Synthesis, in vitro antimycobacterial evaluation and docking studies of some new 5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one schiff bases

Article abstract of DOI:10.1016/j.bmcl.2015.12.083

Development of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) has been considered as major health burden, globally. In order to develop novel, potential molecules against drug resistant TB, twenty two (22) new 3-substituted-7-benzyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a-k) and 3-substituted-7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a-k) derivatives were designed and synthesized by using appropriate synthetic protocols. Pantothenate synthetase (PS) was considered as the target for the molecular docking studies and evaluated the binding pattern at active site, as PS plays a significant role in the biosynthesis of pantothenate in Mycobacterium tuberculosis (MTB). The preliminary in vitro antibacterial screening of test compounds was carried out against two strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The antimycobacterial screening was performed against MTB H37Rv and an isoniazid-resistant clinical isolate of MTB. The compounds 6b, 6c, 6d, 6k, 7b, 7c, 7d and 7k exhibited promising antibacterial activity MIC in the range of 15-73 μM against all bacterial strains used and compounds 6d and 7b showed antimycobacterial activity (IC₅₀<340 μM in LRP assay) and (MIC <9 μM in broth microdilution method).

Full text of DOI:10.1016/j.bmcl.2015.12.083

Accepted Manuscript
Synthesis, In Vitro Antimycobacterial Evaluation and Docking Studies of Some
New 5,6,7,8-Tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4(3H)-one
Schiff Bases
Narender Malothu, Jaswanth S. Bhandaru, Umasankar Kulandaivelu, Malathi
Jojula, Raghuram Reddy Adidala, K.R. Umadevi, V.N. Azger Dusthackeer,
Venkat Rao Kaki, Raghuram R. Akkinepally
PII:
S0960-894X(15)30397-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.083
BMCL 23444
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 July 2015
17 December 2015
24 December 2015
Please cite this article as: Malothu, N., Bhandaru, J.S., Kulandaivelu, U., Jojula, M., Adidala, R.R., Umadevi, K.R.,
Azger Dusthackeer, V.N., Kaki, V.R., Akkinepally, R.R., Synthesis, In Vitro Antimycobacterial Evaluation and
Docking Studies of Some New 5,6,7,8-Tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4(3H)-one Schiff Bases,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.083
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Synthesis, In Vitro Antimycobacterial Evaluation and Docking
Studies of Some New 5,6,7,8-Tetrahydropyrido[4',3':4,5]thieno[2,3-
d]pyrimidin-4(3H)-one Schiff Bases
Narender Malothua, Jaswanth S Bhandarua, Umasankar Kulandaivelub, Malathi Jojulac,
Raghuram Reddy Adidalac, Umadevi K Rd, Azger Dusthackeer V Nd, Venkat Rao Kakie,
Raghuram R Akkinepallya,*
aMedicinal Chemistry Division, University College of Pharmaceutical Sciences, Kakatiya
b
University, Warangal, India; Medicinal Chemistry Division, Vaagdevi College of Pharmacy,
c
Warangal, India; Department of Microbiology, Sri Shivani College of Pharmacy, Warangal,
d
India; Bacteriology Division, National Institute for Research in Tuberculosis (NIRT), Chennai,
e
India; Pharmaceutical Chemistry Division, School of Pharmacy, International Medical
University, Kualalumpur, Malaysia.
*Corresponding Author:
Prof. A. Raghuram Rao
Department of Medicinal Chemistry
University College of Pharmaceutical Sciences
Kakatiya University, Warangal – 506 009
Phone: +918790432456
E-mail address: raghumed@kakatiya.ac.in
1

Abstract
Development of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis
(TB) has been considered as major health burden, globally. In order to develop novel, potential
molecules against drug resistant TB, twenty two (22) new 3-substituted-7-benzyl-5,6,7,8-
tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a-k) and 3-substituted-7-benzyl-
2-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a-k) derivatives
were designed and synthesized by using appropriate synthetic protocols. Pantothenate synthetase
(PS) was considered as the target for the molecular docking studies and evaluated the binding
pattern at active site, as PS plays a significant role in the biosynthesis of pantothenate in
Mycobacterium tuberculosis (MTB). The preliminary in vitro antibacterial screening of test
compounds was carried out against two strains of Gram-positive (Bacillus subtilis and
Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsialla pneumoniae)
bacteria. The antimycobacterial screening was performed against MTB H37Rv and an isoniazid-
resistant clinical isolate of MTB. The compounds 6b, 6c, 6d, 6k, 7b, 7c, 7d and 7k exhibited
promising antibacterial activity MIC in the range of 15-73 µM against all bacterial strains used
and compounds 6d and 7b showed antimycobacterial activity (IC50 >340 µM in LRP assay) and
(MIC >9 µM in broth microdilution method).
Key words: Pyrido[4',3':4,5]thieno(2,3-d)pyrimidine-4-(3H)-one, antibacterial activity, MIC,
LRP assay, Mycobacterium tuberculosis.
2

Identification and development of promising new chemical entities (NCEs) for infectious
diseases is an urgent need, in the light of enhanced prevalence. Multidrug regimen, long duration
of therapy and existence of multidrug résistance (MDR) are problems associated with grave
consequences in Tuberculosis (TB). This infectious disease is considered as a serious global
public health threat. About a third of world population gets infected with TB every year. It has
become more prevalent due to a variety of factors such as conjunction with AIDS, lowered
immunity and widespread drug resistance. World Health Organization estimated that globally
about 5% (0.48 million) of TB patients developed MDR-TB in 2013. Among them about 9%
were extensively drug resistant. There are as many as about 2, 10,000 deaths in that year.1 The
current TB therapy is limited due to several complications with existing drugs. The difficulty in
development of new drugs in TB therapy can be understood by the fact that no new drug could
be introduced in clinics in the last five decades. Only recently, two molecules, bedaquiline and
delamanide have been approved as new anti-TB drugs.2
Recent literature suggests that pantothenate biosynthetic pathway is a potential drug
target for the development of new drugs for TB. The absence of this pathway in mammalian cells
made it more attractive. This factor is essential for growth of persistent and virulent bacteria.
Pantothenate is a major precursor for the biosynthesis of coenzyme A and acyl carrier protein,
and also essential for many intracellular processes including fatty acid metabolism, cell
signaling, synthesis of polyketides and non-ribosomal peptides. The biosynthetic pathway of
pantothenate involves four steps catalyzed by enzymes encoded by panB, panC, panD and panE
genes. PanC encodes a pantothenate synthetase (PS), which catalyzes the last step of
pantothenate biosynthesis via formation of pantoyl adenylate intermediate (b), the ATP-
dependent condensation of pantoate (a) and β-alanine to form pantothenate (c) (Figure 1).3
3

Figure 1. Reaction catalyzed by pantothenate synthetase in biosynthesis of pantothenate.
Some efforts to synthesize and evaluate specific inhibitors of Mycobacterium
tuberculosis pantothenate synthetase (MTB-PS) are reported in literature such as 6-benzyl-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide derivatives (A), 6-acetyl-4,5,6,7-tetra
hydrothieno[2,3-c]pyridine-3-carboxamide derivatives (B), 2-methyl-N'-benzylideneimidazon
[1,2-a]pyridine-3-carbohydrazide derivatives (C), 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-
c]pyridine derivatives, 5-tert-butyl-N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carbox
amide (Figure 2) etc.4-6 It reveals that the five membered heterofused pyridines were found to be
an essential pharmacophore for exhibiting antimycobacterial activity. In addition, they are also
reported to exhibit a diverse range of biological activities.7-11
4

Figure 2. MTB-PS inhibitors.
In order to develop novel MTB-PS inhibitors as potent antimycobacterial agents,
molecular docking approach has been attempted in pursuit of identifying the potential of our
design by taking the advantage of the availability of 3D-structure of the target protein (PDB:
3IVX). Based on the presence of structural features of one of the lead molecules A,12 two series
of molecules 3-substituted-7-benzyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-
4(3H)-ones (6a-k) and 3-substituted-7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]
thieno[2,3-d]pyrimidin-4(3H)-ones (7a-k) were designed and successfully achieved their
synthesis.
The synthesis of title compounds 6a-k and 7a-k was carried out as depicted in Scheme 1.
The required key intermediate 3 was synthesized by classical Gewald reaction using N-benzyl-4-
piperidone (1), elemental sulphur and ethylcyanoacetate (2) by employing a known procedure
which afforded the product in 85% of yield.13 The formation of 3 was confirmed by physical and
spectral data, and
was converted to an iminoester by heating under reflux in
triethylorthoformate/ triethylorthoacetate, independently which resulted in 4a and 4b
respectively.14 Without further purification these two intermediates (4a and 4b) were cyclized by
using hydrazine hydrate at room temperature in ethanol which afforded the respective products,
5a (65%) and 5b (71%).15 The IR spectra of 5a and 5b showed characteristic bands at 3413,
3304 of N-H stretching for primary amine (3-NH2) and 1672 of C=O stretching for carbonyl
5

group at 4th position of pyrimidine ring. The 1H NMR spectrum showed a singlet at δ = 8.95 ppm
in compound 5a assignable for single proton (2-H of pyrimidinone) and a singlet at δ = 2.59 ppm
in compound 5b assignable for three protons (2-CH3 of pyrimidinone). The presence of a singlet
at the δ = 4.8-4.9 ppm assignable for two protons of free amine (3-NH2), was observed in both of
the spectra.
Scheme 1. General scheme for synthesis of compounds. Reagents and conditions: i, S8,
Morpholine, EtOH, reflux, 2 h; ii, triethylorthoformate/triethylorthoacetate, reflux, 2-4 h; iii,
NH2NH2 H2O, EtOH, rt, overnight; iv, R1CHO, EtOH, CH3COOH, reflux, 12-16 h.
In the final step compounds 5a and 5b were condensed with differently substituted
aromatic aldehydes using catalytic amounts of glacial acetic acid to obtain the respective schiff’s
bases as title compounds 6a-k and 7a-k.16 Formation of 6a-k and 7a-k were confirmed by
6

physical and spectral data. The IR spectra of compound 6a-k and 7a-k showed characteristic
bands at 1642-1684 (C=O stretching) and 1541-1610 (C=N stretching of -N=CH). The
disappearance of both bands at 3413 and 3304 (-NH stretching for primary amine 3-NH2) was
1
conspicuous which revealed the imine formation. The H NMR spectrum for compounds 6a-k
showed characteristic singlet at δ = 8.58-10.18 ppm assignable to imine proton (-N=CH) and
another sharp singlet at δ = 8.21-9.12 ppm assignable to 2-H of pyrimidinone. Compounds 7a-k
showed singlet at δ = 8.58-9.48 ppm assignable to imine proton (-N=CH) and another singlet at δ
= 2.53-2.61 ppm assignable to three methyl protons (2-CH3) of pyrimidinone. The MS spectra
showed the molecular ion (M+ and M++1) peaks as base peak (100% intensity)) for all the
compounds. The presence of isotopic peaks (M+, M++1 and M++2 etc.) in all halogenated
compounds further confirmed the presence of halogen as a substituent, in compounds 6b, 6c, 6k,
7b, 7c and 7k. All the synthesized products 6a-k and 7a-k were characterized based on their
physical and spectral data.
All the title compounds 6a-k and 7a-k were evaluated for a preliminary antibacterial
screening against two strains each of Gram-positive (Bacillus subtilis and Staphylococcus
aureus) and Gram-negative (Escherisia coli and Klebsialla pneumoniae) bacteria to assess the
antibacterial susceptibility of the test compounds.17 In the antibacterial screening the compounds
6a, 6b, 6c, 6d, 6k, 7a, 7b, 7c, 7d and 7k exhibited minimum inhibitory concentration (MIC)
values in the range 15-62 µg/ mL and they exhibited less activity against K. pneumonia MTCC
109 (MIC-31 to 62 µg/ mL). Para substitution on arylideneimino ring with groups like Cl, CN, F
and OCH3 influenced the inhibitory potencies against both Gram-positive and Gram-negative
bacterial strains. The in vitro antibacterial activity (MIC values) data of all the compounds was
summarized in Table 1.
7

TABLE 1
Antibacterial activity data of test compounds 6a-k & 7a-k
MIC (µM)
Comp.
S. aureus B. subtilis
E. coli
K. pneumoniae
Code
MTCC 96 MTCC 441 MTCC 443
MTCC 109
155
71
6a
6b
6c
6d
6e
6f
6g
6h
6i
77
34
73
35
34
149
255
139
36
158
66
77
34
73
35
34
149
255
139
150
158
32
77
16
73
16
16
149
255
139
150
158
66
147
72
72
149
255
139
150
158
66
149
69
71
70
69
144
248
135
297
153
64
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
74
33
71
34
74
33
71
34
74
15
71
15
69
33
33
144
248
135
297
309
64
144
248
135
297
309
15
144
248
135
297
309
64
7j
7k
Std
Ctrl
<1
NA
<1
NA
<1
NA
<1
NA
Std = Streptomycin; Ctrl = DMF in water (1:3); NA = Not active.
Selected compound 6d, 6e, 6i, 7b and 7d were evaluated for antimycobacterial activity using
Luciferase Reporter Phase (LRP) assay on MTB H37Rv and one isoniazid-resistant (INH-
resistant) clinical isolate at National Institute for Research in Tuberculosis (NIRT), Chennai.18
The percentage growth inhibition was recorded at the concentrations of 200, 100 and 50 µg/ mL
(Table 2). Compounds 6d and 7b exhibited IC50 at a lower concentration (below 200 µg/ mL).
Compounds 6e and 7d were found moderately active at this concentration. Looking at the these
8

encouraging results, further biological assays were conducted to determine their MIC values on
different strains of Mycobacterium.
TABLE 2
Antimycobacterial activity data of 6d, 6e, 6i, 7b & 7d by LRP assay
Comp.
Code
6d
Concentration
(µM)
117
CFU
(Col/ mL)
% inhibition
0
9.7
69.89
0
15
16.9
0
235
470
1x 107
116
232
465
6e
6i
9.3 x 106
123
246
492
0
0
8 x 106
8.7 x 106
7.3 x 106
1 x 106
111
223
446
29.6
40.8
99.8
0
7b
113
7d
224
455
6.5
9.8
0
1.4 x 107
1 x 107
-
Ctrl
Ctrl = DMF in water (1:3)
Since our attempt was to know the inhibitory potencies of title compounds 6b, 6d, 6e, 6i,
7b and 7d against both the strains, the MIC ranges were determined in liquid cultures by using
broth microdilution method.19 These compounds were tested at the concentrations of 1250, 625,
312, 156, 78, 39, 19, 9, 4, 2 and 1 µg/ mL in Middlebrook 7H9 medium supplemented with
OADC. Interestingly, two compounds 6d and 7b showed MIC ranges at a concentration of >9
µM (4 µg/ mL) for 14 and 21 days respectively for both the strains (Table 3). The biochemical
screening clearly indicated the absence of viable and identifiable bacteria after 28 days of
incubation. This indicated the bactericidal activity of all the test compounds in addition to
9

potential activity against INH-resistant bacteria. The inhibitory potencies of test compounds were
found more in liquid cultures. The variation in inhibition might be due to the nature of LRP assay
which could be considered as a screening procedure for rapidly testing large number of
compounds. Results of these investigations indicate that some of the test compounds exhibited
significant antimycobacterial activity along with antibacterial activity against all the tested
strains.
TABLE 3
Antimycobacterial activity data of 6b, 6d, 6e, 6i, 7b & 7d by broth
microdilution method
MIC (µM)
Comp.
MTB H37Rv
14 days 21 days
Clinical isolate of MTB
Code
14 days
21 days
6b
6d
20
9
20
9
20
9
20
9
6e
6i
7b
20
22
8
20
22
8
20
22
8
20
22
8
7d
20
0.28
2
4
0.09
NA
20
0.28
2
4
0.09
NA
20
>8
2
4
2
20
>8
2
4
2
Std1
Std2
Std3
Std4
Ctrl
NA
NA
Std1 = Isoniazid; Std2 = Streptomycin; Std3 = Ethambutol.
Std4 = Rifampicin; Ctrl = DMF in water (1:3); NA = Not active.
The binding pattern with target protein was analyzed by using Molegro Virtual Docker
(MVD) software.20 The docking results were evaluated to know the extent and nature of ligand-
protein interactions at the active site. The docking scores of the molecules were taken as key
parameter to evaluate the best docking interactions. Molecules 6b, 6d, 7b and 7d showed higher
docking scores -124.69, -138.87, -130.12 and -130.72 respectively. Binding mode of the ligand
10

molecules with crystal structure of MTB-PS was analyzed and docking poses of ligands 6b, 6d,
7b and 7d at active site are represented in Figure 3.
A
B
Figure 3. The typical binding interactions of ligands 6d (A), 7b (B) (white, stick model, N in
blue, O in red and S in yellow) with active site of MTB-PS, hydrogen bond interactions (blue
dotted lines) of C=O with residue of Gly158.
In this study, all the ligands showed strong hydrogen bond interaction of carbonyl (C=O)
group with Gly158 (Table 4). Further the cyano (CN) group in molecule 6d showed interaction
with Val187. The methyl group of 7b (at 2nd position) showed interaction with His44. Presence
of these additional interactions in molecules 6d and 7b might be the reason for showing good
binding interactions, and it was also comparable with the experimental MIC and MIC50 values.
11

TABLE 4
H-bond interactions of 6b, 6d, 7b & 7d with amino acid residues of MTB-PS
Comp.
code
6b
PDB Atom
Name
Distance
(A°)
Residue Atom ID
Ligand Name
Gly158
1163
N (Donor)
O (Acceptor)
N (Acceptor)
O (Acceptor)
O (Acceptor)
O (Acceptor)
2.434
3.074
3.022
2.707
2.789
Gly158
Gly158
Gly158
1163
1163
1163
N (Donor)
N (Donor)
N (Donor)
6d
7b
7d
In addition to these interactions, all the molecules showed steric interactions
of N-benzyl moiety of the basic ring system (pyrido[4',3': 4,5]thieno[2,3-d]pyrimidin-4(3H)-one)
with amino acid residues Val37, Pro38 and Phe157 (Table 5), in general.
TABLE 5
Steric interactions of 6b, 6d, 7b and 7d with amino acid residues of MTB-PS
Comp.
Residues involved in steric interactions
code
Pro38, Thr39, Met40, Ser65, Ile66, Phe157, Glu159, Lys160,
Val 184, Pro185, Thr186
Val37, Pro38, Thr39, Phe156, Phe157, Gly46, Leu50, Pro185,
Thr186, Val187
Val 37, Pro 38, Thr39, Phe157, Gln164, His44, Pro185, Thr186,
Val 187
Val37, Pro38, Phe156, Phe157, Gln164, Thr186, Val187
6b
6d
7b
7d
The present study describes the design of twenty two new analogues of 5,6,7,8-
tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-ones and all of them were successfully
synthesized in quantitative yields by using appropriate methods. Some of the test compounds 6b,
6c, 6d, 6k, 7b, 7c, 7d and 7k showed impressive antibacterial activity (MIC in the range of 15-
73 µM) and compounds 6d and 7b further showed significant antimycobacterial activity on MTB
H37Rv and one INH-resistant strain (IC50 in the range of >340 µM by LRP assay and MIC in the
12

range of >9 µM by broth microdilution method). In liquid cultures compounds 6b, 6e, 6i, and 7b
along with 6d and 7b also showed good activity (MIC- >22 µM) against both the strains. The
molecular docking results on MTB-PS correlated well with the in vitro antimycobacterial
activity. Further investigations are underway on enzyme inhibition, cytotoxicity and
pharmacokinetic properties to confirm these results.
Acknowledgements
One of the authors (MN) thanks UGC-BSR (No.F.7-106/2007) for providing a merit
scholarship. The authors are thankful to the Principal, UCPSc, Kakatiya University for providing
facilities and the Management, Sri Shivani College of Pharmacy, Warangal for permitting to
carry out biological activity. We are also thankful to the Dr. P. Shanthan Rao and Dr. B.
Narsaiah, Scientists, Flouroorganic Division, IICT, Hyderabad for helpful suggestions.
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S. M. Eur. J. Med. Chem. 2012, 51, 145.
16. Al-Taisan, K. M.; Al-Hazimi, H. M. A.; Al-Shihry, S. S. Molecules 2010, 15, 3932.
17. Antibacterial activity: All the test compounds were assayed in vitro for antibacterial
activity against two different strains of Gram-positive (S. aureus MTCC 96 and B. subtilis
MTCC 441) and Gram-negative (E. coli MTCC 443 and K. pneumoniae MTCC 109)
bacteria. The MIC was determined by the broth microdilution technique using21 Mueller-
Hinton nutrient broth and the MIC values were also tested for well-known antibiotic
streptomycin to compare the antibacterial activity of test compounds. The stock solution
(4 mg/ mL) of test compounds was prepared in dimethyl formamide (DMF). The stock
solution was sterilized by passing through a 0.2 mm polycarbonate sterile membrane
(Nuclepore) filters. Further the serial dilution of test compounds was carried out and the
following concentration was used: 1000, 500, 250, 125, 62, 31, 15, 7, 3 and 1 µg/ mL.
Test compounds at various concentrations were added to culture medium in a sterilized
borosilicate test tube and different bacterial strains were inoculated at 106 cfu/ mL
concentration. The tubes were incubated at 37 °C for 24 h and then examined for the
presence or absence of visible growth of the test organisms. All experiments were
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performed in triplicate. The MIC values were obtained from the lowest concentration of
the test compound where the tubes remained clear, indicated that the bacterial growth was
completely inhibited at this concentration.
18. Luciferase Reporter Phage (LRP) assay:22 The MTB H37Rv and clinical isolate of
MTB were grown in Middlebrook 7H9 complete medium 12 with and without test
sample for 3 days at 37 °C. LRP assay was done using concentrations of 200,100 and 50
μg/ mL of test samples. 50 µL bacterial suspension equivalent to MacFarlands no.2
standard was added to 400 μL of 7H9 with and without the test compound. For each
sample, two drug-free controls and two drug concentrations were prepared and this set up
was incubated for 72 h at 37 °C. After incubation, 50 μL of the high titer luciferase
reporter phage (phAETRC202) and 40 μL of 0.1 M CaCl2 were added to all the vials and
this set up was incubated at 37°C for 4 h. After incubation, 100μL of the mixture was
taken from each tube into a Luminometer cuvette and an equal amount of working D-
luciferin (0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was added. The
Relative Light Units (RLU) was measured after 10s of integration in the Luminometer.
Duplicate readings were recorded for each sample and the mean was calculated. The
percentage reduction in the RLU was calculated for each test sample and compared with
control.
19. Broth microdilution method: The MIC for MTB H37Rv and isoniazid (INH) resistant
clinical isolate of MTB was determined using a broth microdilution method23 in
Middlebrook 7H9 medium supplemented with OADC, with a final inoculum of 1 x 107
cfu/ mL. The compounds were dissolved in DMF (1.25 mg/ mL) and used as a stock
solution. Concentrations ranging from 1 to 1250 µg/ mL were used to assess the
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effectiveness of the compounds. After inoculation the microtiter tubes were incubated at
37 °C for 72 h, and the growth inhibition was recorded for 14 and 21 days respectively.
The MIC value represents the lowest dilution of the compound at which no bacterial
growth was detected.
20. Molecular docking: The molecular docking studies were carried using Molegro Virtual
Docker (MVD) tools24 on targeted protein (PS). The crystal structure of PS was retrieved
from protein data bank (PDB: 3IVX). The 3D structures of ligand molecules were drawn
using ChemDraw Ultra 12.0 version and then these structures were minimized by using
MOPAC (semi-empirical quantum mechanics), Jop Type with 100 iterations and
minimum RMS gradient of 0.01, and saved as MDL molfile (.mol).
21. Shetty, N. S.; Lamani, R. S.; Khazi, I. A. M. J. Chem. Sci. 2009, 121, 301.
22. (a). Shawar, R. M.; Humble, D. J.; van Dalfsen, J. M.; Stover, C. K.; Hickey, M. J.;
Steele, S.; Mitscher, L. A.; Baker, W. Antimicrob. Agents Chemother. 1997, 41, 570; (b).
Prabu, A.; Seenivasan, P.; Kumar, V. Asian J. Med. Sci. 2014, 5(3), 54; (c). Dusthackeer,
A.; Kumar, V.; Subbian, S.; Sivaramakrishnan, G.; Zhu, G.; Subramanyam, B.; Hassan,
S.; Nagamaiah, S.; Chan, J.; Rama, N.P. J Microbiol. Methods 2008, 73, 18.
23. Tran, T.; Saheba, E.; Arcerio, A. V.; Chavez, V.; Li, Q.-Y.; Martinez, L. E.; Primm, T. P.
Bioorg. Med. Chem. 2004, 12, 4809.
24. Boyapati, S.; Kulandaivelu, U.; Sangu, S.; Vanga, M. R. Arch. Pharm. Chem. Life Sci.
2010, 10, 570.
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