New bithiazolyl hydrazones: Novel synthesis, characterization and antitubercular evaluation

Article abstract of DOI:10.1016/j.bmcl.2016.11.056

New bithiazolyl hydrazones (6a–l) have been first time synthesized by carrying novel one pot cyclocondensation of 5-acyl thiazoles (1a–b), thiosemicarbazide (2) and substituted phenacyl chlorides (4a–f) in freshly prepared ionic liquid, diisopropyl ethyl ammonium acetate (DIPEAc) at room temperature. The newly synthesized compounds have been evaluated for their antitubercular activity and the compounds 3b, 6a, 6b, 6d, 6e, 6f, 6g, and 6l have displayed noticeable antitubercular activity compared to Rifampicin with tolerable cytotoxicity. All these compounds were also screened for their antibacterial activity and found that, compounds 6j and 6k have exhibited a very good antibacterial activity. Molecular docking study has shown better harmony with the evaluation trend shown by these compounds under in vitro antitubercular screening.

Full text of DOI:10.1016/j.bmcl.2016.11.056

Accepted Manuscript
New bithiazolyl hydrazones: novel synthesis, characterization and antitubercu-
lar evaluation
Mahendra B. Bhalerao, Sambhaji T. Dhumal, Amarsinh R. Deshmukh, Laxman
U. Nawale, Vijay Khedkar, Dhiman Sarkar, Ramrao A. Mane
PII:
S0960-894X(16)31208-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.056
BMCL 24451
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 August 2016
22 October 2016
21 November 2016
Please cite this article as: Bhalerao, M.B., Dhumal, S.T., Deshmukh, A.R., Nawale, L.U., Khedkar, V., Sarkar, D.,
Mane, R.A., New bithiazolyl hydrazones: novel synthesis, characterization and antitubercular evaluation,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.056
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New bithiazolyl hydrazones: novel synthesis, characterization and
antitubercular evaluation
Mahendra B. Bhalerao a, Sambhaji T. Dhumal a, Amarsinh R. Deshmukh a, Laxman U. Nawale b,
Vijay Khedkar c, Dhiman Sarkar b, Ramrao A. Mane a,*
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad
431004, Maharashtra, India.
bCombi-Chem Bio Resource Center, CSIR-National Chemical Laboratory, Pune 411008,
Maharashtra, India.
cSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa.
*Corresponding author Email: manera2011@gmail.com
Abstract
New bithiazolyl hydrazones (6a-l) have been first time synthesized by carrying novel one pot
cyclocondensation of 5-acyl thiazoles (1a-b), thiosemicarbazide (2) and substituted phenacyl
chlorides (4a-f) in freshly prepared ionic liquid, diisopropyl ethyl ammonium acetate (DIPEAc)
at room temperature. The newly synthesized compounds have been evaluated for their
antitubercular activity and the compounds 3b, 6a, 6b, 6d, 6e, 6f, 6g, and 6l have displayed
noticeable antitubercular activity compared to Rifampicin with tolerable cytotoxicity. All these
compounds were also screened for their antibacterial activity and found that, compounds 6j and
6k have exhibited a very good antibacterial activity. Molecular docking study has shown better
harmony with the evaluation trend shown by these compounds under in vitro antitubercular
screening.
Keywords: Thiosemicarbazones, Bithiazole, Ionic liquid, Antitubercular Activity, Antibacterial
Activity, Cytotoxicity, Molecular Docking.
Tuberculosis (TB), a contagious disease responsible for high mortality worldwide, is now
coexisting with human immunodeficiency virus (HIV) and causing serious threat to mankind.1,2
Nowadays, the tuberculosis strains are found to become resistant to existing first line as well as
second line multi drugs and therefore treating multi drug resistant (MDR) and extremely drug
resistant (XDR) strains are becoming challenge for medicinal chemists.3 Recently, new drug
diarylquinoline (bedaquiline) has been introduced for treating the MDR-TB strains. However,
these efforts are not completely satisfying the present threat of TB treatments. Therefore, in
search of safer and potentially active new antitubercular agents attempts are found to generate
library of new entities having various biodynamic heteryl scaffolds and active pharmacophores.4
Literature also reveals that, thiazole scaffold is well explored and it is a key constituent of
various medicaments.5-11 It is also reported that, thiazoles having other heteryl scaffolds and
1

pharmacophores viz. pyridyl,12 aryl,13,14 thiazolyl,2 amino,15 hydrazolyl,16 triazolyl,17
piperidinyl18,19 and thiosemicarbazonyl20,21 have displayed noticeable antitubercular activity.
Thiosemicarbazones and hydrazones/hydrazides have long attracted attention, due to their
biological and pharmacological properties. Thiosemicarbazides are a key component of drugs
like, triapine (anticancer), metisazone (small pox), MAIQ (anticancer), isonicotinaldehyde
thiosemicarbazone (antitubercular), thiacetazone (antitubercular), and ambazone (antiseptic)
(Fig. 1).20 Hydrazones are well explored as prodrugs for antitubercular treatment.22-24
HN
N
H3C
NH NH2
S
NH
N
S
O
N
H2N
Isonicotinaldehyde thiosemicarbazone
(Antitubercular)
Thiacetazone
(Antitubercular)
HO
S
N
O
H
N
N
OH
O
N
N
OH
H
N
N
N
N
N
S
S
N
N
S
S
O
Cl
O
Micrococcinic acid
(Antibiotic)
Dasatinib
(antineoplastic agent)
Figure 1. Thiazoles, amino thiazoles and thiosemicarbazones based drug candidates.
It is reviewed that, there is scanty information on the bithiazolyl scaffolds bridged with
hydrazolyl pharmacophore in single molecular architectural frame. Therefore, it was thought to
design and synthesize such molecular frameworks to achieve synergistic effect to get the entities
with potential antitubercular activity. Hence, in the present work, it was decided to synthesize
new bithiazolyl hydrazones (6a-l) and evaluate them for their antitubercular, cytotoxic, and
antibacterial activities.
The synthesis of title bithiazolyl hydrazones (6) was initially attempted in two steps, path
first and path second (Scheme 1).25 To overcome the drawbacks of these paths and to obtain
better yields of the title products, here then efforts were directed to carry one pot
cyclocondensation of freshly prepared and readily available, 5-acyl thiazoles (1a, 1b)
2

thiosemicarbazide (2) and substituted phenacyl chlorides (4a-f) in reflux ethanol (Scheme 2,
Route A).26 It was observed that though there was a little enhancement in the yields but time
required for completion of cyclocondensation was pretty long i.e. more than 4 h (Table S2). To
overcome these drawbacks and to practice green principles, here we then attempted this one pot
cyclocondensation in various ionic liquids27 at room temperature (Table S1). After screening the
ionic liquids, it was found that diisopropyl ethyl ammonium acetate (DIPEAc)28 was a suitable
and novel medium for carrying the cyclocondensation leading to title products with excellent
yields (Table S1, entry 7). The advantage of DIPEAc is that, it is stable, easily synthesized, cost
effective, and recyclable.28
Hence, a mixture of three components, 5-acyl thiazoles (1a-b), thiosemicarbazide (2),
and phenacyl chlorides (4a-f) (Scheme 2, Route B)29 was dissolved in freshly prepared non-
volatile organic solvent, DIPEAc and the solution was stirred at room temperature for about 30
min. Then, the products (6a-l) were isolated with enhanced yields, 82-96 % (Table S2).
The rate acceleration and enhancement of the yields of bithiazolyl hydrazones (6a-l)
would be due to in situ successive condensations of 5-acyl thiazoles (1a-b), thiosemicarbazide
(2), and phenacyl chlorides (4a-f). Initially, in situ phenacyl chlorides might be rapidly
cyclocondensed with thiosemicarbazide yielding the intermediates, 2-hydrazinyl thiazoles (5a-f)
which then subsequently be condensed in situ with available 5-acyl thiazoles, giving the titled, 1-
(1-(2-phenyl/
methyl-4-methylthiazol-5-yl)
ethylidene)-2-(4-substitutedphenylthiazol-2-yl)
hydrazines (6a-l).
Here in this protocol DIPEAc might be displaying dual role. Its significant role could be a
safer and non-volatile medium having ability to dissolve all the reactants at room temperature,
generating their saturated solutions forming homogeneous mass. This initial high concentration
of the reactants in the reaction solution might be helping for the rate acceleration of initial
cyclocondensation and subsequent condensation. The other role of DIPEAc might be catalytical
and the plausible mechanism, responsible for rate acceleration is depicted in the Fig. 2.
3

Path first:
N
O
R
S
Cl
N
N
S
NH
R
(4)
N
S
Ethanol,
N
+
H2NHN
NH2
S
N
R
S
Ethanol, Reflux, Stir, 1 h
Reflux, Stir, 1.5 h
HN
O
S
H2N
(2)
(1a, 1b)
(6a, 6b)
(3a, 3b)
Where,
R = -CH3, -Ph
Path second:
N
N
R
O
R
S
S
S
O
N
Cl
+
Ethanol,
(1a, 1b)
Ethanol, Reflux, Stir, 2 h
N
NH
H2N N
NH2
H
Reflux, Stir, 1 h
NHNH2
S
S
N
(2)
(4)
(5)
(6a, 6b)
Where,
R = -CH3, -Ph
Scheme 1. Synthesis of 1-(1-(2,4-dimethylthiazol-5-yl) ethylidene)-2-(4-phenylthiazol-2-yl)
hydrazine (6).
N
Ethanol,
Route A Reflux, 4h
R1
S
O
S
N
N
Cl
NH
+
NH2
+
H2N
N
R1
H
S
O
R2
S
N
(1a-b)
(2)
(4a-f)
Route B
DIPEAc, rt,
Stir, 30 min.
(6a-l)
R2
Where, R1 = -CH3, -Ph
R2 = -H, -F, -Cl, -Br, -CH3, -OCH3
Scheme 2. One pot synthesis of 1-(1-(2-Phenyl/methyl-4-methylthiazol-5-yl) ethylidene)-2-(4-
substitutedphenylthiazol-2-yl) hydrazines (6a-l) using ethanol and DIPEAc.
4

N
H
N
H
O
N
Cl
H
(4a-f)
O
R2
HO
N
S
H
S
H
H2N N
C
H2N N C N H
H
HCl
N
H
H
H2NHN
R2
S
(2)
O
H
O
O
O
O
O
H2O
R2
O
O
N
N
H
R1
S
N
H
N
HO
N
H N N
H
NH
S
S
R1
N
N H
NH
(5a-f)
S
N
O
R1
H2O
S
O
O
S
N
(1a-b)
H
R2
(6a-l)
N
R2
Figure 2. Plausible mechanism of successive one pot condensations leading to 1-(1-(2-
Phenyl/methyl-4-methylthiazol-5-yl) ethylidene)-2-(4-substitutedphenylthiazol-2-yl) hydrazines
(6a-l), carried in DIPEAc ionic liquid.
All the newly synthesized compounds were evaluated for their in vitro antitubercular activity
against Mycobacterium tuberculosis H37Ra (ATCC 25177) and Mycobacterium bovis Bacillus
Calmette-Guerin (BCG) (ATCC 35743) at dormant (12 days incubation) and active (8 days
incubation) stages at concentrations of 0.7, 1.5, 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL. Activity
against M. bovis was estimated through nitrate reductase (NR) assay, reading absorbance at 540
nm.30 XTT reduction menadione assay (XRMA) was performed to determine the inhibition of M.
tuberculosis.31 The absorbance of XRMA was measured at 470 nm.
In vitro studies against M. tuberculosis H37Ra and M. bovis BCG revealed notable
antitubercular activity of the entities 3b, 6a, 6b, 6d, 6e, 6f, 6g, and 6l (Table 1). Among these
5

eight active compounds, 1-(1-(4-methyl-2-phenylthiazol-5-yl) ethylidene) thiosemicarbazide
(3b) and 2-(4-(4-methoxyphenyl) thiazol-2-yl)-1-(1-(4-methyl-2-phenylthiazol-5-yl) ethylidene)
hydrazine (6l) were found to be highly effective to inhibit both M. tuberculosis H37Ra and M.
bovis BCG in active and dormant states, with MICs ranging from 1.78 to 14 μg/mL. MIC (<15
μg/mL) of thiazoles corroborate their well-known antimicrobial nature.32 However, the activity is
not profound as that of rifampicin (MIC 0.8 µg/mL).
Amongst the tested compounds, compound 3b has thiosemicarbazonyl pharmacophore and
phenyl moiety on thiazole ring came out as the most active compound against M. tuberculosis
H37Ra (MIC in active stage, 1.78 g/mL and in dormant stage, 2.05 g/mL) and M. bovis BCG
(MIC in active stage, 3.04 g/mL and in dormant stage, 3.46 g/mL). The compound 6l has
phenyl moiety in one of the thiazoles and 4-methoxy phenyl in other thiazole ring system has
displayed better inhibition against M. tuberculosis H37Ra with MIC of 6.87 g/mL in the active
stage and 7.15 g/mL in dormant stage.
The compounds 6b, 6d, and 6l exhibited prominent antitubercular activity against M. bovis
BCG with MICs in the range of 3.04-13.51 g/mL and 3.46-14 g/mL in the active and dormant
stage respectively. From the standpoint of structure activity relationship, it was observed that the
electronic environment of substituents on phenyl ring at R2 position had no significant effect on
biological activity as the active compounds had both electron withdrawing as well as donating
substituents on them.
The pattern was similar in ex vivo THP-1 infection model assay with thiazolyl
thiosemicarbazones (3a, 3b) and bithiazolyl hydrazones (6a-l), having highest efficiency to
inhibit M. tuberculosis (Table 1). MIC of dormant stage mycobacteria was observed to be
greater than its active stage.
In view of this, the overall antitubercular activity exhibited by these thiazolyl
thiosemicarbazones (3a, 3b) and bithiazolyl hydrazones (6a-l) is significant, although they
possess lower potencies as compared to that of standard drug, Rifampicin. Further experiments
were carried out with the most effective compounds (3b, 6g and 6l).
Generally hydrazone derivatives are considered to be nontoxic.33 Therefore, additionally
all thiazolyl thiosemicarbazones (3a, 3b) and bithiazolyl hydrazones (6a-l) were evaluated for
their in vitro cytotoxic effect against three human cancer cell lines, MCF-7, HCT116 and THP-1
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with paclitaxel
6

Table 1
Antitubercular activities (Ex vivo, in vitro) and cytotoxicity profile (In vitro) against THP-1(Leukemia) human cancer cell line of
thiazolyl thiosemicarbazones (3a, 3b) and bithiazolyl hydrazones (6a-l)
Ex vivo
antitubercular
activity (MIC90,
µg/mL)
Cytotoxicity
In vitro antitubercular activity
(IC50, µg/mL)
In vitro antitubercular activity
profile
(GI50,
µg/mL)
THP-1
(MIC90, µg/mL)
Compound
MTB H37Ra
MTB H37Ra
M. bovis BCG
MTB H37Ra
M. bovis BCG
(Active) (Dormant) (Active) (Dormant) (Active) (Dormant) (Active) (Dormant) (Active) (Dormant) (Leukemia)
3a
>100
>100
0.21
0.12
0.82
2.67
11.91
0.15
11.07
0.88
0.17
>100
0.12
0.18
0.16
0.078
0.51
-
0.19
0.78
2.00
2.04
9.02
0.66
8.35
1.24
0.98
>100
0.24
0.14
0.075
0.075
0.75
-
4.42
0.94
11.63
6.00
1.95
1.6
5.09
0.97
3.81
6.04
3.06
2.51
9.67
8.41
7.95
>100
26.58
>100
5.29
2.82
0.81
-
>100
1.78
25.34
25.27
>100
44.15
>100
74.07
9.46
>100
>100
>100
>100
6.87
0.51
-
30.54
>100
>100
>100
81.51
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.86
>100
>100
>100
0.1374
3b
1.94
3.67
2.05
3.04
3.46
6a
32.54
57.02
>100
47.88
74.32
48.23
15.58
>100
>100
>100
>100
12.31
0.5
23.41
44.27
>100
48.84
82.82
46.17
11.97
>100
>100
>100
>100
9.38
16.47
53.75
>100
43.73
65.76
20.49
12.76
>100
>100
57.74
>100
7.15
40.36
9.29
17.21
10.42
16.96
10.6
6b
6c
16.05
11.42
54.76
67.33
21.2
6d
6e
17.93
7.37
8.75
>100
16.98
>100
5.21
2.43
0.45
-
83.17
24.04
18.71
>100
83.68
>100
57.49
14
6f
6g
6h
>100
77.85
>100
19.66
13.51
0.45
6i
6j
6k
6l
Rifampicin
Paclitaxel
0.7
0.75
0.81
-
-
-
-
-
RIF: Rifampicin; MIC90: minimum inhibitory concentration for 90% (or greater) inhibition (µg/mL). GI50 indicates concentration to inhibit
50% growth of cells
7

as a positive control.34,35 The corresponding GI50 and GI90 values of cell lines (MCF-7,
HCT 116 and THP-1) have been calculated using OriginPro Software and are listed in Table S3.
GI50 (>100 µg/mL) values of all compounds (except compounds 3b and 6j) against cell lines
indicate that they are potent and specific inhibitors against MTB. The compounds, 3b and 6j
have shown antiproliferative activity with GI50 values in between 25.99 to 81.51μg/mL against
MCF-7, HCT 116 and THP-1 cell lines.
Selectivity index describes the selectivity of thiazolyl thiosemicarbazones (3a, 3b) and
bithiazolyl hydrazones (6a-l) toward human cell lines against M. tuberculosis (Table 2 and S4).
It reflects the amount of agent/drug is effective against mycobacteria without harming the host
cells. GI50 of these compounds against the tested cell lines was >100 μg/mL, except 3b and 6j.
Among these derivatives, 3b showed the highest selectivity index (9–49) in dormant and active
stage. Thus, a higher value of SI indicates that the compound can be used as a therapeutic agent.
Moreover, the antimycobacterial activity of an agent is considered to be specific when selectivity
index >10.36 In our report 3b, 6g and 6l exhibited high selectivity index, indicating their potential
as antitubercular agent and these should be investigated further.
Table 2
Selectivity index (SI) of thiazolyl thiosemicarbazone (3b) and bithiazolyl hydrazones (6g and 6l)
on human cell lines against active as well as dormant stage of M.tb. H37Ra and M. bovis BCG
SI on MCF-7
SI on HCT 116
SI on THP-1
Compound
Active Stage
Against
H37Ra
Against
BCG
9
Against
H37Ra
Against
BCG
16
Against
H37Ra
Against
BCG
27
3b
6g
15
27
46
11
15
5
7
11
15
5
7
11
15
5
7
6l
RIF
196
222
196
222
196
222
Dormant Stage
3b
6g
49
8
29
5
49
29
40
8
24
5
8
5
6l
14
133
7
14
133
7
14
133
7
RIF
123
123
123
8

For investigating the specificity of (3a, 3b) and (6a-l) compounds, all the compounds
were screened for their anti-bacterial activity against four bacteria strains (Gram-negative strains:
Escherichia coli, Pseudomonas aeruginosa; Gram-positive strains: Staphylococcus aureus,
Bacillus subtilis) and it was observed that, two compounds (6j, 6k) have displayed more than
90% inhibition in a compound concentration of 3 µg/ml (Table 3 and S5). The compounds (3a,
3b and 6a-l) exhibited MIC values >30 µg/ml. The compounds having good anti-bacterial
activities were further analyzed for determining the Minimal Inhibitory Concentration (MIC).
The calculation of MIC values for active anti-bacterial compounds was carried using Dose
response curve. The compounds 6j and 6k showed a very good anti-bacterial activity against E.
coli, S. aureus, P. aeruginosa and B. subtilis, with MIC values varying from 1.05 to 2.94 (Table
S6).
Table 3
Antibacterial activities of bithiazolyl hydrazones (6j-l) against E. coli, P. aeruginosa, S. aureus,
B. subtilis
E. coli
Gram-negative
MIC (µg/mL)
P. aeruginosa
S. aureus
Gram-positive
MIC (µg/mL) MIC (µg/mL)
B. subtilis
Compound
MIC (µg/mL)
6j
6k
6l
1.14
1.43
>30
1.46
1.05
1.53
>30
4.36
1.84
2.46
>30
1
1.95
2.94
>30
10.32
1.35
Ampicillin
Kanamycin
1.62
0.49
>30
Figure 3. Comparison of MIC in (g/ml) of compounds with some antibiotics.
9

The MICs of 6j and 6k compounds were found to be least as compared to others, not
showing selectivity towards three human cancer cell lines, nil activity towards M. tb. and BCG
shows that 6j and 6k can further be considered as potential anti-bacterial agent.
Molecular docking is a powerful tool in understanding different type of binding interactions
of a molecule to the biological receptor. Therefore to rationalize the biological data and to
investigate the possible interactions of the synthesized compounds (3a-b and 6a–l), molecular
docking study was performed with the target enzyme, decaprenylphosphoryl-β-D-ribose-2-
epimerase (DprE1) of M. tuberculosis. The crystal structure of M. tuberculosis DprE1 enzyme,
complexed with its inhibitor (pdb code: 4FDO) was used for the docking study.37,38
The objective of this work was to introduce bithiazolyl hydrazones as a potential scaffold
against M. tuberculosis targeting DprE1. In the absence of available resources for the
experimental enzyme-based assays, molecular docking has emerged as a very important tool to
identify the targets for different ligands and the associated thermodynamic interactions with the
target enzyme.39-41 DprE1 is a highly conspicuous target being inhibited by multiple scaffold
which motivated us to evaluate the binding affinity of the title class of molecules (bithiazolyl
hydrazones) against this enzyme.42-44 The significant correlation obtained between the docking
scores and the in vitro antitubercular activity provides us with an opportunity to adopt the
structure-based drug design approach i.e. molecular docking to optimize the title scaffold for this
target DprE1. In order to carry out enzyme-based assay, we are utilizing the outcomes of current
docking study to optimize this scaffold for designing more specific and tighter binding
compounds. Once we arrive at compounds with very potent activity in the in vitro assay (which
is one requirement to perform enzyme-based assay) we will perform assay against DprE1. In the
future communication we will present the outcome of our designed bithiazolyl hydrazones based
on molecular docking approach with enzyme-based activity against DprE1.
Molecular docking study revealed that all the bithiazolyl hydrazones could perfectly fit
into the active site of DprE1 with varying degree of affinities adopting a very similar orientation
and at co-ordinates very close to that of the native ligand. Their complexation was stabilized by a
network of steric and electrostatic interactions. The Glide docking score for each of these
analogues ranged from -7.31 to -6.00 with a significant correlation between the docking score
and the obtained biological activity. A plot of the antitubercular activity expressed as IC50 values
versus the glide docking score of NAH derivatives is shown in Figure 4. The active compounds
having higher scores while those with relatively low active are also predicted to have a lower
10

score (Table S7). The more negative value of docking score and binding energy signify a good
binding affinity of the ligand towards the target and vice versa. The docking score for the native
ligand was found to be -7.95.
Figure 4. Correlation plot of the glide docking score vs activity against Dormant M. tuberculosis
H37Ra (IC50) for the bithiazolyl hydrazone derivatives.
A detailed per-residue interaction analysis between the docked bithiazolyl hydrazone
derivatives and the residues lining the active site of the enzyme was carried out to identify the
most significantly interacting the residues and the type of thermodynamic elements (bonded and
non-bonded interactions) governing the binding of these molecules to the target. However in
order to maintain the brevity of text, this analysis has been elucidated only for one of the most
active analog (6l) while the results for the remaining compounds are summarized in (Table S7)
and their binding modes are provided in the supplementary material as Fig. 1S-13S.
The lowest energy docked conformation of the most active bithiazolyl hydrazone (6l) into
the active site of DprE1 enzyme (Fig. 5) showed, the inhibitor binds at the same site as the native
ligand with a significantly higher binding affinity. The compound showed the highest binding
affinity with a docking score of -7.84. This higher binding affinity can be explained in terms of
the specific bonded and non-bonded per residue interactions with the residues lining the active
site of DprE1.
11

Figure 5. Binding mode of 6l into the active site of DprE1 enzyme (the π-π stacking interaction
has been represented using green lines).
Analysis of the binding pose of (6l) revealed that it is stabilized within the active site of
DprE1 through an extensive network of favorable van der Waals interactions with Lys418 (-
2.792 Kcal/mol), Tyr415 (-1.883 Kcal/mol), Cys387 (-2.662 Kcal/mol), Val365 (-4.113
Kcal/mol), Gly321 (-1.974 Kcal/mol), Leu317 (-4.706 Kcal/mol), Thr118 (-3.046 Kcal/mol),
Tyr60 (-2.426 Kcal/mol), Trp16 (-2.083 Kcal/mol) through the bithiazolyl hydrazone scaffold.
While a similar network of interaction observed with the Ala417 (-1.664 Kcal/mol), Asn385 (-
1.844 Kcal/mol), Lys367 (-1.984 Kcal/mol), Gln336 (-1.929 Kcal/mol), His132 (-2.590
Kcal/mol), Ile131 (-1.904 Kcal/mol), Gly117 (-2.820 Kcal/mol), Pro116 (-1.893 Kcal/mol),
Ser59 (-1.951 Kcal/mol), Arg58 (-3.444 Kcal/mol) lining the active site via the aromatic
substitution on both terminal of the scaffold contributed to the binding affinity. Similarly the
compound was engaged in a set of relatively few but significant electrostatic interactions as well
observed with Lys418 (-4.413 Kcal/mol), Asp389 (-1.935 Kcal/mol), Glu322 (-1.833 Kcal/mol),
Leu317 (-1.307 Kcal/mol) and Gly117 (-1.518 Kcal/mol) residues. The enhanced binding
affinity of 6l can also be attributed to a close pi-pi (π- π) stacking interactions observed between
the aromatic ring and His132 residue (2.871Å) which serve as an "anchor", intensely determining
the 3D orientation of the ligand in the active site. These strong thermodynamic interactions of 6l
with DprE1 account for its good in silico binding and provide a clue for the significant in vitro
antitubercular activity.
A similar binding mode and network of interactions was observed for the other
bithiazolyl hydrazones (please refer supplementary material) as well but decreasing gradually
with their observed antitubercular activity. The per residue ligand interaction energy distribution
12

for all these bithiazolyl hydrazones showed that the primary driving forces for mechanical
interlocking was the steric complementarity between the ligand and the active site residues of
DprE1 as is reflected in the relatively higher number of favorable van der Waals interaction over
other components contributing to the overall binding affinity. Overall, it is evident from this
docking simulation and more specifically from the per-residue interaction analysis that these
molecules have promising affinity for the DprE1 enzyme making them pertinent starting points
for structure-based drug design.
New thiazolyl thiosemicarbazones (3a and 3b) and bithiazolyl hydrazones (6a-l) have
been conveniently synthesized. A novel one pot multi component synthetic protocol, mediated
and catalyzed by diisopropyl ethyl ammonium acetate (DIPEAc), operating at room temperature
have been first time developed for obtaining the title products (6a-l) with better to excellent
yields. Thiosemicarbazones (3a and 3b) and the hydrazones (6a-l) having thiazolyl motif were
screened for their in vitro antitubercular and cytotoxicity activities and observed that most of
them have displayed prominent activities. The compounds 3b, 6g, 6j, 6k, and 6l have shown
excellent inhibitory activity against active as well as dormant M. tuberculosis H37Ra (ATCC
25177) and M. bovis Bacillus Calmette-Guerin (BCG) (ATCC 35743) and found to have lower
toxicity. In the current report compounds 3b, 6g, and 6l exhibited a moderate selectivity index
(SI) of >10, indicating their potential as an antitubercular agent. The compounds 3b and 6j have
displayed notable antiproliferative activity against MCF7, HCT 116 and THP-1 human cancer
cell lines. Compounds 6j and 6k have shown excellent antibacterial activity against strains E.
coli, S. aureus, P. aeruginosa, and B. subtilis. The theoretical predictions from the molecular
docking study were found to be in harmony with the trend observed in the in vitro antitubercular
activity. A detailed per-residue interaction analysis was carried to gain an insight into the various
thermodynamic components involved in the binding of these molecules within the enzyme
cavity. These findings suggest that systematic alterations around these leads through iterative
synthesis and docking could gradually lead to identification of potent antitubercular candidates.
Acknowledgements
Authors are thankful to Professor D. B. Ingle for his invaluable discussions and guidance.
Authors are grateful to Combi-Chem Bio Resource Center, CSIR-National Chemical Laboratory,
Pune for providing screening data of newly synthesized compounds. One of the authors, MBB is
also grateful to Council of Scientific and Industrial Research, New Delhi for awarding Senior
13

Research Fellowship and Dr. Babasaheb Ambedkar Marathwada University, Aurangabad for
providing necessary facilities to carry the work. Authors also thank Schrödinger Inc. for
providing the Demo license of Schrödinger Suite which has tremendously helped in this study.
References and notes
1. (a) Global Tuberculosis Report 2014, World Health Organization 2014.
http://www.who.int/tb/ publications/global report/en/; (b) Smith, I. Clin. Microbiol. Rev.
2003, 16, 463; (c) “Treatment of Tuberculosis Guidelines”, WHO, Geneva 2010, 30
(www.who.int/tb/).
2. Abhale, Y. K.; Sasane, A. V.; Chavan, A. P.; Deshmukh, K. K.; Kotapalli, S. S.;
Ummanni, R.; Sayyad, S. F.; Mhaske, P. C. Eur. J. Med. Chem. 2015, 94, 340.
3. Moraski, G. C.; Markley, L. D.; Chang, M.; Cho, S.; Franzblau, S. G.; Hwang, C. H.;
Boshoff, H.; Miller, M. J. Bioorg. Med. Chem. 2012, 20, 2214.
4. Andries, K.; Verhasselt, P.; Guillemont, J.; Gohlmann, H. W.; Neefs, J. M.; Winkler, H.;
Ven Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; Williams, P.; de Chaffoy, D.; Huitric,
E.; Hoffner, S.; Cambau, E.; Truffot-Pernot, C.; Lounis, N.; Jarlier, V. A. Science 2005,
307, 223.
5. (a) Milne, G. W. A. In. Ashgate (Ed.), Handbook of Antineoplastic Agents, Gower,
London, UK, 2000; (b) Das, J.; Chen, P.; Norris, D.; Padmanabha, R.; Lin, J.; Moquin, R.
V.; Shen, Z.; Cook, L. S.; Doweyko, A. M.; Pitt, S.; Pang, S.; Shen, D. R.; Fang, Q.; de
Fex, H. F.; McIntyre, K. W.; Shuster, D. J.; Gillooly, K. M.; Behnia, K.; Schieven, G. L.;
Wityak, J.; Barrish, J. C. J. Med. Chem. 2006, 49, 6819.
6. De Souza, M. V. N.; De Almeida, M. V. Quím. Nova 2003, 26, 366.
7. Pasqualotto, A. C.; Thiele, K. O.; Goldani, L. Z. Curr. Opin. Investig. Drugs 2010, 11,
165.
8. Fox, L. M.; Saravolatz, L. D. Clin. Infect. Dis. 2005, 40, 1173.
9. Lednicer, D.; Mitscher, L. A.; George, G. I. Organic Chemistry of Drug Synthesis 1990,
4, 95.
10. Knadler, M. P.; Bergstrom, R. F.; Callaghan, J. T.; Rubin, A. Drug Metab. Dispos. 1986,
14, 175.
11. Nauen, R.; Ebbinghaus-Kintscher, U.; Salgado, V. L.; Kaussmann, M. Pest. Biochem.
Physiol. 2003, 76, 55.
14

12. Mjambili, F.; Njoroge, M.; Naran, K.; De Kock, C.; Smith, P. J.; Mizrahi, V.; Warner, D.;
Chibale, K. Bioorg. Med. Chem. Lett. 2014, 24, 560.
13. Telvekar, V. N.; Bairwa, V. K.; Satardekar, K.; Bellubi, A. Bioorg. Med. Chem. Lett.
2012, 22, 649.
14. Roy, K. K.; Singh, S.; Sharma, S. K.; Srivastava, R.; Chaturvedi, V.; Saxena, A. K.
Bioorg. Med. Chem. Lett. 2011, 21, 5589.
15. Meissner, A.; Boshoff, H. I.; Vasan, M.; Duckworth, B. P.; Barry, C. E.; Aldrich, C. C.
Bioorg. Med. Chem. 2013, 21, 6385.
16. Ozadali, K.; Tan, O. U.; Yogeeswari, P.; Dharmarajan, S.; Balkan, A. Bioorg. Med.
Chem. Lett. 2014, 24, 1695.
17. Shiradkar, M. R.; Murahari, K. K.; Gangadasu, H. R.; Suresh, T.; Kalyan, C. A.; Panchal,
D.; Kaur, R.; Burange, P.; Ghogare, J.; Mokale, V.; Raut, M. Bioorg. Med. Chem. 2007,
15, 3997.
18. Jeankumar, V. U.; Renuka, J.; Santosh, P.; Jonnalagadda, V. S.; Sridevi, P.; Suryadevara,
P.; Yogeeswari, P.; Sriram, D. Eur. J. Med. Chem. 2013, 70, 143.
19. Aridoss, G.; Amirthaganesan, S.; Kim, M. S.; Kim, J. T.; Jeong, Y. T. Eur. J. Med.
Chem. 2009, 44, 4199.
20. Jallapally, A.; Addla, D.; Yogeeswari, P.; Sriram, D.; Kantevari, S. Bioorg. Med. Chem.
Lett. 2014, 24, 5520 and references cited there in.
21. Pavan, F. R.; Maia, P. I. da S.; Leite, S. R. A.; Deflon, V. M.; Batista, A. A.; Sato, D. N.;
Franzblau, S. G.; Leite, C. Q. F. Eur. J. Med. Chem. 2010, 45, 1898.
22. Kocyigit-Kaymakcioglu, B.; Unsalan, S.; Kucukquzel, S. G.; Sener, G.; Rollas, S. Eur. J.
Drug Metab. Pharmacokinet. 2007, 32, 197.
23. Gulerman, N. N.; Oruc, E. E.; Kartal, F.; Rollas, S. Eur. J. Drug Metab. Pharmacokinet.
2000, 25, 103.
24. Kaymakciog lu, B.; Aktan, Y.; Suzen, S.; Gokhan, N.; Koyunog lu, S.; Erol, K.;
Yesjxada, A.; Rollas, S. Eur. J. Drug Metab. Pharmacokinet. 2005, 30, 255.
25. General procedure for the preparation of 1-(1-(2-methyl/phenyl-4-methylthiazol-5-yl)
ethylidene) thiosemicarbazides (3a, 3b). A mixture of 5-acyl thiazole (4.6 mmol) and
thiosemicarbazide (4.6 mmol) was refluxed in dry ethanol for 1.5 h. The progress of the
reaction was monitored by thin layer chromatography (TLC). After completion of the
reaction, reaction mass was allowed to cool at room temperature and then the reaction
15

mass was poured on crushed ice. The obtained solid mass was filtered, washed with water
and dried. The crude products were crystallized using dry ethanol.
1-(1-(2,4-Dimethylthiazol-5-yl) ethylidene) thiosemicarbazide (3a). ield 60 yellow
powder mp 156-158 C. FTIR (KBr pellet) cm-1: 3442 (N-H str.), 1570 (C=N str.
1
hydrazolyl), 1086 (C-S-C str., aromatic); H-NMR (400 MHz, CDCl3): δ 2.30 (s, 2H, -
CH3), 2.58 (s, 3H, -CH3), 2.67 (s, 3H, -CH3), 8.66 (1H, br, NH) ppm; HRMS (ESI, m/z):
calcd for C8H13N4S2 (M + H+) 229.0503; found: 229.05761.
1-(1-(4-Methyl-2-phenylthiazol-5-yl) ethylidene) thiosemicarbazide (3b). ield 58
1
yellow powder mp 160-162 C. H-NMR (400 MHz, CDCl3): δ 2.39 (s, 3H, -CH3), 2.60
(s, 3H, -CH3), 7.43-7.93 (m, 5H, Ar-H), 8.39 (1H, br, NH), 10.48 (1H, br, NH) ppm;
HRMS (ESI, m/z): calcd for C13H15N4S2 (M + H+) 291.066; found: 291.07326.
General procedure for the synthesis of 1-(1-(2-phenyl/methyl-4-methylthiazol-5-yl)
ethylidene)-2-(4-substitutedphenylthiazol-2-yl) hydrazines (6). A mixture of thiazolyl
thiosemicarbazone (3) (4.4 mmol) and phenacyl chloride (4) (4.4 mmol) was allowed to
reflux in dry ethanol for 1 h. The progress of the reaction was monitored by thin layer
chromatography (TLC). After completion of the reaction, the reaction mixture was
allowed to cool at room temperature and poured on crushed ice and then neutralized by
aqueous ammonia. The obtained solid compound was filtered, washed with water and
dried. The crude compound was crystallized using DMF-Ethanol.
26. General procedure for one pot synthesis of 1-(1-(2-Phenyl/Methyl-4-methylthiazol-5-yl)
ethylidene)-2-(4-substitutedphenylthiazol-2-yl) hydrazines (6) using ethanol as a solvent.
5-Acyl thiazole (1) (4.6 mmol), thiosemicarbazide (2) (4.6 mmol) and phenacyl chloride
(4) (4.6 mmol) were refluxed in ethanol (5 ml) for 4 h. The progress of the reaction was
monitored by TLC. After completion of the reaction, the reaction mass was poured on
crushed ice and neutralized by aqueous ammonia. The obtained solid was filtered,
washed with water and dried. The crude compound was crystallized using DMF-Ethanol.
27. Zhao, H.; Malhotra, S. V. Aldrichimica Acta 2002, 35, 75.
28. Khillare, L. D.; Bhosle, M. R.; Deshmukh, A. R.; Mane, R. A. Res Chem Intermed 2015,
41, 8955.
29. General procedure for one pot synthesis of 1-(1-(2-Phenyl/Methyl-4-methylthiazol-5-yl)
ethylidene)-2-(4-substitutedphenylthiazol-2-yl) hydrazines (6) using diisopropyl ethyl
ammonium acetate (DIPEAc). 5-Acyl thiazole (1) (4.6 mmol), thiosemicarbazide (2) (4.6
16

mmol) and phenacyl chloride (4) (4.6 mmol) were dissolved in freshly prepared
diisopropyl ethyl ammonium acetate (DIPEAc) (5 ml) and stirred at room temperature for
30 min. After stirring the reaction mixture for 30 min., the reaction mass was poured on
crushed ice and neutralized by aqueous ammonia. The obtained solid was filtered,
washed with water and dried. The crude compound was crystallized using DMF-Ethanol.
1-(1-(2,4-Dimethylthiazol-5-yl) ethylidene)-2-(4-phenylthiazol-2-yl) hydrazine (6a).
ield 96 brown powder mp 154-156 C. FTIR (KBr pellet) cm-1: 1550 (C=N str.
1
hydrazolyl), 1100 (C-S-C str., aromatic) H-NMR (400 MHz, CDCl3): δ 2.38 (s, 3H, -
CH3), 2.62 (s, 3H, -CH3), 2.67 (s, 3H, -CH3), 6.81 (s, 1H, thiazolyl), 7.41-7.73 (m, 5H,
Ar-H) ppm; 13C-NMR (100 MHz, CDCl3): δ 169.55, 164.84, 159.13, 153.64, 148.31,
133.61, 129.31, 129.18, 125.76, 125.76, 114.27, 102.38, 19.19, 18.65, 18.50 ppm; HRMS
(ESI, m/z): calcd for C16H17N4S2 (M + H+) 329.0816; found: 329.0889.
2-(4-(4-Fluorophenyl) thiazol-2-yl)-1-(1-(2,4-dimethylthiazol-5-yl) ethylidene) hydrazine
(6b). ield 94 brown powder mp 172-174 C. FTIR (KBr pellet) cm-1: 3448 (N-H str.),
1
1567 (C=N str. hydrazolyl), 1093 (C-S-C str., aromatic), 1131 (C-F str.); H-NMR (400
MHz, CDCl3): δ 2.12 (s, 3H, -CH3), 2.59 (s, 3H, -CH3), 2.63 (s, 3H, -CH3), 6.79 (s, 1H,
thiazolyl), 7.04 (d, 2H, Ar-H, J = 8 Hz), and 7.69 (d, 2H, Ar-H, J = 8 Hz) ppm; 13C-NMR
(100 MHz, CDCl3): δ 169.75, 163.74, 163.46, 161.49, 150.23, 141.62, 130.96, 127.67,
127.61, 115.66, 115.49, 103.46, 19.12, 18.34, 16.92 ppm; HRMS (ESI, m/z): calcd for
C16H16FN4S2 (M + H+) 347.0722; found: 347.0790.
2-(4-(4-Chlorophenyl) thiazol-2-yl)-1-(1-(2,4-dimethylthiazol-5-yl) ethylidene) hydrazine
(6c). Yield 85 yellow powder mp 164-166 C. FTIR (KBr pellet) cm-1: 3321 (N-H str.),
1
1550 (C=N str. hydrazolyl), 1091 (C-S-C str.), 731 (C-Cl str.); H-NMR (400 MHz,
CDCl3): δ 2.16 (s, 3H, -CH3), 2.61 (s, 3H, -CH3), 2.65 (s, 3H, -CH3), 6.87 (s, 1H,
thiazolyl), 7.32 (d, 2H, Ar-H, J = 8 Hz), and 7.67 (d, 2H, Ar-H, J = 8 Hz) ppm; 13C-NMR
(100 MHz, CDCl3): δ 170.59, 163.80, 161.17, 150.03, 141.95, 133.62, 130.00, 128.86,
128.86, 127.18, 127.18, 115.64, 104.28, 19.17, 18.38, 16.96 ppm. HRMS (ESI, m/z):
calcd for C16H16ClN4S2 (M + H+) 363.0427; found: 363.0625.
2-(4-(4-Bromophenyl) thiazol-2-yl)-1-(1-(2,4-dimethylthiazol-5-yl) ethylidene) hydrazine
(6d). ield 90 green powder mp 176-178 C. FTIR (KBr pellet) cm-1: 3380 (N-H str.),
1
1548 (C=N str. hydrazolyl), 1096 (C-S-C str.), 723 (C-Br str.); H-NMR (400 MHz,
CDCl3): δ 2.18 (s, 3H, -CH3), 2.61 (s, 3H, -CH3), 2.65 (s, 3H, -CH3), 6.86 (s, 1H,
17

thiazolyl), 7.48-7.60 (m, 4H, Ar-H) ppm; 13C-NMR (100 MHz, CDCl3): δ 170.04,
169.65, 164.00, 153.30, 142.69, 131.86, 131.86, 130.08, 127.42, 127.42, 122.06, 114.25,
104.16, 19.14, 18.44, 17.21 ppm; HRMS (ESI, m/z): calcd for C16H16BrN4S2 (M + H+)
406.9922; found: 406.9994.
1-(1-(2,4-Dimethylthiazol-5-yl) ethylidene)-2-(4-p-tolylthiazol-2-yl) hydrazine (6e).
ield 82 brown powder mp 188-190 C. FTIR (KBr pellet) cm-1: 3335 (N-H str.), 1553
1
(C=N str. hydrazolyl), 1103 (C-S-C str.); H-NMR (400 MHz, CDCl3): δ 2.11 (s, 3H, -
CH3), 2.29 (s, 3H, -CH3), 2.54 (s, 3H, -CH3), 2.61 (s, 3H, -CH3), 6.73 (s, 1H, thiazolyl),
7.13 (d, 2H, Ar-H, J = 8 Hz), and 7.57 (d, 2H, Ar-H, J = 8 Hz) ppm; 13C-NMR (100
MHz, CDCl3): δ 169.85, 164.07, 161.17, 150.56, 144.12, 138.41, 130.11, 129.53, 129.53,
125.77, 125.77, 115.94, 102.29, 21.25, 19.14, 18.44, 17.74 ppm; HRMS (ESI, m/z): calcd
for C17H19N4S2 (M + H+) 343.0973; found: 343.1046.
2-(4-(4-Methoxyphenyl)
thiazol-2-yl)-1-(1-(2,4-dimethylthiazol-5-yl)
ethylidene)
hydrazine (6f). ield 93 brown powder mp 202-204 C. FTIR (KBr pellet) cm-1: 3420
(N-H str.), 1550 (C=N str. hydrazolyl), 1098 (C-S-C str.), 1021 (C-O str.); 1H-NMR (400
MHz, CDCl3): δ 2.09 (s, 3H, -CH3), 2.58 (s, 3H, -CH3), 2.63 (s, 3H, -CH3), 3.76 (s, 3H, -
OCH3), 6.69 (s, 1H, thiazolyl), 6.87 (d, 2H, Ar-H, J = 8 Hz), and 7.64 (d, 2H, Ar-H, J = 8
Hz) ppm; 13C-NMR (100 MHz, CDCl3): δ 169.41, 163.69, 159.43, 150.30, 150.02,
142.38, 133.40, 130.48, 127.27, 127.16, 114.66, 114.04, 114.04, 101.79, 55.24, 19.09,
18.34, 17.18 ppm; HRMS (ESI, m/z): calcd for C17H19N4OS2 (M + H+) 359.0922; found:
359.0995.
1-(1-(4-Methyl-2-phenylthiazol-5-yl) ethylidene)-2-(4-phenylthiazol-2-yl) hydrazine
(6g). ield 92 brown powder mp 196-198 C. FTIR (KBr pellet) cm-1: 3450 (N-H str.),
1
1549 (C=N str. hydrazolyl), 1095 (C-S-C str.); H-NMR (400 MHz, CDCl3): δ 2.43 (s,
3H, -CH3), 2.70 (s, 3H, -CH3), 6.82 (s, 1H, thiazolyl), 7.36-7.94 (m, 10H, Ar-H) ppm;
13C-NMR (100 MHz, CDCl3): δ 169.39, 165.88, 161.46, 153.14, 146.35, 133.12, 130.41,
129.15, 129.15, 129.01, 129.01, 129.01, 129.01, 126.00, 126.00, 125.74, 125.74, 113.80,
102.45, 19.19, 18.49 ppm; HRMS (ESI, m/z): calcd for C21H19N4S2 (M + H+) 391.0973;
found: 391.1046.
2-(4-(4-Fluorophenyl) thiazol-2-yl)-1-(1-(4-methyl-2-phenylthiazol-5-yl) ethylidene)
hydrazine (6h). ield 89 yellow powder mp 192-194 C. 1H-NMR (400 MHz, CDCl3):
δ 2.22 (s, 3H, -CH3), 2.68 (s, 3H, -CH3), 6.77 (s, 1H, thiazolyl), 7.05 (d, 2H, Ar-H, J = 4
18

Hz), 7.40 (t, 3H, Ar-H, J = 4 Hz), 7.72 (d, 2H, Ar-H, J = 8 Hz), and 7.92 (d, 2H, Ar-H, J
= 8 Hz) ppm. 13C-NMR (100 MHz, CDCl3): δ 169.62, 165.16, 163.87, 161.41, 152.10,
148.80, 142.77, 133.27, 130.23, 130.23, 128.97, 128.97, 127.00, 127.00, 126.39, 126.39,
115.86, 115.64, 103.17, 18.95, 17.29 ppm. HRMS (ESI, m/z): calcd for C21H18FN4S2 (M
+ H+) 409.0879; found: 409.09514.
2-(4-(4-Chlorophenyl) thiazol-2-yl)-1-(1-(4-methyl-2-phenylthiazol-5-yl) ethylidene)
hydrazine (6i). ield 90 brown powder mp 186-188 C. FTIR (KBr pellet) cm-1: 3470
(N-H str.), 1564 (C=N str. hydrazolyl), 1089 (C-S-C str.), 725 (C-Cl str.); 1H-NMR (400
MHz, CDCl3): δ 2.21 (s, 3H, -CH3), 2.73 (s, 3H, -CH3), 6.90 (s, 1H, thiazolyl), 7.36 (d,
2H, Ar-H, J = 4 Hz), 7.44 (t, 3H, Ar-H, J = 4 Hz), 7.73 (d, 2H, Ar-H, J = 8 Hz), 7.96 (d,
13
2H, Ar-H, J = 8 Hz), and 8.05 (s, 1H, =N-N-H) ppm; C-NMR (100 MHz, CDCl3): δ
169.52, 164.96, 162.61, 151.83, 150.18, 141.51, 133.61, 133.38, 130.15, 128.96, 128.96,
128.86, 128.86, 127.20, 127.20, 126.40, 126.40, 115.94, 104.39, 18.86, 16.95 ppm;
HRMS (ESI, m/z): calcd for C21H18ClN4S2 (M + H+) 425.0583; found: 425.0656.
2-(4-(4-Bromophenyl) thiazol-2-yl)-1-(1-(4-methyl-2-phenylthiazol-5-yl) ethylidene)
hydrazine (6j). ield 94 tan powder mp 182-184 C. FTIR (KBr pellet) cm-1: 3430 (N-
1
H str.), 1552 (C=N str. hydrazolyl), 1088 (C-S-C str.), 664 (C-Br str.); H-NMR (400
MHz, CDCl3): δ 2.28 (s, 3H, -CH3), 2.75 (s, 3H, -CH3), 6.93 (s, 1H, thiazolyl), 7.45 (t,
3H, Ar-H, J = 4 Hz), 7.52 (d, 2H, Ar-H, J = 4 Hz), 7.66 (d, 2H, Ar-H, J = 8 Hz), and 7.96
13
(d, 2H, Ar-H, J = 8 Hz) ppm; C-NMR (100 MHz, CDCl3): δ 169.41, 167.41, 165.01,
151.85, 148.35, 133.61, 133.36, 131.80, 131.80, 128.96, 128.96, 128.96, 128.96, 128.00,
126.41, 126.41, 121.79, 116.16, 104.55, 18.88, 16.88 ppm; HRMS (ESI, m/z): calcd for
C21H18BrN4NaS2 (M + H+) 490.9999; found: 490.9980.
1-(1-(4-Methyl-2-phenylthiazol-5-yl) ethylidene)-2-(4-p-tolylthiazol-2-yl) hydrazine
(6k). ield 85 yellow powder mp 196-198 C. FTIR (KBr pellet) cm-1: 3350 (N-H
str.), 1550 (C=N str. hydrazolyl), 1091 (C-S-C str.); 1H-NMR (400 MHz, CDCl3): δ 2.16
(s, 3H, -CH3), 2.36 (s, 3H, -CH3), 2.73 (s, 3H, -CH3), 6.86 (s, 1H, thiazolyl), 7.20 (d, 2H,
Ar-H, J = 4 Hz), 7.44 (t, 3H, Ar-H, J = 4 Hz), 7.68 (d, 2H, Ar-H, J = 8 Hz), and 7.96 (d,
2H, Ar-H, J = 8 Hz) ppm; 13C-NMR (100 MHz, CDCl3): δ 169.63, 167.06, 164.85,
151.65, 141.47, 137.74, 133.43, 130.10, 129.39, 129.39, 128.95, 128.95, 126.39, 126.39,
126.39, 125.92, 125.92, 118.72, 103.14, 21.24, 18.78, 16.95 ppm; HRMS (ESI, m/z):
calcd for C22H21N4S2 (M + H+) 405.1129; found: 405.1202.
19

2-(4-(4-Methoxyphenyl) thiazol-2-yl)-1-(1-(4-methyl-2-phenyl thiazol-5-yl) ethylidene)
hydrazine (6l). ield 93 brown powder mp 184-186 C. HRMS (ESI, m/z): calcd for
C22H21N4OS2 (M + H+) 421.1079; found: 421.1151.
30. Khan, A.; Sarkar, D. J Microbiol Methods. 2008, 73, 62.
31. Singh, U.; Akhtar, S.; Mishra, A.; Sarkar, D. J Microbiol Methods 2011, 84, 202.
32. Turan-Zitouni, G.; Ozdemir, A.; Kaplancikli, Z. A.; Benkli, K.; Chevallet, P.; Akalin, G.
Eur J Med Chem. 2008, 43, 981.
33. Pinheiro, A. C.; Kaiser, C. R.; Nogueira, T. C.; Carvalho, S. A.; da Silva, E. F.; Feitosa
Lde, O.; Henriques, M. D.; Candéa, A. L.; Lourenço, M. C.; de Souza, M. V. Med Chem.
2011, 6, 611.
34. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
35. Ciapetti, G.; Cenni, E.; Pratelli, L.; Pizzoferrato, A. Biomaterials 1993, 14, 359.
36. Hartkoorn, R. C.; Chandler, B.; Owen, A. et al. Tuberculosis 2007, 87, 248.
37. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
38. Stanley, S. A.; Grant, S. S.; Kawate, T.; Iwase, N.; Shimizu, M.; Wivagg, C.; Silvis, M.;
Kazyanskaya, E.; Aquadro, J.; Golas, A.; Fitzgerald, M.; Dai, H.; Zhang, L.; Hung, D. T.
ACS Chem. Biol. 2012, 7, 1377.
39. Saha, R.; Alam, Md. M.; Akhter, M. RSC Adv. 2015, 5, 12807.
40. Bano, S.; Alam, M. S.; Javed, K.; Dudeja, M.; Das, A. K.; Dhulap, A. Eur. J. Med. Chem.
2015, 95, 96.
41. Shaikh, M. H.; Subhedar, D. D.; Nawale, L.; Sarkar, D.; Khan, F. A. K.; Sangshetti, J. N.;
Shingate, B. B. Med. Chem. Commun. 2015, 6, 1104.
42. Chikhale, R.; Menghani, S.; Babu, R.; Bansode, R.; Bhargavi, G.; Karodia, N.;
Rajasekharan, M. V.; Paradkar, A.; Khedekar, P. Eur. J. Med. Chem. 2015, 96, 30.
43. Brecik, M.; Centarova, I.; Mukherjee, R.; Kolly, G. S.; Huszar, S.; Bobovska, A.;
Kilacskova, E.; Mokosova, V.; Svetlikova, Z.; Sarkan, M.; Neres, J.; Kordulakova, J.;
Cole, S. T.; Mikusova, K. ACS Chem. Biol. 2015, 10, 1631.
44. Manjunatha, U. H.; Smith, P. W. Bioorg. Med. Chem. 2015, 23, 5087.
20

Graphical Abstract
New bithiazolyl hydrazones: novel synthesis, characterization and
antitubercular evaluation
Mahendra B. Bhalerao a, Sambhaji T. Dhumal a, Amarsinh R. Deshmukh a, Laxman U. Nawale b,
Vijay Khedkar c, Dhiman Sarkar b, Ramrao A. Mane a*
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad
431004, Maharashtra, India.
bCombi-Chem Bio Resource Center, CSIR-National Chemical Laboratory, Pune 411008,
Maharashtra, India.
cSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa.
*Corresponding author Email: manera2011@gmail.com
21

Highlights
 New bithiazolyl hydrazones have been synthesized by one pot novel synthetic route.
 Bithiazolyl hydrazones displayed noticeable antiTB activity compared to Rifampicin.
 Compounds, 3b, 6g, 6j, 6k, and 6l have shown excellent antiTB activity.
 Molecular docking study has shown better harmony with the evaluation trend.
22