Synthesis, in vitro, and in vivo (Zebra fish) antitubercular activity of 7,8-dihydroquinolin-5(6H)-ylidenehydrazinecarbothioamides

Article abstract of DOI:10.1016/j.bioorg.2020.103626

We, herein, describe the synthesis of a series of novel aryl tethered 7,8-dihydroquinolin-5(6H)-ylidenehydrazinecarbothioamides 4a–v, which showed in vitro and in vivo antimycobacterial activity against Mycobacterium tuberculosis (Mtb) H37Rv. The intermed

Full text of DOI:10.1016/j.bioorg.2020.103626

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Synthesis, in vitro, and in vivo (Zebra fish) antitubercular activity of 7,8-dihy-
droquinolin-5(6H)-ylidenehydrazinecarbothioamides
Sandeep kumar Marvadi, Vagolu Siva Krishna, Goverdhan Surineni,
Rudraraju Srilakshmi Reshma, Balasubramanian Sridhar, Dharmarajan
Sriram, Srinivas Kantevari
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S0045-2068(19)31149-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.103626
YBIOO 103626
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Please cite this article as: S. kumar Marvadi, V. Siva Krishna, G. Surineni, R. Srilakshmi Reshma, B. Sridhar, D.
Sriram, S. Kantevari, Synthesis, in vitro, and in vivo (Zebra fish) antitubercular activity of 7,8-
dihydroquinolin-5(6H)-ylidenehydrazinecarbothioamides, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.103626
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Synthesis, in vitro, and in vivo (Zebra fish) antitubercular activity of 7,8-
dihydroquinolin-5(6H)-ylidenehydrazinecarbothioamides
Sandeep kumar Marvadi,a Vagolu Siva Krishna,b Goverdhan Surineni,a Rudraraju Srilakshmi
Reshma,b Balasubramanian Sridhar,c,d Dharmarajan Sriram,b Srinivas Kantevari,*a,d
a Fluoro and Agrochemicals Division, CSIR- Indian Institute of Chemical Technology,
Hyderabad-500007, Telangana, INDIA.
bMedicinal Chemistry and Antimycobacterial Research Laboratory, Pharmacy Group, Birla
Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad-
500078, Telangana, India.
c Division of X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Uppal Road,
Hyderabad 500007, India.
dAcademy of Scientific and innovative Research, CSIR- Indian Institute of Chemical Technology,
Hyderabad-500 007, Telangana, India.
1 | P a g e

Abstract:
We, herein, describe the synthesis of a series of novel aryl tethered 7,8-dihydroquinolin-5(6H)-
ylidenehydrazinecarbothioamides 4a–v, which showed in vitro and in vivo antimycobacterial
activity against Mycobacterium tuberculosis (Mtb) H37Rv. The intermediates dihydro-6H-
quinolin-5-ones 3a-v were synthesized from β-enaminones, reacting with cyclochexane-1,3-
dione/5,5-dimethylcyclohexane-1,3-dione and ammonium acetate using a modified Bohlmann-
Rahtz reaction conditions. They were further reacted with thiosemicarbazide to give the respective
hydrazine carbothioamides 4a-v. All the new analogues 4a–v, were characterized by their NMR
and mass spectral data analysis. Among the twenty-two compounds screened for in vitro
antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC27294), two
compounds, 4e and 4j, exhibited the highest inhibition with an MIC of 0.39µg/mL. Compounds
4a, 4g, and 4k were found to inhibit Mtb at an MIC of 0.78µg/mL. Hydrazinecarbothioamides 4a-
k, exhibited enhanced activity than dihydroquinolinones 3a-k. The observed increase in potency
provides a clear evidence that hydrazinecarbothioamide is a potential pharmacophore, collectively
imparting synergistic effect in enhancing antitubercular activity of the dihydroquinolinone core.
The in vivo (Zebra fish) antimycobacterial screening of the in vitro active molecules led to the
identification of a hit compound, 4j, with significant activity in the Mtb nutrient starvation model
(2.2-fold reduction). Docking studies of 4j showed a hydrogen bond with the P156 residue of the
protein.
Keywords:
Dihydroquinoline; hydrazinecarbothioamides; Mycobacterium tuberculosis; Nutrient Starvation;
Zebra fish; Molecular docking.
2 | P a g e

1. Introduction
Tuberculosis (TB) is considered to be an inordinately spreading contagious disease, which
arises due to Mycobacterium tuberculosis (Mtb) infection, and claims life of more people than
malaria or HIV/AIDS [1]. As per the global TB report released by the World Health Organization
(WHO), around 1.5 million people died and 11 million new TB cases were registered [2-7].
Further, the rapid emergence of multi-drug resistant (MDR) and extremely drug resistant (XDR)
and totally drug resistant TB (TDR) tuberculosis cases are of major concern. It is evident from the
literature/clinical data that the existing pool of drugs such as isoniazid (INH), rifampicin (RIF),
and fluoroquinolones are becoming less sensitive (Figure 1) [8-10]. The new generation quinoline
based drug Bedaquiline, approved by FDA for medical use in 2012, suffers from cardiovascular
adverse effects [11,12]. Another drug Delamanid, a mycolic acid biosynthesis inhibitor has been
approved for treating MDR-TB in South Korea, Europe and Japan [13].
HO
CH3
H
O
O2N
O
N
N
NH2
O
OH O
N
O
OH OH
O
N
O
O
NH
N
N
O
N
Isoniazid (INH)
O
O
OH
O
N
OCF3
Delamanid
Rifampicin (RIF)
O
O
O
N
F
F
OH
N
OH
OH
Br
H
N
N
N
HN
O
O
N
HN
H
Ciprofloxacin (CIF)
Bedaquiline
Moxifloxacin (MOXI)
Figure 1. Representative anti-tubercular drugs.
In the recent years, molecules bearing thiosemicarbazones have been investigated extensively for
their various pharmacological properties like antiviral, antifungal, antineoplastic,
3 | P a g e

antimycobacterial, and antimalarial effects. The versatility of sulphur and nitrogen atoms to act as
donors allows them to bring in a great variety of coordination modes with bacterial protein. Many
derivatives containing thiosemicarbazone moiety have been synthesized and evaluated for their
antibacterial activity. Thiacetazone, (TAC; p-acetamidobenzaldehyde thiosemicarbazones also
known as thioacetazone), is one of the oldest and cheapest second line drug used for tuberculosis
treatment (Figure 2). But this drug shows only bacteriostatic activity, and develops resistance
easily during tuberculosis therapy. It also has cross-resistance to ethionamide. Owing to its high
frequency of Stevens-Johnson syndrome, thioacetazone is forbidden to use in patients with human
immunodeficiency virus (HIV). To counter such affects, newer agents such as SRI-286 [14], SRI-
224 and KBF-611 [15] bearing thiosemicarbazide are in clinical trials for treating tuberculosis
(Figure 2). Although Thiacetazone (TAC) thought to inhibit mycolic acid biosynthesis, the exact
mechanism of action has proven elusive. Recent studies of Jackson et al [16, 17] throw light on
the mechanistic aspects of thioacetazone and other carbothiamide analogues and led to the
speculation that there could be other enzymes playing role in the inhibition against Mycobacterium
tuberculosis. As part of global efforts to develop an efficient antitubercular drug, our group is
focused on the development of new dihydroquinolone based chemical entities as potential
antitubercular agents. In view of the importance of the thiosemicarbazide moiety, we envisaged to
hybridize dihydro-6H-quinolin-5-one with thiosemicarbazide pharmacophore. In the present
paper, a series of novel 7,8-dihydroquinoline-5(6H)ylidene)hydrazinecarbothioamide derivatives,
4a-v, were synthesized and evaluated for the in vitro inhibitory effect on pathogenic bacterium,
Mycobacterium tuberculosis H37Rv. Among the 22 new derivatives, five compounds 4e, 4j (MIC
0.39µg/mL), and 4a, 4g, 4k (MIC 0.78µg/mL) resulted as the most promising antitubercular
agents. The in vivo (Zebra fish) antimycobacterial screening of the active molecules led to the
identification of an important hit compound 4j, which may be pursued for further development.
S
S
S
N
N
N
N
F
CH3
N
H2N
N
H2N
N
H
N
NH2
O
N
H
H
H
NH2
N
HN
NH2
H3C
N
H
S
S
O
N
O
NH
CH3
Thioacetazone
Isonicotinaldehyde
thiosemicarbazone
KBF-611
SRI-286
SRI-224
Figure 2. Thiosemicarbazide scaffold based antitubercular agents.
4 | P a g e

2. Results and discussion
2.1. Chemistry
Variable
substituent
H2N
N
S
Active pharmacophore
of Thioacetazone
NH
Antitubercular
template
R
N
Figure 3. Molecular structure of hydrazinecarbothioamide derivatives used in the present study.
Broadly, the designed scaffold has three fragments. The first fragment is the
dihydroquinoline pharmacophore from our previous reports, with antitubercular activity [18, 19].
The second fragment, hydrazinecarbothioamide, is a key pharmacophore present in antitubercular
drugs such as thioacetazone. The third fragment is a variably substituted aryl and heteroaryl groups
appended to dihydroquinoline moiety. Maneuvering all these three fragments into single molecular
frame could impart or enhance antitubercular properties into the newer scaffold, 7,8-
dihydroquinolin-5(6H)-ylidenehydrazine carbothioamides (Figure 3).
The target molecules, 4a-v, were synthesized by following a three-step synthetic protocol
described in Scheme 1. β-Enaminones, 2a-k, were prepared by refluxing aryl and heteroaryl
methyl ketones 1a-k, with N,N-DMF-DMA in xylene. Compounds 2a-k were reacted with
cyclochexane-1,3-dione or 5,5-dimethyl-cyclohexane-1,3-dione, and NH4OAc in the presence of
CeCl3.7H2O-NaI in i-PrOH at reflux temperature to result the dihydro-6H-quinolin-5-ones 3a-v in
very good yields [18-28]. Compounds 3a-v were condensed with thiosemicarbazide in methanol
in the presence of catalytic amount of conc. H2SO4 to give desired 7,8-dihydroquinolin-5(6H)-
ylidene)hydrazinecarbothioamide 4a-v in excellent yields (Table 1). All the synthesized
compounds were purified over a silica gel column and were fully characterized by IR, 1H and 13C
NMR, electrospray ionization (ESI), and high-resolution mass spectral (HRMS) data analysis
(Supporting Information). For example, 1H NMR spectral data of compound 4j showed aromatic
protons at δ 10.13 - 7.00, the methylene (-CH2) protons appeared at δ 2.90 (2H) as triplet (J = 6.0
Hz), δ 2.74 (2H) as triplet (J = 6.2 Hz), and δ 1.98 (2H) as
5 | P a g e

H2N
N
S
NH
O
O
O
(ii)
(iii)
(i)
R1
R1
R
CH3
1a-k
R1
R1
R
N
R
N
R
N
2a-k
3a-v
4a-v
R1 = H, CH3
R =
S
S
S
Cl
Br
F
Cl
Br
O
Ph
a
b
c
d
e
f
g
h
i
j
k
Scheme 1. Synthesis of compounds 4a–v. Reagents and conditions: (i) N,N-DMF-DMA, xylene,
reflux, 7 h, 95%; (ii) cyclohexane-1,3-dione/ 5,5-dimethyl cyclohexane-1,3-dione, NH4OAc,
CeCl3.7H2O-NaI, i-PrOH, reflux, 4 h, 86-94%; (iii) NH2CSNHNH2, conc. H2SO4, MeOH, reflux,
4 - 5 h, 76–92%.
Figure 4. ORTEP representation of compound 4j with the thermal displacement ellipsoids drawn
at 30% probability. CCDC 1511250 contains supplementary crystallographic data for the structure.
quintet (J = 6.0 Hz), respectively. 13C NMR spectrum showed methylene carbons at δ 31.6, 25.0,
20.3. HRMS data analysis of 4j displayed a molecular ion peak at m/z 337.03429 [M+H]+
suggesting the molecular formula as C14H14N4ClS2 of the said compound. Additionally, the IR
spectra for the target compounds 4a-v exhibited characteristic absorption bands at 3427-3137 cm-1,
1588-1502 cm-1 and 1089-1026 cm-1 which corresponded to N-H (stretch), N-H (bend) and C=S
respectively. The structure of compound 4j was further confirmed by single crystal X-ray analysis
(Figure 4).
6 | P a g e

2.2. Pharmacological evaluation
2.2.1. Antimycobacterial activity
All the synthesized 7,8-dihydroquinoline-5(6H)ylidene)hydrazinecarbothioamides, 4a-v, were
screened for their in vitro antimycobacterial activity against M. tuberculosis H37Rv (ATCC27294)
strain by using the broth dilution method to determine the Minimum Inhibitory Concentration
(MIC) [29,30]. The tested compounds exhibited an MIC range of 0.39μg/mL-25.0μg/mL (Table
1). Two compounds 4e and 4j, displayed potent antitubercular activity with an MIC of 0.39μg/mL.
Compounds 4a, 4g, and 4k (MIC 0.78 μg/mL) also exhibited potent antitubercular effect on
H37Rv. In addition to these five compounds, another five compounds 4b, 4c, 4f, 4h, and 4i (MIC
1.56 μg/mL) exhibited greater inhibitory activity than standard drugs ethambutol and
pyrazinamide. Two compounds 4d and 4s (MIC 3.125 μg/mL) were found to be equipotent to the
standard drug ethambutol. Among other derivatives, compound 4p exhibited MIC 12.5 μg/mL,
two compounds 4n, and 4o (MIC of 25 μg/mL), and 4l, 4m, 4q, 4r, and 4t-v were not active at an
MIC of 25 μg/mL (Table 1). Compounds 4a-k inhibited M. tuberculosis at much lower
concentrations (MIC 0.39-3.13 μg/mL) compared to the compounds 4l-v (MIC 3.13- >25.0μg/mL)
implying that unsubstituted 1,3-cyclohexanedione is more active than its dimethylated counterpart.
Although most of the compounds 4a-k significantly inhibited Mtb, the MIC is slightly varied with
respect to the substituent on phenyl ring or thiophene ring coupled to quinoline core. The
substituent on quinoline core with respect to potency is varied in the order- 4e (naphthyl), 4j
(chlorothiophenyl) with an MIC value of 0.39μg/mL > 4a (phenyl), 4g (4-chlorophenyl), 4k
(bromothiophenyl) with an MIC value of 0.78μg/mL > 4b (4-methylphenyl),
Table 1. Antituberculosis evaluation of 4a–v against M.tuberculosis H37Rv and cytotoxicity.
MIC (µg/mL)
Cytotoxicity
% Inhibition at 50 µg/mL
Entry
Product
R
R1 Yield (%)a
4a-v
0.78
1.56
1.56
3a-v
12.5
12.5
12.5
1
2
3
4
5
4a
4b
4c
4d
4e
a
b
c
d
e
H
H
H
H
H
92
89
86
88
82
41.02
32.42
28.08
42.78
20.36
3.13 >25.0
0.39 >25.0
7 | P a g e

6
4f
4g
f
g
h
i
H
80
83
81
85
86
83
85
76
81
79
84
82
82
78
80
82
81
---
---
---
---
1.56
NT
24.64
37.15
34.28
27.58
18.24
21.08
NT
7
H
0.78 >25.0
8
4h
H
1.56
1.56
0.39
0.78
NT
9
4i
H
12.5
6.25
6.25
10
11
12
13
14
15
16
17
18
18
20
21
22
23
24
25
26
4j
j
H
4k
k
a
b
c
d
e
f
H
4l
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
>25.0 >25.0
>25.0 >25.0
4m
4n
NT
25.0
12.5
NT
4o
25.0 >25.0
NT
4p
12.5
1.56
NT
36.94
NT
4q
>25.0
4r
g
h
i
>25.0 12.5
3.13 12.5
>25.0 6.25
NT
4s
39.48
NT
4t
4u
j
>25
>25
0.1
3.13
3.13
NT
4v
k
NT
Isoniazid
--- ---
NT
Rifampicin --- ---
Ethambutol --- ---
Pyrazinamide --- ---
0.2
NT
3.13
50
NT
NT
a Isolated yield; NT – not tested.
4c (4-methoxyphenyl), 4f (4-fluorophenyl), 4h (4-bromophenyl), and 4i (thiophenyl) with an MIC
value of 1.56μg/mL > 4d (4-biphenyl) with an MIC value of 3.13 μg/mL. On the other hand,
compounds 4l-v, with gem-dimethyl group, irrespective of the aryl substituent, showed significant
reduction in antitubercular potency. Compound 4s (bromophenyl, MIC-3.13μg/mL) showed good
activity among compounds 4l-v. It was interesting to note that the panel of compounds 4a-k,
containing hydrazinecarbothioamide, exhibited enhanced activity by several folds than
dihydroquinolinones 3a-k without hydrazinecarbothioamide, even when the other frameworks and
substituents are same (Entries 1-11, Table 1). This observed increase in potency provides clear
evidence that hydrazinecarbothioamide, consisting a free thiourea moiety along with ionizable
proton, is a potential pharmacophore, collectively imparting synergetic effect on enhancing
antitubercular activity to dihydroquinolinone core. To our surprise, compounds 4l-v with gem-
dimethyl group (Entries 12-22, Table 1), and hydrazinecarbothioamide could not induce
synergistic effect and in few cases (Entries 16, 21, and 22, Table 1) dihydro-6H-quinolin-5-ones
8 | P a g e

without hydrazinecarbothioamide were more potent. The overall structure activity relationship
(SAR) of the synthesized compounds 4a-v is presented in Figure 5.
4a-k
Compounds
with
hydrazinecarbothioamide
group has better MIC value
Thiourea
H2N
N
S
3a-k
than compounds
without
hydrazinecarbothioamide group
NH
Ionizable Hydrogen
Substituents on phenyl
4a-k
Compounds
without
4a-h
group
thiophine ring
influenced MIC value
or on
R
N
gem-dimethyl group has
better activity than
4i-k
4l-v
compounds
Figure 5: SAR of dihydroquinolin-5(6H)-ylidene)hydrazinecarbothioamide derivatives 4a–v.
2.2.2. In vitro cytotoxicity
The active analogues from antitubercular assay (MIC ≤ 12.5 µg/mL) was assessed for in
vitro cytotoxicity at 50μg/mL concentration against Human Embryonic Kidney (HEK-293T) cells
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [31, 32].
Percentage inhibition of cells is reported in Table 1. The most promising antitubercular
compounds 4e, 4j, and 4a, 4g, 4k, and 4b, 4c, 4f, 4h, 4i exhibited 20.36%, 18.24%, and 41.02%,
37.15%, 21.08%, and 32.42%, 28.08%, 24.64%, 34.28%, 27.58% inhibition, respectively. The
results clearly indicated that the most potent analogues 4e and 4j with high antitubercular activity
are less toxic to Human Embryonic Kidney (HEK-293T) cells and are suitable for further
mechanistic evaluations. The ratio between cytotoxicity and antimycobacterial activity in vitro
enabled the determination of selectivity index (SI). Compounds that exhibited SI values greater
than 10 in all four cell lines were considered nontoxic [20].
2.2.3. Mycobacterium tuberculosis H37Rv nutrient starved model.
The nutrient starvation model, developed by Betts et. al., has successfully been utilized by
medicinal chemists to test compounds as antimycobacterial agents. The model mimics bacterial
9 | P a g e

characteristics in its persistent state causing bacterial growth arrest by nutrient starvation,
minimizing aerobic metabolism while maintaining viability. We performed this assay with the
most active compounds to identify the log reduction in the bacterial population and the results are
show in Figure 6. Here, the dormancy in bacteria is induced by starving the culture in phosphate
buffer saline (PBS) for 6 weeks. The dormant culture was tested with compounds 4e, 4g, 4j, and
4k at a concentration of 10 µM based on their MIC data [33,34]. Compounds 4e, 4g, 4j, and 4k
showed a log reduction of 0.7, 2.1, 2.2, and 0.9, respectively. By comparison, standards INH and
MOXI shown a log reduction of 1.5, and 2.0 respectively, compared to the control. The study
reveals that the inhibition of dormant mycobacterium by new compounds can be beneficial towards
the development of lead molecules to combat tuberculosis.
7
6
5
4
3
2
1
0
l
j
I
e
k
g
H
4
o
X
4
4
4
r
N
t
I
O
n
M
o
C
Figure 6. Activity profile of 4e, 4g, 4j and 4k in nutrient starvation model. Bacterial count was
estimated through MPN (most probable number) assay and significance plot was developed by
adopting two-way ANOVA (p < .0001, using GraphPad Prism Software).
2.2.4. In vivo activity testing assay on Mycobacterium marinum infected zebra fish
Despite the extensive in vitro evaluation of Mycobacterium tuberculosis inhibitors, validation of
the data in the animal models is essential. Of the several animal models developed for
Mycobacterium tuberculosis infection, the most versatile one has been the mouse model. However,
it has limitations like failure in caseating granulomas observed in humans. In this scenario, zebra
fish infection model, using M. marinum, a genetic relative of M. tuberculosis is utilized
complementary to the above models [35]. Activity and dosage of antibiotics in zebra fish is close
10 | P a g e

to that of the dosage in humans. This could be the most cost-effective and trustworthy model for
researchers to study the in vivo activity of new drug compounds [35]. In this study, zebra fish was
infected with M. marinum which genetically belongs to MTB family. Active molecule 4j was
tested for in vivo inhibitory activity using adult zebra fish model. Standard compounds INH and
MOXI have shown reductions of 2.8 and 2.6 in log scale and compound 4j has shown 2.3 log
reductions against control (Figure 7).
8
7
6
5
4
3
2
1
0
Control
4j
INH
MOXI
Figure 7. Bacterial count estimation (Mean ± S.E.M., n = 6) for control and treated groups by
zebra fish model conducted by using MPN assay. Statistical significance has been analyzed by
Two-way ANOVA using Graph Pad Prism Software.
2.3. Molecular docking studies
With the fact that hydrazinecarbothioamide is a key pharmacophore in thioacetazone (TAC) drug,
its mechanism of action is still uncertain. Among the target studies TAC is shown to remove
Mycobacterium tuberculosis interfering in mycolic biosynthesis pathway, the precise number of
enzymes involved in this process remains unknown.[16, 17] To understand the probable binding
mode of the most active compound 4j, molecular docking studies were performed against
Mycobacterium tuberculosis enzymes, enoyl reductase (InhA), PDB ID: 4TZK;
Decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1), PDB ID: 4P8N; Alcohol dehydrogenase
(ADH), PDB ID: 2VHW; (3R)-hydroxyacyl-ACP dehydratase (HadAB), PDB ID:4RV2; Lysine-
ɛ aminotransferase (LAT), PDB ID: 2CJD; Topoisomerase (DNA Gyrase), PDB ID: 5BS8 and
Glutamate recemase (MurI), PDB ID: 5H7J. The results of docking studies are described in Table
11 | P a g e

S1 (supporting information). In silico studies of 4j showed good binding energy (-8.313) to the
target enzyme enoyl reductase (InhA), whereas co-crystallized inhibitor 1-cyclohexyl-N-(3,5-
dichlorophenyl)-5-oxopyrrolidine-3-carboxamide showed -10.155 kcal/mol. The binding mode of
the target enzyme is confirmed from X-ray with a resolution of 1.62 Å [35,36]. The docking results
were drawn based on the docking score, hydrogen bonding and Van der Waals interactions of the
ligand with the enzyme (Figure 8). The docking results showed that compound 4j bonded via
hydrogen bonding with the P156 residue of the enzyme. InhA is known to catalyze the NADH-
dependent reduction of enoyl-ACP in the biosynthesis of fatty and mycolic acids, an essential
component of the membrane and cell wall of M. tuberculosis.[37] Hence the antitubercular
property of compound 4j could be due to the inhibition of InhA enzyme.
O
N
(d)
Cl
H
N
O
Cl
Figure 8: Binding mode of compound 4j to Mtb InhA. (a) The binding mode of 4j after docking
(yellow carbons). (b) The binding of 4j with target after 50 ns of molecular dynamics simulation
with a transparent surface for Mtb InhA in the background. (c) Comparison, interactions of test
compound 4j with Mtb InhA docking pose (yellow carbon) and after 50 ns of molecular dynamics
simulation (cyan carbons). Amino acid side-chains are shown as thin sticks. Ligand bonds to
residues are shown as green dashed lines. (d) The chemical structure of 1-cyclohexyl-N-(3,5-
dichlorophenyl)-5-oxopyrrolidine-3-carboxamide.
2.4. Molecular Dynamics (MD) Simulations
12 | P a g e

Molecular dynamics (MD) simulation was run for receptor with 4j complex. The inclusion
complex was soaked in explicit solvent water, and MD simulation was run at a temperature of 300
K for a total time period of 50 ns, during which 5208 structural frames of receptor and ligand
complex were enumerated, was saved in the trajectory. To access the stability of complex
structures computed during MD simulation, the structures from the trajectory aligned to receptor
atoms of the first frame and root mean square deviation (RMSD) was calculated for receptor and
individual ligand with respect to the initial frame. The complex gets stabilized in the initial phase
of MD simulation. So the analysis of the structural stability was carried out on the final phase of
50 ns. Test compound 4j orientation changed slightly after dynamics and formed an additional
hydrogen bond with Q214 residue while retaining the interactions with P156. The key residue
Y158 bonded via van der Waals interaction with in the 4 Å regions throughout the 50ns simulations
time. Average RMSD values of receptor and 4j were observed to be 1.9Å and 2.1Å respectively
(Figure 9) [39,40].
Figure 9: Receptor and compound 4j RMSD in 50ns time
3. Conclusion
In conclusion, the present study describes the synthesis and evaluation of dihydroquinolin-
5(6H)-ylidene)hydrazinecarbothioamide derivatives 4a–v against M. tuberculosis H37Rv.
Compounds 4a-k were found to exhibit good antitubercular inhibition (MIC<3.13 µg/mL). Two
compounds 4e and 4j exhibited the highest inhibition with an MIC of 0.39µg/mL and low
cytotoxicity. Hydrazinecarbothioamides 4a-k exhibited enhanced activity by several times than
dihydroquinolinones 3a-k without hydrazinecarbothioamide. The observed increase in potency
13 | P a g e

provides clear evidence that hydrazinecarbothioamide is a potential pharmacophore and when
coupled to dihydroquinoline, imparts increased antitubercular properties to the molecule.
Compounds 4l-v with gem-dimethyl group and hydrazinecarbothioamide could not induce
synergistic effect. The in vivo (Zebra fish) antimycobacterial screening of active molecules led to
the identification of a hit compound 4j and exhibited a significant bacterial log reduction in the
nutrient starvation model (2.2-fold reduction). Docking studies of 4j showed a hydrogen bond
between the molecule and P156 residue of the target protein. It may be said that the antitubercular
property of the molecule could be via the inhibition of InhA enzyme. As part of our global efforts
to identify new antitubercular agents the data presented here may serve as foundation for the
identified lead compound 4j for further preclinical and clinical investigations.
4. Experimental Section
All the starting materials and other reagents of the best grade were commercially available
and were used without further purification. TLC was performed on Merck 60 F-254 silica gel
plates. Spots were visualized by UV light. All melting points were measured by CINTEX
programmable melting point apparatus and are uncorrected. 1H and 13C NMR spectra of samples
in CDCl3 and DMSO-d6 were recorded on 75, 100, 300, 400, and 500 MHz spectrometers using
tetramethylsilane as the internal standard. Chemical shifts are reported in parts per million (ppm)
downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), brs (broad
singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). Coupling constants are reported in
hertz (Hz). HRMS analyses were acquired on single quadruple and carried out using the ESI
techniques at 70 eV. Wherever required column chromatography was performed using silica gel
of 60–120 mm with hexane and ethyl acetate as eluents.
4.1. General Procedure for the synthesis of dihydro-6H-quinolin-5-ones (3a-v)
The dihydro-6H-quinolin-5-one (3a-v) were synthesized based on a literature method as following:
aryl and heteroaryl methylketones (1a-k, 5.23 mmol) were treated with dimethylformamide-
dimethylacetal (DMF-DMA) ( 20.92 mmol) in refluxing xylene for 7 h to form β-enaminones
(2a-k), and reacted with cyclohexane-1,3-dione/5,5-dimethylcyclohexane-1,3-dione (0.16 g, 1.2
mmol) ammonium acetate (2.0 mmol) in 2-propanol (5 mL) were added CeCl3.7H2O (0.2 mmol),
NaI (0.2 mmol) and refluxed for 4 h (monitored by TLC). The reaction mixture was cooled to
room temperature; a solid precipitate was filtered and washed with cold 2-propanol. The combined
solvent was evaporated, and the crude residue obtained was subjected to column chromatography
14 | P a g e

(hexane: ethyl acetate) to obtain dihydro-6H-quinolin-5-one derivatives (3a-v) as solids in very
good yields.
4.1.1. 2-(4-fluorophenyl)-7,8-dihydroquinolin-5(6H)-one (3f)
Light yellow solid, yield 88%, mp: 90-92 °C; 1H NMR (400 MHz, CDCl3):  8.31 (d, J = 8.1 Hz,
1H, Ar-H), 8.10-8.02 (m, 2H, Ar-H), 7.65 (d, J = 8.1 Hz, 1H, Ar-H), 7.21-7.13 (m, 2H, Ar-H),
3.20 (t, J = 6.2 Hz, 2H, CH2), 2.71 (t, J = 6.1 Hz, 2H, CH2), 2.22 (qt, J = 6.2 Hz, 2H, CH2); 13C
NMR (100 MHz, CDCl3):  196.6, 163.3 (d, J = 250.1 Hz, 1C), 163.0, 158.2, 134.9, 133.7 (d, J =
2.2 Hz, 1C), 128.7 (d, J = 8.8 Hz, 2C), 125.6, 117.5, 115.0 (d, J = 22.0 Hz, 2C), 37.7, 32.1, 21.2;
MS (ESI): m/z 242 [M+H]+; HRMS (ESI) calcd for C15H13ONF [M+H]+ 242.09773; found:
242.09757.
4.1.2. 2-(4-bromophenyl)-7,8-dihydroquinolin-5(6H)-one (3h)
Light yellow solid, yield 84%, mp: 124-126 °C; 1H NMR (400 MHz, CDCl3):  8.31 (d, J = 8.1
Hz, 1H, Ar-H), 7.96-7.90 (m, 2H, Ar-H), 7.66 (d, J = 8.1 Hz, 1H, Ar-H), 7.63-7.57 (m, 2H, Ar-
H), 3.20 (t, J = 6.2 Hz, 2H, CH2), 2.71 (t, J = 7.0 Hz, 2H, CH2), 2.22 (qt, J = 6.2 Hz, 2H, CH2);
13C NMR (100 MHz, CDCl3):  178.7, 157.1, 154.6, 145.3, 137.1, 133.2, 131.2, 128.2, 125.8,
122.7, 117.6, 45.6, 37.9, 30.3, 27.8; MS (ESI): m/z 302 [M+H]+; HRMS (ESI) calcd for
C15H13ONBr [M+H]+ 302.01845; found: 302.01750.
4.1.3. 2-(4-fluorophenyl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-one (3q)
Light yellow solid, yield 90%, mp: 96-98 °C; 1H NMR (400 MHz, CDCl3):  8.30 (d, J = 8.1 Hz,
1H, Ar-H), 8.10-8.03 (m, 2H, Ar-H), 7.66 (d, J = 8.1 Hz, 1H, Ar-H), 7.22-7.14 (m, 2H, Ar-H),
3.09 (s, 2H, CH2), 2.57 (s, 2H, CH2), 1.14 (s, 6H, 2CH3); 13C NMR (100 MHz, CDCl3):  196.8,
163.5 (d, J = 250.1 Hz, 1C), 161.7, 158.9, 134.7, 133.9 (d, J = 2.2 Hz, 1C), 128.8 (d, J = 8.8 Hz,
2C), 128.8, 124.9, 117.6, 115.2 (d, J = 21.2 Hz, 2C), 51.4, 46.0, 32.3, 27.7; MS (ESI): m/z 270
[M+H]+; HRMS (ESI) calcd for C16H17N4S [M+H]+ 270.12933; found: 270.12887.
4.2. General Procedure for the synthesis of dihydroquinolin-5(6H)-ylidene)hydrazine
carbothioamide derivatives (4a-v)
A mixture of dihydro-6H-quinolin-5-one (3a-v) (1.0 mmol), thiosemicarbazide (1.1 mmol) and
catalytic amount of sulphuric acid in methanol (10 mL) were refluxed for 4-5 h. After completion,
(monitored on TLC), the reaction product was cooled, precipitate was filtered and the dried residue
was recrystalized by using methanol to afford compound (4a-v) as solids in excellent yields.
4.2.1. (E)-2-(2-phenyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothioamide (4a)
15 | P a g e

Colorless solid, yield 0.42 g, 92%, mp: 246-248 °C; 1H NMR (300 MHz, DMSO-d6):  9.62 (br s,
1H, NH), 8.42 (d, J = 8.3 Hz, 1H, Ar-H), 8.14-8.03 (m, 2H, Ar-H), 7.73 (br s, 1H, NH), 7.67-7.54
(m, 2H, Ar-H), 7.52-7.41 (m, 3H, Ar-H), 3.04 (t, J = 5.8 Hz, 2H, CH2), 2.73 (t, J = 6.4 Hz, 2H,
CH2), 2.07 (qt, J = 5.8 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3+DMSO-d6):  178.6, 158.4,
155.8, 146.0, 138.0, 133.0, 128.6, 128.1, 126.2, 125.9, 117.8, 31.9, 24.8, 20.4; IR (KBr) 3423,
3244, 3147, 2948, 1578, 1497, 1456, 1447, 1283, 1105, 1081, 839, 749, 695 cm-1; MS (ESI): m/z
297 [M+H]+. HRMS (ESI) calcd for C16H17N4S [M+H]+ 297.11627; found: 297.11684.
4.2.2. (E)-2-(2-p-tolyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothioamide (4b)
Colorless solid, yield 0.40 g, 89%, mp: 260-262 °C; 1H NMR (300 MHz, DMSO-d6):  10.25 (br
s, 1H, NH), 8.85 (d, J = 8.3 Hz, 1H, Ar-H), 8.29 (br s, 1H, NH), 8.04 (br s, 1H, NH), 8.01-7.94
(m, 2H, Ar-H), 7.76 (d, J = 8.3 Hz, 1H, Ar-H), 7.46-7.31 (m, 2H, Ar-H), 3.06 (t, J = 5.8 Hz, 2H,
CH2), 2.78 (t, J = 6.2 Hz, 2H, CH2), 2.42 (s, 3H, CH3), 2.02 (qt, J = 5.8 Hz, 2H, CH2); 13C NMR
(75 MHz, CDCl3+DMSO-d6):  178.7, 157.1, 154.2, 144.7, 139.5, 135.6, 133.2, 129.1, 127.0,
126.8, 118.8, 30.6, 24.8, 20.7, 20.0; IR (KBr) 3404, 3381, 3202, 3137, 2945, 1584, 1506, 1455,
1412, 1293, 1082, 815, 751, 674 cm-1; MS (ESI): m/z 311 [M+H]+. HRMS (ESI) calcd for
C17H19N4S [M+H]+ 311.13205; found: 311.13249.
4.2.3. (E)-2-(2-(4-methoxyphenyl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio-
amide (4c)
Colorless solid, yield: 0.38 g, 86%; mp; 238-240 0C 1H NMR (300 MHz, DMSO-d6): δ 10.22 (br
s, 1H, NH), 8.72 (d, J = 8.3 Hz, 1H, Ar-H), 8.28 (br s, 1H, NH), 8.08-7.97 (m, 3H, Ar-H), 7.69
(d, J = 8.3 Hz, 1H, Ar-H), 7.10-6.96 (m, 2H, Ar-H), 3.85 (s, 3H, OCH3), 2.98 (t, J = 5.4 Hz, 2H,
CH2), 2.76 (t, J = 6.0 Hz, 2H, CH2), 1.98 (qt, J = 5.6 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 +
DMSO-d6): δ 178.7, 160.5, 157.7, 154.6, 145.7, 134.8, 129.4, 128.3, 126.1, 118.0, 114.0, 55.1,
31.3, 25.0, 20.3; IR (KBr) 3422, 3245, 3149, 2931, 1625, 1603, 1588, 1504, 1455, 1243, 1175,
1026, 822, 673 cm-1; MS (ESI): m/z 327 [M+H]+ ; HR-MS (ESI) Calcd for C17H19N4OS [M+H]+:
327.12691, found: 327.12741.
4.2.4. (E)-2-(2-(biphenyl-4-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothioamide
(4d)
Colorless solid, yield: 0.38 g, 88%; mp: 252-254 0C 1H NMR (300 MHz, DMSO-d6): δ 10.32 (br
s, 1H, NH), 8.80 (d, J = 8.3 Hz, 1H, Ar-H), 8.36 (br s, 1H, NH), 8.29-8.17 (m, 3H, Ar-H), 7.87 (d,
J = 8.3 Hz, 1H, Ar-H), 7.83-7.71 (m, 4H, Ar-H), 7.56-7.45 (m, 2H, Ar-H), 7.40-7.32 (m, 1H, Ar-
16 | P a g e

H), 2.96 (t, J = 5.6 Hz, 2H, CH2), 2.75 (t, J = 6.0 Hz, 2H, CH2), 1.93 (qt, J = 5.6 Hz, 2H, CH2);
13C NMR (75 MHz, DMSO-d6): δ 178.7, 158.6, 154.8, 146.2, 140.7, 139.3, 136.9, 134.0, 128.9,
127.6, 127.1, 126.8, 126.5, 118.0, 32.0, 25.1, 20.5; IR (KBr) 3407, 3223, 3139, 1587, 1496, 1455,
1293, 1105, 1080, 847, 761, 693 cm-1; MS (ESI): m/z 373 [M+H]+; HR-MS (ESI) Calcd for
C22H21N4S [M+H]+: 373.14847, found: 373.14814.
4.2.5.
(E)-2-(2-(naphthalen-2-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio
amide (4e)
Colorless solid, yield: 0.36 g, 82%; mp: 224-226 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.33 (br
s, 1H, NH), 8.91 (d, J = 8.2 Hz, 1H, Ar-H), 8.66 (br s, 1H, NH), 8.38 (br s, 1H, NH), 8.30-8.14
(m, 2H, Ar-H), 8.09-7.92 (m, 4H, Ar-H), 7.60-7.53 (m, 2H, Ar-H), 3.05 (t, J =5.5 Hz, 2H, CH2),
2.79 (t, J = 6.0 Hz, 2H, CH2), 1.99 (qt, J = 5.5 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 + DMSO-
d6): δ 178.8, 156.8, 154.4, 145.3, 135.0, 134.1, 133.2, 132.7, 128.5, 128.1, 127.3, 127.0, 126.8,
126.4, 126.3, 124.2, 119.1, 31.2, 25.0, 20.2; IR (KBr) 3249, 3144, 1578, 1494, 1449, 1249, 1214,
1109, 995, 847, 752, 672 cm-1; MS (ESI): m/z 347 [M+H]+ ; HR-MS (ESI) Calcd for C20H19N4S
[M+H]+: 347.13250, found: 347.13249.
4.2.6.
(E)-2-(2-(4-fluorophenyl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio
amide (4f)
Colorless solid, yield: 0.36 g, 80%; mp: 232-234 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.25 (br
s, 1H, NH), 8.74 (d, J = 8.4 Hz, 1H, Ar-H), 8.32 (br s, 1H, NH), 8.21-8.05 (m, 3H, Ar-H), 7.74 (d,
J = 8.4 Hz, 1H, Ar-H), 7.24 (m, 2H, Ar-H), 2.96 (t, J = 5.8 Hz, 2H, CH2), 2.76 (t, J = 6.2 Hz, 2H,
CH2), 1.96 (qt, J = 5.8 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 + DMSO-d6) δ: 178.6, 162.7 (d,
J = 247.0 Hz, 1C), 158.3, 154.2, 145.9, 134.4 (d, J = 2.2 Hz, 1C), 133.7, 128.5 (d, J = 8.2 Hz, 2C),
126.2, 117.6, 115.1 (d, J = 21.4 Hz, 2C), 31.9, 25.0, 20.4; IR (KBr) 3234, 3140, 1586, 1503, 1456,
1292, 1227, 1159, 1079, 826, 772, 673 cm-1; MS (ESI): m/z 315 [M+H]+; HR-MS (ESI): Calcd
for C16H16N4FS [M+H]+ 315.10696, found: 315.10742.
4.2.7.
(E)-2-(2-(4-chlorophenyl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio
amide (4g)
Colorless solid , yield: 0.37 g, 83%. mp: 266-268 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.24 (br
s, 1H, NH), 8.73 (d, J = 8.3 Hz, 1H, Ar-H), 8.31 (br s, 1H, NH), 8.20-8.03 (m, 3H, Ar-H), 7.75 (d,
J = 8.3 Hz, 1H, Ar-H), 7.56-7.46 (m, 2H, Ar-H), 2.97 (t, J = 5.8 Hz, 2H, CH2), 2.76 (t, J = 6.2 Hz,
2H, CH2), 1.97 (qt, J = 5.8 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.7, 158.4,
17 | P a g e

154.0, 145.8, 136.7, 134.0, 133.7, 128.3, 128.0, 126.6, 117.8, 32.0, 25.0, 20.4; IR (KBr) 3424,
3237, 3145, 2949, 1579, 1496, 1453, 1413, 1285, 1215, 1091, 844, 754 cm-1; MS (ESI): m/z 331
[M+H]+; HR-MS (ESI) Calcd for C16H16N4ClS [M+H]+: 331.07795, found: 331.07787.
4.2.8.
(E)-2-(2-(4-bromophenyl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio
amide (4h)
Colorless solid, yield: 0.35 g, 81%; mp: 278-280 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.28 (br
s, 1H, NH), 8.76 (d, J = 8.3 Hz, 1H, Ar-H), 8.33 (br s, 1H, NH), 8.18-7.98 (m, 3H, Ar-H), 7.78 (d,
J = 8.3 Hz, 1H, Ar-H), 7.72-7.62 (m, 2H, Ar-H), 2.96 (t, J = 5.8 Hz, 2H, CH2), 2.76 (t, J = 6.2 Hz,
2H, CH2), 1.95 (qt, J = 5.8 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.7, 158.5,
154.0, 145.8, 137.1, 133.8, 131.3, 128.3, 126.7, 122.7, 117.8, 31.9, 25.0, 20.4; IR (KBr) 3423,
3228, 3144, 1594, 1579, 1453, 1291, 1215, 1082, 846, 769, 663 cm-1; MS (ESI): m/z 375 [M+H]+;
HR-MS (ESI) Calcd for C16H16N4BrS [M+H]+: 375.02610, found: 375.02736.
4.2.9. (E)-2-(2-(thiophen-2-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothioamide
(4i)
Colorless solid, yield: 0.39 g, 85%; mp: 226-228 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.16 (br
s, 1H, NH), 8.61 (d, J = 8.2 Hz, 1H, Ar-H), 8.28 (br s, 1H, NH), 8.03 (br s, 1H, NH), 7.75 (dd, J =
3.5, 0.8 Hz, 1H, Ar-H), 7.61 (d, J = 8.2 Hz, 1H, Ar-H), 7.54 (dd, J = 4.9, 0.8 Hz, 1H, Ar-H), 7.13
(dd, J = 4.9, 3.5 Hz, 1H, Ar-H), 2.86 (t, J = 5.7 Hz, 2H, CH2), 2.70 (t, J = 6.3 Hz, 2H, CH2), 1.91
(qt, J = 5.7 Hz, 2H, CH2); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.6, 158.4, 151.1, 145.9,
144.0, 133.6, 128.4, 128.0, 126.0, 125.4, 116.6, 31.7, 25.0, 20.4; IR (KBr) 3415, 3248, 3148, 2948,
1577, 1492, 1456, 1428, 1279, 1082, 858, 751, 663 cm-1; MS (ESI): m/z 303 [M+H]+; HR-MS
(ESI) Calcd for C14H15N4S2 [M+H]+: 303.07308, found: 303.07326.
4.2.10. (E)-2-(2-(5-chlorothiophen-2-yl)-7,8-dihydroquinolin-5(6H)ylidene)hydrazinecarbo
thioamide (4j)
Colorless solid, yield: 0.38 g, 86%. mp: 236-238 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.13 (br
s, 1H, NH), 8.59 (d, J = 8.3 Hz, 1H, Ar-H), 8.22 (br s, 1H, NH), 7.93 (br s, 1H, NH), 7.58 (d, J =
8.3 Hz, 1H, Ar-H), 7.53 (d, J = 3.9 Hz, 1H, Ar-H), 7.00 (d, J = 3.9 Hz, 1H, Ar-H), 2.90 (t, J = 6.0
Hz, 2H, CH2), 2.74 (t, J = 6.2 Hz, 2H, CH2), 1.98 (qt, J = 6.0 Hz, 2H, CH2); 13C NMR (75 MHz,
CDCl3 + DMSO-d6): δ 178.7, 158.5, 150.2, 145.8, 143.2, 133.8, 131.0, 128.0, 126.6, 125.2, 116.1,
31.6, 25.0, 20.3; IR (KBr) 3416, 3222, 3134, 2944, 1586, 1502, 1458, 1430, 1294, 1097, 838, 794,
18 | P a g e

764 cm-1; MS (ESI): m/z 337 [M+H]+; HR-MS (ESI) Calcd for C14H14ClN4S2 [M+H]+: 337.03414,
found: 337.03429.
4.2.11. (E)-2-(2-(5-bromothiophen-2-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbo
thioamide (4k)
Colorless solid, yield: 0.35 g, 83%. mp: 218-220 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.27 (br
s, 1H, NH), 8.74 (d, J = 8.5 Hz, 1H, Ar-H), 8.33 (br s, 1H, NH), 8.18 (br s, 1H, NH), 7.80-7.69
(m, 2H, Ar-H), 7.30-7.24 (m, 1H, Ar-H), 2.85 (t, J = 5.7 Hz, 2H, CH2), 2.71 (t, J = 6.0 Hz, 2H,
CH2), 1.88 (qt, J = 5.7 Hz, 2H, CH2); 13C NMR (75 MHz, DMSO-d6): δ 178.7, 158.6, 150.1, 145.9,
145.5, 134.4, 131.9, 126.8, 126.7, 116.6, 115.0, 31.5, 25.1, 20.4; IR (KBr) 3415, 3223, 3136, 2948,
1585, 1502, 1457, 1425, 1292, 1099, 827, 795, 663 cm-1; MS (ESI): m/z 380 [M+H]+; HR-MS
(ESI) Calcd for C14H14BrN4S2 [M+H]+: 380.98369, found: 380.98378.
4.2.12. (E)-2-(7,7-dimethyl-2-phenyl-7,8-dihydroquinolin-5(6H)ylidene)hydrazinecarbothio
amide (4l)
Colorless solid, yield: 0.38 g, 85%; mp: 230-232 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.17 (br
s, 1H, NH), 8.65 (d, J = 8.2 Hz, 1H, Ar-H), 8.22 (br s, 1H, NH), 8.08-7.94 (m, 3H, Ar-H), 7.66
(d, J = 8.2 Hz, 1H, Ar-H), 7.48-7.34 (m, 3H, Ar-H), 2.83 (s, 2H, CH2), 2.59 (s, 2H, CH2), 1.02 (s,
6H, 2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.6, 157.0, 155.9, 145.4, 138.0, 133.0,
128.7, 128.2, 126.3, 125.4, 117.6, 45.6, 37.9, 30.3, 27.8; IR (KBr) 3409, 3249, 3149, 2952, 1588,
1493, 1441, 1289, 1079, 853, 782, 688 cm-1; MS (ESI): m/z 325 [M+H]+; HR-MS (ESI) Calcd for
C18H21N4S [M+H]+: 325.14840, found: 325.14814.
4.2.13. (E)-2-(7,7-dimethyl-2-p-tolyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazinecarbothio
amide (4m)
Colorless solid, yield: 0.34 g, 78%; mp: 244-246 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.28 (br
s, 1H, NH), 8.76 (d, J = 8.2 Hz, 1H, Ar-H), 8.33 (br s, 1H, NH), 8.13 (br s, 1H, NH), 8.08-7.98
(m, 2H, Ar-H), 7.77 (d, J = 8.2 Hz, 1H, Ar-H), 7.36-7.26 (m, 2H, Ar-H), 2.84 (s, 2H, CH2), 2.63
(s, 2H, CH2), 2.36 (s, 3H, CH3), 1.00 (s, 6H, 2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ
178.7, 157.1, 155.9, 145.7, 138.6, 135.3, 133.3, 129.1, 126.3, 125.3, 117.5, 45.7, 38.0, 30.4, 27.9,
20.8; IR (KBr) 3426, 3251, 3154, 2954, 1589, 1493, 1452, 1440, 1293, 1183, 1075, 818, 750, 673
cm-1; MS (ESI): m/z 339 [M+H]+; HR-MS (ESI) Calcd for C19H23N4S [M+H]+: 339.16405, found:
339.16379.
19 | P a g e

4.2.14. (E)-2-(2-(4-methoxyphenyl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)ylidene)hydra
zinecarbothioamide (4n)
Colorless solid, yield: 0.35 g, 81%; mp; 226-228 0C 1H NMR (300 MHz, DMSO-d6): δ 10.14 (br
s, 1H, NH), 8.60 (d, J = 8.2 Hz, 1H, Ar-H), 8.20 (br s, 1H, NH), 8.03-7.88 (m, 3H, Ar-H), 7.59
(d, J = 8.2 Hz, 1H, Ar-H), 6.95-7.05 (m, 2H, Ar-H), 3.80 (s, 3H, OCH3), 2.80 (s, 2H, CH2), 2.57
(s, 2H, CH2), 1.01 (s, 6H, 2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.6, 160.1, 156.8,
155.5, 145.6, 133.2, 130.3, 127.8, 124.7, 117.0, 113.7, 54.9, 45.6, 37.9, 30.4, 27.8; IR (KBr) 3427,
3250, 3154, 2956, 1579, 1492, 1453, 1293, 1247, 1179, 1075, 847, 751, 673 cm-1; MS (ESI): m/z
355 [M+H]+ ; HR-MS (ESI) Calcd for C19H23N4OS [M+H]+: 355.15919, found: 355.15871.
4.2.15. (E)-2-(2-(biphenyl-4-yl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazine
carbothioamide (4o)
Colorless solid, yield: 0.33 g, 79%; mp: 218-220 0C 1H NMR (300 MHz, DMSO-d6): δ 10.32 (br
s, 1H, NH), 8.83 (d, J = 8.2 Hz, 1H, Ar-H), 8.37 (br s, 1H, NH), 8.29-8.17 (m, 3H, Ar-H), 7.88 (d,
J = 8.2 Hz, 1H, Ar-H), 7.84-7.67 (m, 4H, Ar-H), 7.58-7.48 (m, 2H, Ar-H), 7.46-7.38 (m, 1H, Ar-
H), 2.87 (s, 2H, CH2), 2.65 (s, 2H, CH2), 1.01 (s, 6H, 2CH3); 13C NMR (75 MHz, DMSO-d6): δ
178.7, 157.1, 155.1, 145.5, 140.7, 139.2, 136.7, 133.8, 128.8, 127.6, 127.1, 126.7, 126.5, 125.9,
118.0, 45.4, 37.9, 30.4, 27.8; IR (KBr) 3425, 3250, 3150, 2955, 1579, 1493, 1452, 1282, 1075,
830, 753, 665 cm-1; MS (ESI): m/z 401 [M+H]+; HR-MS (ESI) Calcd for C24H25N4S [M+H]+:
401.17995, found: 401.17944.
4.2.16.
(E)-2-(7,7-dimethyl-2-(naphthalen-2-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydra
zinecarbothioamide (4p)
Colorless solid, yield: 0.36 g, 84%; mp: 242-244 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.27 (br
s, 1H, NH), 8.78 (d, J = 8.2 Hz, 1H, Ar-H), 8.64 (br s, 1H, NH), 8.37-8.23 (m, 2H, Ar-H), 8.13 (br
s, 1H, NH), 8.04-7.85 (m, 4H, Ar-H), 7.56-7.48 (m, 2H, Ar-H), 2.89 (s, 2H, CH2), 2.64 (s, 2H,
CH2), 1.04 (s, 6H, 2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.7, 157.2, 155.5, 145.5,
135.4, 133.3, 133.1, 132.8, 128.4, 127.9, 127.2, 126.4, 126.1, 125.76, 125.70, 124.1, 118.1, 45.7,
37.9, 30.4, 27.8; IR (KBr) 3411, 3241, 3144, 2950, 1584, 1496, 1448, 1291, 1077, 855, 750, 673
cm-1; MS (ESI): m/z 375 [M+H]+ ; HR-MS (ESI) Calcd for C22H23N4S [M+H]+: 375.16444, found:
375.16379.
4.2.17. (E)-2-(2-(4-fluorophenyl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazine
carbothioamide (4q)
20 | P a g e

Colorless solid, yield: 0.36 g, 82%; mp: 244-246 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.26 (br
s, 1H, NH), 8.74 (d, J = 8.5 Hz, 1H, Ar-H), 8.32 (br s, 1H, NH), 8.19-8.09 (m, 3H, Ar-H), 7.74 (d,
J = 8.5 Hz, 1H, Ar-H), 7.29-7.20 (m, 2H, Ar-H), 2.82 (s, 2H, CH2), 2.61 (s, 2H, CH2), 1.06 (s, 6H,
2CH3); 13C NMR (75 MHz, DMSO-d6) δ: 178.7, 162.8 (d, J = 246.4 Hz, 1C), 157.2, 154.7, 145.6,
134.5 (d, J = 2.2 Hz, 1C), 133.6, 128.7 (d, J = 8.8 Hz, 2C), 125.6, 117.7, 115.4 (d, J = 21.4 Hz,
2C), 45.6, 37.9, 30.4, 27.8; IR (KBr) 3401, 3223, 3146, 2953, 1584, 1496, 1452, 1291, 1214, 1073,
748, 670 cm-1; MS (ESI): m/z 343 [M+H]+; HR-MS (ESI): Calcd for C18H20N4FS [M+H]+
343.13911, found: 343.13872.
4.2.18.(E)-2-(2-(4-chlorophenyl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)hydrazine
carbothioamide (4r)
Colorless solid, yield: 0.34 g, 82%. mp: 248-250 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.32 (br
s, 1H, NH), 8.81 (d, J = 8.3 Hz, 1H, Ar-H), 8.37 (br s, 1H, NH), 8.26-8.12 (m, 3H, Ar-H), 7.84 (d,
J = 8.3 Hz, 1H, Ar-H), 7.62-7.52 (m, 2H, Ar-H), 2.84 (s, 2H, CH2), 2.64 (s, 2H, CH2), 1.00 (s, 6H,
2CH3); 13C NMR (75 MHz, DMSO-d6): δ 178.8, 157.5, 154.6, 145.8, 136.9, 134.1, 133.7, 128.7,
128.3, 126.1, 118.1, 45.7, 38.0, 30.5, 27.9; IR (KBr) 3406, 3248, 3148, 2954, 1587, 1497, 1450,
1288, 1089, 853, 751 cm-1; MS (ESI): m/z 359 [M+H]+; HR-MS (ESI) Calcd for C18H20N4ClS
[M+H]+: 359.10932, found: 359.10917.
4.2.19.
(E)-2-(2-(4-bromophenyl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)
hydrazine carbothioamide (4s)
Colorless solid, yield: 0.29 g, 78%; mp: 226-228 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.20 (br
s, 1H, NH), 8.66 (d, J = 8.5 Hz, 1H, Ar-H), 8.24 (br s, 1H, NH), 8.04-7.95 (m, 3H, Ar-H), 7.68 (d,
J = 8.5 Hz, 1H, Ar-H), 7.60-7.52 (m, 2H, Ar-H), 2.81 (s, 2H, CH2), 2.58 (s, 2H, CH2), 1.00 (s, 6H,
2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.7, 157.1, 154.6, 145.3, 137.1, 133.2, 131.2,
128.2, 125.8, 122.7, 117.6, 45.6, 37.9, 30.3, 27.8; IR (KBr) 3427, 3249, 3147, 2956, 2337, 1577,
1496, 1449, 1281, 1074, 823, 764, 663 cm-1; MS (ESI): m/z 403 [M+H]+; HR-MS (ESI) Calcd for
C18H20N4BrS [M+H]+: 403.05950, found: 403.05866.
4.2.20. (E)-2-(7,7-dimethyl-2-(thiophen-2-yl)-7,8-dihydroquinolin-5(6H)-ylidene)hydrazine
carbothioamide (4t)
Colorless solid, yield: 0.43 g, 80%; mp: 236-238 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.28 (br
s, 1H, NH), 8.73 (d, J = 8.3 Hz, 1H, Ar-H), 8.35 (br s, 1H, NH), 8.19 (br s, 1H, NH), 7.87 (dd, J =
3.9, 0.9 Hz, 1H, Ar-H), 7.75 (d, J = 8.3 Hz, 1H, Ar-H), 7.65 (dd, J = 4.9, 0.9 Hz, 1H, Ar-H), 7.17
21 | P a g e

(dd, J = 4.9, 3.9 Hz, 1H, Ar-H), 2.77 (s, 2H, CH2), 2.61 (s, 2H, CH2), 0.99 (s, 6H, 2CH3); 13C NMR
(75 MHz, DMSO-d6): δ 178.7, 157.3, 151.7, 145.7, 144.1, 133.4, 128.8, 128.4, 125.8, 125.4,
116.6, 45.3, 37.9, 30.4, 27.8; IR (KBr) 3406, 3248, 3146, 2952, 1586, 1495, 1453, 1288, 1104,
1078, 845, 710, 663 cm-1; MS (ESI): m/z 331 [M+H]+; HR-MS (ESI) Calcd for C16H19N4S2
[M+H]+: 331.10422, found: 331.10456.
4.2.21. (E)-2-(2-(5-chlorothiophen-2-yl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)hy
drazinecarbothioamide (4u)
Colorless solid, yield: 0.32 g, 82%. mp:243-245 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.30 (br
s, 1H, NH), 8.76 (d, J = 8.2 Hz, 1H, Ar-H), 8.36 (br s, 1H, NH), 8.21 (br s, 1H, NH), 7.81-7.73
(m, 2H, Ar-H), 7.19 (d, J = 4.1 Hz, 1H, Ar-H), 2.75 (s, 2H, CH2), 2.60 (s, 2H, CH2), 0.98 (s, 6H,
2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.6, 157.0, 150.7, 145.2, 143.1, 133.1, 131.1,
127.5, 125.6, 124.5, 115.8, 45.2, 37.8, 30.2, 27.7; IR (KBr) 3404, 3242, 3146, 1589, 1496, 1455,
1288, 1106, 1031, 850, 789, 756, 638 cm-1; MS (ESI): m/z 365 [M+H]+; HR-MS (ESI) Calcd for
C16H18N4S2Cl [M+H]+: 365.06623, found: 365.06559.
4.2.22. (E)-2-(2-(5-bromothiophen-2-yl)-7,7-dimethyl-7,8-dihydroquinolin-5(6H)-ylidene)hy
drazinecarbothioamide (4v)
Colorless solid, yield: 0.30 g, 81%. mp: 252-254 0C; 1H NMR (300 MHz, DMSO-d6): δ 10.18 (br
s, 1H, NH), 8.63 (d, J = 8.5 Hz, 1H, Ar-H), 8.24 (br s, 1H, NH), 8.01 (br s, 1H, NH), 7.78-7.62
(m, 2H, Ar-H), 7.12 (d, J = 3.8 Hz, 1H, Ar-H), 2.73 (s, 2H, CH2), 2.56 (s, 2H, CH2), 0.99 (s, 6H,
2CH3); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 178.6, 157.0, 150.6, 145.8, 145.2, 133.1, 131.0,
125.6, 125.4, 115.8, 114.5, 45.1, 37.8, 30.2, 27.7; IR (KBr) 3405, 3240, 3146, 2955, 1575, 1494,
1454, 1283, 1105, 1072, 848, 756, 663 cm-1; MS (ESI): m/z 409 [M+H]+; HR-MS (ESI) Calcd for
C16H18N4S2Br [M+H]+: 409.01561; found: 409.01508.
4.3. Antitubercular evaluation assay
All the compounds were further screened for in vitro antimycobacterial activity against M.
Tuberculosis H37Rv strain by microplate Alamar blue assay method. Briefly, the inoculum was
prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5%
glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted to a
McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution
was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-
22 | P a g e

fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl
7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on each
plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation.
The plate was covered, sealed in plastic bags and incubated at 37oC in normal atmosphere. After
7 days incubation, 30 ml of Alomar blue solution was added to each well, and the plate was re-
incubated overnight. A change in color from blue (oxidized state) to pink (reduced) indicated the
growth of bacteria, and the MIC was defined as the lowest concentration of drug that prevented
this change in color.
4.4. Evaluation of cytotoxicity
The in vitro cytotoxicity of the antitubercular active compounds with MIC ≤12.5µg/mL were
assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against
growth inhibition of HEK-293 (Human Embryonic Kidney) cells at 50 μg/mL concentration. Cell
lines were maintained at 37 oC in a humidified 5% CO2 incubator (Thermo scientific). Detached
the adhered cells and followed by centrifugation to get cell pellet. Fresh media was added to the
pellet to make a cell count using haemocytometer and plate 100μL of media with cells ranging
from 5,000-6,000 per well in a 96-well plate. The plate was incubated overnight in CO2 incubator
for the cells to adhere and regain its shape. After 24 h cells were treated with the test compounds
at 50μg/mL diluted using the media to deduce the percentage inhibition on normal cells. The cells
were incubated for 48 h to assay the effect of the test compounds. Zero hour reading was noted
down with untreated cells and also control with 1% DMSO to subtract further from the 48 h
reading. After 48 h incubation, cells were treated by MTT (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) dissolved in PBS (5 mg/mL) and incubated for 3-4 h at 37 oC. The
formazan crystals thus formed were dissolved in 100μL of DMSO and the viability was measured
at 540 nm on a multimode reader (Spectra max). The values were further calculated for percentage
inhibition which in turn helps us to know the cytotoxicity of the test compounds.
4.5. Nutrient starvation model
M. tuberculosis (H37Rv strain) culture (O.D. of 0.8-1.0) grown in Middlebrook 7H9 broth
supplemented with OADC (growth supplement) was centrifuged and pellet was washed twice with
PBS (Phosphate Buffer Saline, HiMedia) to remove broth completely. Further pellet was re-
23 | P a g e

suspended in PBS in sealed bottles and incubated at 37 °C for six weeks. During this time bacteria
will go into dormant stage followed by inhibitory testing with standard drugs like Ionized (INH),
and Moxifloxacin (MOX) along with synthesized compounds for 7 days at a concentration of 10
µg/mL. Next step is to dilute cell suspensions 10-fold up to 10-6 using Middlebrook 7H9 broth
with 10% OADC and 100 µl of each dilution was plated in sterile 48 well plates containing 450
ml of Middlebrook 7H9 broth with 10% OADC in triplicates. The plates were kept for incubation
at 37 °C for four weeks. Wells with visible bacterial growth were counted as positive, and most
probable number (MPN) values were calculated using standard statistical methods [33,34].
4.6. In vivo activity assay in zebrafish
Most active compound from in vitro assays was taken for testing at in vivo level. M. marinum
strain (ATCC BAA-535) (genetically a close relative of M. tuberculosis) incubated at 30 °C in
Middlebrook 7H9 broth was infected to adult zebrafish as follows. Fishes were initially weighed
and divided accordingly into control and treatment groups (n = 6). All the groups were infected by
intraperitoneal route with 20 µl of bacterial culture (around 0.75 million bacterial cells) for a week
maintaining fish chamber at 25 °C. After the infection has attained, fishes were administered with
test drug orally for a 7 day treatment period with 10 mg/kg dose (for INH 5 mg/kg). Further after
treatment period was completed the fishes were sacrificed (fishes were suspended for few minutes
in Kanamycin to kill the external bacteria on fishes and to minimize the interference of any other
bacteria), the tissue was homogenized and sample was prepared in Middlebrook 7H9 broth. The
plates were checked for bacterial count using MPN assay method [35].
4.7. Molecular docking studies in silico
In silico analysis were performed in Dell Precision T7610 workstation (8 processors; 8 GB RAM;
NVIDIA 3GB graphics; Maestro 9.8, Schrodinger, New York, U.S.A) workstation running on
Redhat 6.1 Linux environment. The structure of ligand was drawn in ChemDraw Ultra 6.0. The
3D coordinate file of target protein was retrieved from protein data bank (PDB). Molecular
docking studies were performed against Mycobacterium tuberculosis enzymes, enoyl reductase
(InhA), PDB ID: 4TZK; Decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1), PDB ID: 4P8N;
Alcohol dehydrogenase (ADH), PDB ID: 2VHW; (3R)-hydroxyacyl-ACP dehydratase (HadAB),
PDB ID:4RV2; Lysine-ɛ aminotransferase (LAT), PDB ID: 2CJD; Topoisomerase (DNA Gyrase),
24 | P a g e

PDB ID: 5BS8 and Glutamate recemase (MurI), PDB ID: 5H7J. The protein was prepared with
the help of Protein Preparation Wizard of Schrödinger Suite 9.8. The prepared protein was
optimized and minimized using algorithm OPLS_2005 (optimized potential for liquid simulations)
force field and grid was generated using the Glide Grid Generation panel in Glide. The co-
crystallized inhibitor 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide and
test compounds were energy minimized using LigPrep module. The minimized test compounds
were docked using Glide XP docking calculations. The XP Glide scoring function was used to get
the best ranked compounds and the specific interactions like H-bonds, π-cation and π-π stacking
and van der Waals were analyzed using XP visualizer in Glide module [36,38].
4.8. Molecular Dynamics simulations
The Desmond module from Schrodinger was used for running MD simulations with periodic
boundary conditions. Receptor and ligand complex was immersed in an orthorhombic simulation
box, with the TIP4P explicit water model using the System Builder Panel with the minimum
thickness of a solvent layer, 10 Å. In order to neutralize the system, counter ions were added.
Before equilibration and long production of MD simulations, the systems were minimized and pre-
equilibrated using relaxation routine implemented in Desmond. Whereas, program ran six steps
composed a) energy minimization was used by hybrid method of steepest descent and limited-
memory Broyden-Fletcher- Goldfarb-Shanno (LBFGS) algorithm with a maximum steps of 2000
including preliminary 10 steps of steepest descent with solute restrained, b) Energy minimization
for 2000 steps without solute restraints, c) 12ps simulation in NVT ensemble (temperature 10K)
restraining nonhydrogen solute atoms, d) 12ps simulation in the NPT ensemble (temperature 10K)
restraining non-hydrogen solute atoms, e) 24ps simulation in the NPT ensemble restrained along
with solute non-hydrogen atoms (temperature 300K) and f) 24ps simulation in the NPT ensemble
(temperature 300K) with no restraints respectively. The temperatures and pressures in the short
initial simulations were checked by applied Berendsen thermostats and barostats algorithms. The
equilibrated system was simulated for 50ns with a time step of 2fs, NPT ensemble was used a
Nose-Hoover thermostat at 300K and Martyna-Tobias- Klein barostat at 1.01325 pressure bar. A
time step of 1.2fs was used. Saving energy and structure enumerated for every 9.6 ps during
simulation, the MD trajectory was generated. Finally to analyze trajectory simulation, a simulation
interaction diagram tool was used [39, 40].
25 | P a g e

4.9 X-ray Crystallography: X-ray data for the compound 4j was collected at room temperature
using a Bruker Smart Apex CCD diffractometer with graphite monochromated MoK radiation
(=0.71073Å) with ω-scan method. Preliminary lattice parameters and orientation matrices were
obtained from four sets of frames. Integration and scaling of intensity data were accomplished
using SAINT program [41]. The structure was solved by direct methods using SHELXS97 and
refinement was carried out by full-matrix least-squares technique using SHELXL97 [42].
Anisotropic displacement parameters were included for all non-hydrogen atoms. The N-bound H
atom was located in difference Fourier maps, and their positions and isotropic displacement
parameters were refined. All other H atoms were located in difference Fourier maps and
subsequently geometrically optimized and allowed for as riding atoms, with C-H = 0.93- 0.97 Å,
with Uiso(H) = 1.5Ueq(C) for methyl H or 1.2Ueq(C). The methyl groups were allowed to rotate but
not to tip.
Crystal Data for 4j: C16H19N4OS3Cl (M =415.01 g/mol): monoclinic, space group P21/c (no. 14),
a = 17.1430(6) Å, b = 9.4670(4) Å, c = 12.4260(8) Å, β = 101.909(2)°, V = 1973.24(17) Å3, Z =
4, T = 294.15 K, μ(Mo Kα) = 0.523 mm-1, Dcalc = 1.3969 g/cm3, 22404 reflections measured
(4.86° ≤ 2Θ ≤ 56.74°), 4755 unique (Rint = 0.0180, Rsigma = 0.0144) which were used in all
calculations. The final R1 was 0.0489 (I>2σ(I)) and wR2 was 0.1440 (all data). CCDC 1511250
contains supplementary Crystallographic data for the structure. These data can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic
Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email:
deposit@ccdc.cam.ac.uk].
Acknowledgement:
The authors thank Science and Engineering Research Board (SERB), the Department of Science
and Technology, the Government of India (EMR/2017/002946) for financial support. SKM is
thankful to UGC for senior research fellowship. CSIR- IICT communication no. is IICT/Pubs./
2019/076.
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