 43300), and two fungi: Candida albicans (ATCC 90028) and
Cryptococcus neoformans (ATCC 208821). Samples were made up to 10 mg/mL in DMSO
or water and stored frozen at -20°C. An aliquot of each sample was diluted to 320 µg/mL in
water, and plated in 384-well polypropylene plates (PP). Five µL was plated in duplicate
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(n=2) into a 384-well non-binding surface plate (NBS) for each strain or cell type assayed
against. Once cells were added, the final compound concentration was 32 µg/mL and DMSO
concentration was 0.3%. All bacteria were cultured in cation-adjusted Mueller Hinton broth
(CAMHB) at 37°C overnight. A sample of each culture was then diluted 40-fold in fresh
broth and incubated at 37°C for 1.5-3 h. The resultant mid-log phase cultures were diluted
(CFU/mL measured by OD600), then 45 µL was added to each well of the compound
containing plates, giving a cell density of 5×105 CFU/mL and the nominated final compound
concentration. All plates were covered and incubated at 37°C for 18 h without shaking.
Inhibition of bacterial growth was determined by measuring absorbance at 600 nm (OD600),
using a Tecan M1000 Pro monochromator plate reader. The percent growth inhibition was
calculated for each well, using the negative (media only) and positive controls (bacteria
without inhibitors) on the same plate as references. The significance of the inhibition values
was determined by Z-scores, calculated using the average and standard deviation of the
sample wells (no controls) on the same plate. Samples with inhibition value above 50% and
Z-Score above 2.5 for either replicate (n=2 on different plates) were classified as actives. For
antifungal studies, fungal strains were cultured for 3 days on Yeast Extract-Peptone Dextrose
(YPD) agar at 30°C. A yeast suspension of 1 x 106 to 5 x 106 cells/mL (as determined by
OD530) was prepared from five colonies. These stock suspensions were diluted with Yeast
Nitrogen Base (YNB) broth to a final concentration of 2.5 ×10 CFU/mL. Then, 45 µL of the
fungi suspension was added to each well of the compound-containing plates, giving a final
concentration of 32 µg/mL for the tested samples. Plates were covered and incubated at 35°C
for 24 h without shaking. Inhibition of the growth of C. albicans was determined by
measuring absorbance at 530 nm (OD530), while the inhibition of the growth of C.
neoformans was determined by measuring the difference in absorbance between 600 and 570
nm (OD600-570), after the addition of resazurin (0.001% final concentration) and incubation
at 35°C for additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate
reader. The percent growth inhibition was calculated for each well, using the negative (media
only) and positive controls (fungi without inhibitors) on the same plate. The significance of
the inhibition values was determined by Z-scores, calculated using the average and standard
deviation of the sample wells (no controls) on the same plate. Samples with inhibition value
above 50% and Z-Score above 2.5 for either replicate (n=2 on different plates) were
classified as actives. Colistin and vancomycin were used as positive bacterial inhibitor
standards for Gram negative and Gram-positive bacteria, respectively. Fluconazole was used
as the positive fungal inhibitor standard for C. albicans and C. neoformans. The antibiotics
19

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were provided in 4 concentrations, with 2 above and 2 below its MIC value, and plated into
the first 8 wells of column 23 of the 384-well NBS plates. The quality control (QC) of the
assays was determined by the antimicrobial controls and the Z’-factor (using positive and
negative controls). Each plate was deemed to fulfill the quality criteria (pass QC), if the Z’-
factor was above 0.4, and the antimicrobial standards showed full range of activity, with full
growth inhibition at their highest concentration, and no growth inhibition at their lowest
concentration. All antibiotic controls displayed inhibitory values within the expected range.
3.3 Synthesis
3.3.1 1-(4-Chlorophenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4a): In a round
bottom flask 1 mmol. of 1-fluoro-2-nitrobenzene (1) and 1 mmol. of p-chloroaniline (2) in 2
ml of DMSO were heated for 2 h at 130°C. The completion of this step was verified by TLC.
Then 1.5 mmol. of sodium dithionite and 1.2 mmol. of p-anisaldehyde (3) were added and
heating was continued for 1 h. After completion of the reaction (monitored by TLC), water
(10 mL) was added to the mixture and the solution was extracted with EtOAc (3 ×15 ml).
The combined organic layer was washed with brine (5 ml) and dried over anhydrous Na2SO4.
The residue obtained after evaporation of the organic solvent was purified by column
chromatography with petroleum ether and ethyl acetate (7:3) as the mobile phase; this yielded
1
the titled compound as light brown solid. (101.5 mg, 72%); m.p. 174-176ºC; H-NMR (400
MHz, CDCl3): δ = 3.82 (s, 3H), 6.83-6.87 (m, 2H), 7.19-7.28 (m, 4H), 7.31-7.35 (m, 1H),
7.46-7.51 (m, 4H), 7.84-7.86 ppm (d, J = 7.9 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ =
160.68, 152.32, 143.01, 136.94, 135.72, 134.35, 130.93, 130.15, 128.72, 123.23, 123.11,
121.96, 119.93, 113.93, 110.04, 55.30 ppm; IR (KBr): ν = 2924, 1269, 1300, 754 cm-1; MS
(EI, 70eV) m/z (%): 335.0 (100%), 337.0 (35%) [M + H ]+; HPLC tR = 4.03 mins (98.06%).
3.3.2 1-Phenyl-2-(3-hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole (4b): Light brown
solid (84.6 mg, 60%); m.p. 200-202ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.81 (s, 3H), 5.79
(s, 1H), 6.69-6.71 (d, J = 8.4Hz, 1H), 7.02 (dd, J= 2.0, 8.4Hz, 1H), 7.07-7.08 (d, J = 2.1Hz,
1H), 7.13-7.46 (m, 8H), 7.78-7.80 ppm (d, J= 7.9Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ =
152.04, 147.95, 146.94, 142.44, 136.94, 136.82, 129.75, 128.41, 127.35, 122.55, 122.31,
120.40, 118.73, 115.11, 112.72, 109.96, 55.09 ppm; IR (KBr): ν = 3392, 2936, 1305, 1256,
746, 1491 cm-1; MS (EI, 70eV) m/z (%): 317.1 (100) [M + H ]+; HPLC tR = 3.31 mins
(94.35%).
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343
3.3.3 1-(4-Chlorophenyl)-2-(2,4-dimethoxyphenyl)-1H-benzo[d]imidazole (4c): Light
1
brown solid (91.7 mg, 65%); m.p. 96-98ºC; H-NMR (400 MHz, CDCl3): δ = 3.33 (s, 3H),
20

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349
350
3.83 (s, 3H), 6.28-6.29 (d, J=2.2 Hz, 1H), 6.57 (dd, J = 2.2, 5.0 Hz, 1H), 7.17-7.19 (m, 2H),
7.26-7.31 (m, 1H), 7.31-7.32 (m, 2H), 7.34-7.38 (m, 2H), 7.57-7.59 (d, J = 8.4 Hz, 1H), 7.86
ppm (dd, J = 1.3, 8.9 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ = 162.65, 157.89, 151.31,
143.07, 136.04, 135.01, 133.17, 133.06, 129.25, 127.21, 123.16, 122.71, 119.87, 112.14,
109.96, 105.05, 98.37, 55.45, 54.71, 31.95, 29.72 ppm; IR (KBr): ν = 2925, 1494, 1211, 583,
1306 cm-1; MS (EI, 70eV) m/z (%): 365.1 (100%), 367.1 (35%) [M + H ]+; HPLC tR = 3.84
mins (99.25%).
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357
3.3.4 1-(4-Bromophenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4d): Light brown
solid (86.0 mg, 61%); m.p. 186-188ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.82 (s, 3H), 6.84-
6.87 (m, 2H), 7.19-7.27 (m, 4H), 7.31-7.35 (m, 1H), 7.47-7.51 (m, 2H), 7.62-7.65 (m, 2H),
7.84-7.86 ppm (d, J = 7.9 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ = 160.67, 152.27, 143.06,
136.86, 136.24, 133.14, 130.93, 129.01, 123.24, 123.12, 122.31, 121.95, 119.73, 113.94,
110.05, 55.32 ppm; IR (KBr): ν = 3048, 1481, 1253, 1107, 523 cm-1; MS (EI, 70eV) m/z (%):
379.0 (100%), 381.0 (85%) [M + H ]+; HPLC tR = 4.08 mins (99.26%).
358
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365
3.3.5 1-(2,5-dimethylphenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4e): Light
brown solid (80.4 mg, 57%); m.p. 166-168ºC; 1H-NMR (400 MHz, CDCl3): δ = 1.84 (s, 3H),
2.36 (s, 3H), 3.79 (s, 3H), 6.79-6.82 (m, 2H), 6.96-6.98 (d, J = 8.0 Hz, 1H), 7.18-7.20 (m,
1H), 7.22-7.22 (m, 1H), 7.24-7.28 (m, 2H), 7.30-7.32 (m, 1H), 7.54-7.58 (m, 2H), 7.84-7.86
ppm (d, J = 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ = 160.54, 152.20, 142.97, 137.37,
137.20, 135.96, 132.87, 131.43, 130.19, 130.07, 128.90, 122.83, 122.75, 122.66, 119.39,
113.83, 110.41, 55.25, 29.73, 20.91, 17.09 ppm; IR (KBr): ν = 2921, 1476, 1321, 1250 cm-1;
MS (EI, 70eV) m/z (%): 329.1(100) [M + H ]+; HPLC tR= 3.98 mins (96.31%).
366
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3.3.6 1-(3-Chlorophenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4f): White solid
1
(56.4 mg, 40%); m.p. 202ºC; H-NMR (400 MHz, CDCl3): δ = 3.82 (s, 3H), 6.84-6.86 (d,
2H, J = 8.8 Hz), 7.17-7.47 (m, 6H), 7.47-7.47 (m, 1H), 7.49-7.51 (m, 2H), 7.84-7.86 ppm (d,
J = 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ = 160.70, 152.26, 143.03, 130.37, 136.09,
135.41, 130.91, 128.81, 127.55, 125.90, 123.28, 123.17, 121.91, 119.73, 113.94, 110.08,
55.32 ppm; IR (KBr, cm−1): ν = 3064, 1478, 1250, 1181, 613 cm-1; MS (EI, 70eV) m/z (%):
335.0 (100%), 337.1 (35%) [M + H ]+; HPLC tR = 4.04 mins (98.57%).
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3.3.7 1-(4-Fluorophenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4g): Light brown
solid (88.8 mg, 63%); m.p. 156-158ºC; 1H-NMR (400 MHz,CDCl3): δ = 3.81 (s, 3H), 6.82-
6.85 (m, 2H), 7.17-7.25 (m, 2H), 7.26-7.34 (m, 5H), 7.47-7.51 (m, 2H), 7.84-7.86 ppm (d, J =
21

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379
8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ = 163.41, 160.93, 160.61, 152.43, 142.96,
137.23, 133.20, 133.16, 130.89, 129.30, 129.21, 123.15, 123.01, 122.08, 119.66, 117.07,
116.84, 113.87, 110.09, 55.30 ppm; IR (KBr): ν = 2962, 1475, 1266, 1248, 1054 cm-1; MS
(EI, 70eV) m/z (%): 319.1 (100) [M + H ]+; HPLC tR= 3.76 mins (98.17%).
380
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3.3.8 1-(4-Chlorophenyl)-2-(3-methoxyphenyl)-1H-benzo[d]imidazole (4h): Light brown
solid (80.4 mg, 57%); m.p. 90-92ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.74 (s, 3H), 6.90-6.93
(m, 1H), 7.01-7.36 (m, 8H), 7.45-7.49 (m, 2H), 7.87-7.89 ppm (d, J = 8.0 Hz, 1H); 13C-NMR
(100 MHz, CDCl3): δ = 159.48, 152.10, 142.93, 136.96, 135.57, 134.44, 130.83, 130.14,
129.47, 128.66, 123.66, 123.26, 121.08, 120.03, 116.21, 114.23, 110.23, 55.29 ppm; IR
(KBr): ν = 3053, 1491, 1322, 1271, 746 cm-1; MS (EI, 70eV) m/z (%): 335.1 (100%), 337.1
(35%) [M + H ]+; HPLC tR= 4.11 mins (98.20%).
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3.3.9 1-(4-Isopropylphenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4i): Off white
solid (73.3 mg, 52%); m.p. 154-156ºC; 1H-NMR (400 MHz, CDCl3): δ = 1.31-1.32 (s, 6H),
2.99-3.02 (m, 1H), 3.80 (s, 3H), 6.81-6.84 (m, 2H), 7.21-7.26 (m, 4H), 7.29-7.32 (m, 1H),
7.33-7.35 (m, 2H), 7.51-7.53 (m, 2H), 7.84-7.86 ppm (d, J = 7.9 Hz, 1H); 13C-NMR (100
MHz, CDCl3): δ = 160.45, 152.44, 149.34, 142.98, 137.41, 134.71, 130.90, 127.83, 127.26,
122.87, 122.74, 122.50, 119.47, 113.71, 110.46, 55.27, 33.86, 23.96 ppm; IR (KBr): ν =
2939, 1503, 1378, 1281 cm-1; MS (EI, 70eV) m/z (%): 343.1 (100) [M + H ]+; HPLC tR =
4.16 mins (98.69%).
395
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400
401
402
3.3.10 1-(3-Methoxyphenyl)-2-(4-methoxyphenyl)-1H-benzo[d]imidazole (4j): Off white
solid (88.8 mg, 63%); m.p. 152-154ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.78 (s, 3H), 3.81
(s, 3H), 6.82-7.02 (m, 5H), 7.23-7.26 (m, 2H), 7.29-7.34 (m, 1H), 7.38-7.42 (m, 1H), 7.54
(dd, J = 2.1, 6.8 Hz, 2H), 7.84-7.86 ppm (d, J = 8.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ
= 160.61, 160.54, 152.32, 142.90, 138.24, 137.19, 130.83, 130.59, 123.0, 122.86, 122.35,
119.75, 119.53, 114.26, 113.77, 113.09, 110.38, 55.53, 55.28 ppm; IR (KBr): ν = 2959, 1492,
1477, 1251 cm-1; MS (EI, 70eV) m/z (%): 331.1 (100) [M + H ]+; HPLC tR = 3.76 mins
(98.04%).
403
404
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407
3.3.11 1-(4-Chlorophenyl)-2-(3,4-dimethoxyphenyl)-1H-benzo[d]imidazole (4k): Light
brown solid (77.6 mg, 55%); m.p. 162-164ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.88 (s, 3H),
3.97 (s, 3H), 6.85-6.87 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 2.0, 8.4 Hz, 1H), 7.28-7.37 (m, 2H),
7.36-7.45 (m, 4H), 7.57-7.61 (m, 2H), 7.95-7.97 ppm (d, J = 7.9 Hz, 1H); 13C-NMR (100
MHz, CDCl3): δ = 152.54, 150.22, 148.74, 142.96, 137.01, 135.85, 134.41, 130.15, 128.02,
22

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123.32, 123.15, 122.47, 122.07, 119.72, 112.33, 110.67, 110.05, 55.87, 55.80, 29.71 ppm; IR
(KBr): ν = 2923, 1493, 1282, 1312, 765 cm-1; MS (EI, eV) m/z (%): 365.1 (100%), 367.1
(38%) [M + H ]+; HPLC tR = 3.93 mins (97.67%).
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420
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422
3.3.12 2-(4-Methoxyphenyl)-1,4-diphenyl-1H-imidazole (8a): A round bottom flask
charged with1 mmol of phenacyl bromide (5), 1 mmol of aniline (6), 1 mmol of p-
anisaldehyde (7) and 1.5 mmol of ammonium acetate were heated at 130°C for 2 h. After
completion of the reaction (monitored by TLC), the reaction mixture was cooled to room
temperature. The compounds were purified by column chromatography using petroleum
ether and ethyl acetate (7:3) as the mobile phase; this yielded the titled compound as light
brown solid (143.3 mg, 72%); m.p.196-198ºC; 1H-NMR (400 MHz, CDCl3): δ = 3.78 (s, 3H),
6.79 (dd, J = 1.8, 7.0 Hz, 2H), 7.24-7.28 (m, 3H), 7.37-7.43 (m, 8H), 7.87-7.89 ppm (m, 2H);
13C-NMR (100 MHz, CDCl3): δ = 49.99, 108.42, 112.89, 117.67, 119.78, 120.64, 121.68,
122.84, 123.35, 124.23, 124.96, 128.70, 133.39, 136.23, 141.75, 154.52 ppm; IR (KBr): ν =
2933, 1528, 1292, 1274 cm-1; MS (EI, 70ev) m/z (%): 327.1 (100) [M + H ]+, HPLC tR = 3.25
mins (88.85%).
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3.3.13 2-(4-hydroxyphenyl)-1,4-diphenyl-1H-imidazole (8b): Light brown solid (131.3 mg,
66%); m.p. 233-234ºC; 1H-NMR (400 MHz, CDCl3): δ = 1.60 (s, 1H), 6.63 (dd, J = 2.0, 6.7
Hz, 2H), 7.21-7.43 (m, 11H), 7.85-7.87 ppm (m, 2H); 13C-NMR (100 MHz, CDCl3): δ =
157.64, 146.46, 140.15, 138.16, 133.91, 129.66, 129.21, 128.22, 127.81, 126.33, 125.57,
124.38, 120.90, 118.38, 114.87 ppm; IR (KBr): ν = 3422, 2924, 1493,1269, 1172 cm-1; MS
(EI, 70eV) m/z (%): 313.1 (100) [M + H ]+; HPLC tR = 3.61 mins (99.64%).
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430
3.3.14 2-(4-Fluorophenyl)-3-(morpholine-1-ylmethyl)imidazo[1,2-a]pyridine (12a):
Step 1 Synthesis of 4-Fluorophenyl-imidazo[1,2-a]pyridine (11)
431
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In a round bottom flask, 2-aminopyridine (9) (1 mmol.) and 4-fluorophenacyl chloride (10)
(1.5 mmol.) were added in 5 mL of ethanol and refluxed for 4-6 h. After cooling the reaction
mixture, ethanol was removed in vacuo and the residue was treated with saturated NaHCO3
solution (10 ml) and extracted with CHCl3 (3×10 mL). The combined organic phases were
dried over anhydrous Na2SO4. Removal of the solvent under reduced pressure gave the crude
product which was purified by column chromatography using petroleum ether and ethyl
acetate (7:3) as the mobile phase.
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Step 2 Synthesis of 2-(4-fluorophenyl)-3-(morpholine-1-ylmethyl) imidazo[1,2-a]
pyridine (12)
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To a solution of 4-fluorophenyl-imidazo[1,2-a]pyridine (11) (1 mmol) in acetic acid (5 ml),
morpholine (1.5 mmol) and ~ 36% formalin solution (1.5 mmol) were added slowly and
stirred at 25-35°C until the reaction was complete. The reaction mixture was cooled to 0-
10°C and the pH adjusted between 8-9 with 20% aqueous sodium hydroxide solution. The
solid which precipitated out was filtered, washed with water, dried and purified by column
chromatography using petroleum ether and ethyl acetate (7:3) as the mobile phase; this
yielded the title compound as a white solid (173.8 mg, 82%); m.p. 176-178ºC; 1H-NMR (400
MHz, CDCl3) δ = 2.48 ppm (t, J = 4.4 Hz, 4H), 3.68 ppm (t, J = 4.5 Hz, 4H), 3.94 ppm (s,
2H), 6.86-6.87 (m, 1H), 7.13-7.18 (m, 2H), 7.22-7.26 (m, 1H), 7.62-7.64 (d, J = 9.0Hz, 1H),
7.76-7.80 (m, 2H), 8.41-8.43 ppm (d, J = 6.9Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ =
163.02, 161.36, 145.04, 144.93, 144.38, 143.19, 130.56, 130.47, 130.44, 130.17, 130.09,
125.21, 125.09, 124.80, 124.47, 119.44, 117.21, 117.17, 115.79, 115.55, 115.47, 115.34,
115.26, 112.28, 112.05, 66.95, 53.75, 53.18, 52.02 ppm; IR (KBr): ν = 2965, 1501, 1374,
1358, 1112 cm-1; MS (EI, 70eV) m/z (%): 312.1(100) [M + H ]+; HPLC tR = 2.41 mins
(99.68%).
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457
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459
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3.3.15. 2-(4-Fluorophenyl)-7-methyl-3-(morpholine-1-ylmethyl)imidazo[1,2-a]pyridine
(12b): White solid (180.8 mg, 80%); m.p. 196-198ºC; 1H-NMR (400 MHz, CDCl3) δ = 2.42
(s, 3H), 2.46 (t, J = 4.3 Hz, 4H), 3.67 (t, J = 4.5 Hz, 4H), 3.91 (s, 2H), 6.66-6.68 (m, 1H),
7.12-7.16 (m, 2H), 7.38 (s, 1H), 7.75-7.78 (m, 2H), 8.27-8.29 ppm (d, J = 7 Hz, 1H); 13C-
NMR (100 MHz, CDCl3): δ = 163.73, 161.20, 145.51, 144.13, 135.66, 130.78, 130.74,
130.49, 130.41, 124.38, 115.64, 115.50, 115.28, 115.16, 114.63, 66.99, 53.17, 52.05, 21.37
ppm; IR (KBr): ν = 3070, 1503, 1375, 1358, 1114 cm-1; MS (EI, 70eV) m/z (%): 326.1 (100)
[M + H ]+; HPLC tR = 2.57 mins (98.42%).
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3.3.16. 2-(4-Fluorophenyl)-6-methyl-3-(morpholine-1-ylmethyl)imidazo[1,2-a]pyridine
(12c): White solid (169.5 mg, 75%); m.p. 150-151ºC; 1H-NMR (400 MHz, CDCl3): δ = 2.38
(s, 3H), 2.48 (t, J = 4.5 Hz, 4H), 3.68 (t, J = 4.6 Hz, 4H), 3.90 (s, 2H), 7.08 (dd, J = 1.6,
9.1Hz, 1H), 7.12-7.16 (m, 2H), 7.52-7.54 (d, J = 9.1Hz, 1H), 7.77-7.80 (m, 2H), 8.15 ppm (s,
1H); 13C-NMR (100 MHz, CDCl3): δ = 163.74, 161.29, 144.31, 144.14, 130.79, 130.76,
130.50, 130.42, 127.84, 122.65, 121.61, 116.60, 115.50, 115.20, 66.99, 53.20, 51.99, 10.55
ppm; IR (KBr): ν = 2925, 1503, 1377, 1341, 1112 cm-1; MS (EI, 70eV) m/z (%): 326.1(100)
[M + H ]+; HPLC tR = 2.59 mins (98.93%).
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3.3.17 7-Methyl-2-phenyl-3-(morpholine-1-ylmethyl)imidazo[1,2-a]pyridine (12d): White
solid (151.8 mg, 73%); m.p. 138-140ºC; 1H-NMR (300 MHz, CDCl3): δ = 2.36 (s, 3H), 2.46
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(t, J = 9.0 Hz, 4H), 3.66 (t, J = 6.0 Hz, 4H), 3.95 (s, 2H), 6.71-6.75 (m, 1H), 7.37-7.50 (m,
4H), 7.75-7.80 (m, 2H), 8.32-8.36 ppm (d, J = 12.0 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ
= 144.62, 143.38, 134.96, 134.52, 128.20, 128.16, 128.09, 128.05, 127.23, 127.15, 124.62,
115.38, 114.84, 114.77, 114.11, 66.22, 52.66, 51.09, 20.83 ppm; IR (KBr): ν = 2943, 1504,
1358 cm-1; MS (EI, 70eV) m/z (%): 308.2(100) [M + H ]+; HPLC tR = 2.50 mins (98.55%).
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3.3.18 2-Phenyl-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyridine (12e): White solid (128
1
mg, 66%); m.p. 127-128ºC; H-NMR (300 MHz, CDCl3): δ = 1.43-1.61 (m, 10H), 3.92 (s,
2H), 6.79-6.82 (m, 1H), 7.18-7.23 (m, 1H), 7.34-7.38 (m, 1H), 7.44-7,47 (m, 2H), 7.62-7.64
(d, J = 9.0 Hz, 1H), 7.81-7.83 (m, 2H), 8.49-8.51 ppm (d, J = 8.1 Hz, 1H); 13C-NMR (100
MHz, CDCl3): δ = 144.11, 144.03, 143.76, 142.95, 134.50, 134.28, 128.29, 128.22, 128.15,
128.09, 127.37, 127.20, 125.47, 124.86, 124.63, 124.28, 120.32, 116.06, 116.52, 116.39,
111.78, 111.47, 53.58, 52.33, 51.60, 25.59, 23.91 ppm; IR (KBr): ν = 2915, 1504, 1358 cm-1;
MS (EI, 70eV) m/z (%): 292.2(100) [M + H ]+; HPLC tR = 1.72 mins (98.42%).
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3.3.19 7-methyl-2-phenyl-3-(piperidin-1-ylmethyl)imidazo[1,2-a]pyridine (12f): White
1
solid (166.4 mg, 80%); m.p. 70-72ºC; H-NMR (300 MHz, CDCl3): δ = 1.41-1.52 (m, 6H),
1.80 (m, 4H), 2.40 (s, 3H), 3.87 (s, 2H), 6.63 (m, 1H), 7.43-7.5 (m, 4H), 7.80 (m, 2H), 8.36
ppm (m, 1H); 13C-NMR (100 MHz, CDCl3): δ = 145.40, 144.34, 135.30, 134.84, 128.86,
128.34, 127.44, 124.89, 116.51, 115.44, 114.29, 54.22, 52.20, 26.10, 24.44, 21.37 ppm; IR
(KBr): ν = 2937, 1502, 1340, 1361 cm-1; MS (EI, 70eV) m/z (%): 306.2 (100) [M + H ]+;
HPLC tR = 1.86 mins (99.42%).
493
494
495
496
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500
3.3.20 2-Phenyl-3-(morpholine-1-ylmethyl)imidazo[1,2-a]pyridine (12g): White solid
(163.0 mg, 84%); m.p.141-143ºC; 1H-NMR (300 MHz, CDCl3): δ = 2.49 (t, 4H, J = 4.4 Hz ),
3.68 (t, J = 4.6 Hz, 4H), 3.99 (s, 2H), 6.82-6.86 (m, 1H), 7.21-7.26 (m, 1H), 7.36-7.40 (m,
1H), 7.45-7.49 (m, 2H), 7.63-7.66 (m, 1H), 7.79-7.81 (m, 2H), 8.44 ppm (dd, J = 1.0, 5.7Hz,
1H); 13C-NMR (100 MHz, CDCl3): δ = 145.31, 145.10, 134.43, 128.88, 128.57, 128.47,
128.41, 127.78, 125.32, 124.60, 117.25, 115.91, 111.90, 66.98, 53.20, 52.11 ppm; IR (KBr):
ν = 2859, 1499, 1354, 1268 cm-1; MS (EI, 70eV) m/z (%): 294.1(100) [M + H ]+; HPLC tR =
2.30 mins (95.32%).
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4 Conclusions
In our efforts to identify novel antitubercular agents, we adopted BM212 as the lead molecule
and identified imidazoles, benzimidazoles and imidazopyridines as being similar to BM212
based on shape/volume features. With the scaffold hopping approach we designed and
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synthesized a small library of 20 molecules belonging to the three structurally diverse
heterocycles, namely 2,3-disubstituted benzimidazole, 1,2,4-trisubstituted imidazole, and 2,3-
disubstituted imidazopyridine. These molecules were screened against Mycobacterium
tuberculosis and the 2,3-disubstituted benzimidazoles emerged as the most active
antitubercular agents; the most potent molecule (4a) in this series has an MIC of 2.3 µg/ml.
It is reassuring to note that molecule 4a (MIC 2.3 µg/ml) identified in this work is as potent
as the lead molecule BM212 (MIC 0.7 to 1.5 µg/ml), however it is superior to BM212 in that
it shows no toxicity in VERO as well as HEPG2 cell lines and is also not metabolized by rat
liver microsome. The antimicrobial and antifungal activity shows 4a to be selective against
Mycobacterium tuberculosis.
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Acknowledgements
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The authors are thankful to the Council of Scientific and Industrial Research, New Delhi (File
no. 02(0047)/12/EMR-II), Department of Biotechnology, New Delhi (File No.
BT/PR11810/BRB/10/690/2009) and Department of Science and technology, New Delhi
through their FIST program (SR/FST/LSI-163/2003) for providing computational and
infrastructural facilities. Antimicrobial screening was performed by CO-ADD (The
Community for Antimicrobial Drug Discovery) funded by the Wellcome Trust (UK) and The
University of Queensland (Australia). In vitro SRB assay for cytotoxicity studies was done at
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Anti-Cancer Drug screening facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi
Mumbai, India. The HEPG2 toxicity studies was carried out at Genelon Institute of Life
Sciences Pvt. Ltd., Bengaluru, India.
28