2-(2-Hydrazinyl)thiazole derivatives: Design, synthesis and in vitro antimycobacterial studies

Article abstract of DOI:10.1016/j.ejmech.2013.08.054

In an attempt to discover new potent inhibitors for Mycobacterium tuberculosis (Mtb), a series of 2-(2-hydrazinyl)thiazole derivatives with a wide range of substitutions at 2-, 4- and 5-positions were designed by considering Lipinski rule. The designed compounds were synthesized, characterized and evaluated for their inhibitory potential against Mtb, H₃₇Rv, by in vitro assay. The compounds, ethyl-4-methyl-2-[(E)-2-[1-(pyridin-2-yl)ethylidene] hydrazin-1-yl]-1,3-thiazole-5-carboxylate, 4d, and ethyl-2-[(E)-2-[(2- hydroxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-carboxylate, 2i showed noticeable inhibitory activity against Mtb, H₃₇Rv with minimum inhibitory concentration (MIC) of 12.5 μM and 25 μM respectively. An attempt has been made to understand the mechanism of action by binding interactions of these molecules with β-ketoacyl-ACP synthase protein through docking studies. The inhibition constants for compounds 4d and 2i were found to be 1.46 μM and 0.177 μM respectively.

Full text of DOI:10.1016/j.ejmech.2013.08.054

Accepted Manuscript
2-(2-Hydrazinyl)thiazole derivatives: Design, synthesis and in vitro antimycobacterial
studies
Parameshwar Makam, Ramakrishna Kankanala, Amresh Prakash, Tharanikkarasu
Kannan
PII:
S0223-5234(13)00574-6
10.1016/j.ejmech.2013.08.054
EJMECH 6404
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2013
Revised Date: 30 July 2013
Accepted Date: 21 August 2013
Please cite this article as: P. Makam, R. Kankanala, A. Prakash, T. Kannan, 2-(2-Hydrazinyl)thiazole
derivatives: Design, synthesis and in vitro antimycobacterial studies, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.054.
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.

ACCEPTED MANUSCRIPT
Graphical abstract
2-(2-Hydrazinyl)thiazole derivatives: Design, synthesis, characterization and in vitro
antimycobacterial studies
A library of novel 2-(2-hydrazinyl) thiazole derivatives were designed, synthesized and their
inhibition, cytotoxicity and interactions were evaluated against M. tuberculosis, lung cancer
cells and KasA protein respectively.

ACCEPTED MANUSCRIPT
Research Highlights
•
•
•
Library of 2-(2-hydrazinyl)thiazole derivatives were synthesized and characterized.
All the compounds evaluated against M. tuberculosis, H37Rv, by in vitro assays.
2-Pyridine and 2-hydroxyphenyl hydrazinyl compounds showed good anti-TB
activities.
•
•
2-Pyridine containing thiazole derivatives show low cytotoxicity.
Compound 4d interacts with KasA protein similar to thiolactomycin.

ACCEPTED MANUSCRIPT
2-(2-Hydrazinyl)thiazole derivatives: Design, synthesis and in vitro
antimycobacterial studies
Parameshwar Makama, Ramakrishna Kankanalaa, Amresh Prakashb, Tharanikkarasu
Kannana,*
aDepartment of Chemistry, Pondicherry University, Puducherry-605 014, India.
bCentre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar,
New Delhi-110 025, India.
* Corresponding author. Tel: +91-413-265 4708; Fax: +91-413-265 6740.
E -mail address: tharani.che@pondiuni.edu.in; thavasu@gmail.com
Abstract: In an attempt to discover new potent inhibitors for Mycobacterium tuberculosis
(Mtb), a series of 2-(2-hydrazinyl)thiazole derivatives with a wide range of substitutions at 2-,
4- and 5- positions were designed by considering Lipinski rule. The designed compounds
were synthesized, characterized and evaluated for their inhibitory potential against Mtb,
H37Rv, by in vitro assay. The compounds, ethyl-4-methyl-2-[(E)-2-[1-(pyridin-2-
yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-carboxylate, 4d, and ethyl-2-[(E)-2-[(2-
hydroxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-carboxylate, 2i showed
noticeable inhibitory activity against Mtb, H37Rv with minimum inhibitory concentration
(MIC) of 12.5 µM and 25 µM respectively. An attempt has been made to understand the
mechanism of action by binding interactions of these molecules with β-ketoacyl-ACP
synthase protein through docking studies. The inhibition constants for compounds 4d and 2i
were found to be 1.46 µM and 0.177 µM respectively.
Key words: Mycobacterium tuberculosis, H37Rv, tuberculosis, thiazole, β-ketoacyl-ACP
synthase, Lipinski rule.
1

ACCEPTED MANUSCRIPT
1. Introduction
Tuberculosis (TB) is an airborne and extremely contagious ancient disease which is
caused by the Mycobacterium tuberculosis (Mtb) [1]. Although the causative agent for this
disease was discovered by Robert Koch in 1882, the chemotherapy was started in 1944 with
streptomycin, an antibiotic derived from Streptomyces griseus [2]. Besides persistent effort to
eradicate this disease, it continues to be one of the major global health threat. According to
the sixteenth global report of the World Health Organization (WHO), closely 2 million
people died among the 8.8 million incident cases of TB in 2010 and fatalities may reach up to
36 million by 2020 [3]. The progress in eradicating TB is stagnant in high burden countries
due to poverty, synergy with the HIV/AIDS pandemic [4-7] and slow progress in developing
novel and target effective drugs [8, 9]. The progress in developing new therapeutics and their
clinical uses to treat TB are apprehensive due to the disease complexity, prolonged and poor
administration of treatment. Though streptomycin cured TB initially, Mtb developed
resistance towards streptomycin, due to this, other drugs such as para-aminosalicylic acid
(1948), isoniazid (1951), pyrazinamide (1952), cycloserine (1952), ethionamide (1956),
rifampin (1957), kanamycin (1957) and ethambutol (1962) etc. have been developed and the
multidrug therapy, a combination of two or three or four drugs has also been introduced in
the 1970s [10]. The progress in chemotherapy to treat TB was overshadowed by drug
susceptibility to Mtb, as a result, evolution of multi-drug resistant (MDR), extensively drug-
resistant (XDR) strains took place and very recently, totally drug-resistant TB (TDR) has also
been reported [11]. It is very disappointing to note that the practice of using a combination of
four drugs chemotherapy for the duration of six 6 month is still followed in clinical practice
and no approval of new class of drugs for curing drug susceptible TB has been given so far.
Multidrug therapies and the alarming increase of MDR-TB and XDR-TB strains of Mtb pose
the biggest challenge in treating TB [12-15]. Hence, an inevitable progress is mandatory in
developing novel, effective and safe anti-TB drugs which can target MDR-TB and XDR-TB
strains through new biochemical pathways.
In an attempt to generate novel anti-TB drugs, during the last decade, a wide spectrum
of known and novel scaffolds has been tested for their ability to inhibit Mtb.
Fluoroquinolones, rifamycins, oxazolidinones and riminophenazines are some of the known
drugs which have been repurposed to treat TB [15]. On the other hand, the discovery and
development of new chemical entities such as nitroimidazoles, dipiperidine, capuramycin,
benzothiazinone and caprazene nucleoside have also been successfully tested against TB.
Both the repurposed and novel molecules are currently in the pool of clinical trials [16].
2

ACCEPTED MANUSCRIPT
Bedaquiline and diarylquinoline derivatives, which primarily targets ATP synthase have been
recently approved by the FDA to use as part of the combination therapy for adults with MDR
pulmonary TB [17]. In addition to these successful candidates, 2-aminothiazoles,
benzopyranones, phenoxyalkylimidazoles, thieno[2,3-d]pyrimidines, thiophene-1,1-dioxides,
piperidinamines, thioamides, ureas, thioureas, adamantyl amides and amines, 5-nitrofuran-2-
carboxamides and diazolylthio derivatives were also screened and found to have good
inhibition against Mtb, H37Rv strain [18]. Among the promising scaffolds, 2-aminothiazole
scaffold (See Figure 1) is of particular interest due to its target specificity and its enhanced
inhibition of Mtb. Moreover, thiazole derivatives have been constantly evaluated for their
diverse biological activities as anticancer [19] and antitumor [20, 21], antimalarial [22],
antimicrobial [23], anti-inflammatory [24] and anti hypolipidemic [25] agents. The synthesis
of thiazole scaffold can be easily attained by classical Hantzsch thiazole methodologies and
the scope for generating novel thiazole derivatives is also high [26].
<Insert Figure 1>
2-Aminothiazole scaffold is structurally similar to thiolactomycin (TLM), a naturally
occurring and synthetically challenging antibiotic [27]. TLM inhibits β – ketoacyl - ACP
synthase (KasA) in mtFabH fatty acid synthesis and consequently inhibits the cell wall
biosynthesis leading to the death of Mtb [28]. Interestingly, 2-aminothiazole-4–carboxylate
derivatives (see a in Figure 1) are potent inhibitors of KasA protein [29]. Similarly, 2-
aminothiazole derivative (see b in Figure 1) has shown superior biological activity over
thiazolidinones in inhibiting UDP-galactopyranose mutase thwart biochemical pathway to
inhibit Mtb [30]. Nitazoxanide (NTZ) (see c in Figure 1), a drug approved by FDA to treat
protozoal infections [31], 2-aminothiazole-4-carboxylates (see a in Figure 1) and 2-amino-4-
(2-pyridyl) thiazoles (see d in Figure 1) are currently in clinical trials [32]. Very few recent
reports suggest that 2-(2 -hydrazinyl)thiazole derivatives comprising 2-aminothiazole
scaffold have exhibited good activity against Mtb strain, H37Rv [33-35]. As 2-aminothiazole
scaffold shows promising anti-TB activity, in the present investigation, 2 -(2-hydrazinyl)
thiazole derivatives with a wide range of substitutions at 2-, 4- and 5- positions by
considering Lipinski rule of five [36] are designed, synthesized and evaluated for their
inhibitory activity against Mtb, H37Rv.
2. Results and Discussion
To identify new lead molecules to treat Mtb, initially, 2 -(2-hydrazinyl)thiazole
derivatives with both hydrophilic and lipophilic inducing groups have been designed as
3

ACCEPTED MANUSCRIPT
shown in Figure 1 (see f). It is interesting to note that the NTZ (see c in Figure 1) and its
hydrolyzed active metabolite tizoxanide (TIZ) (see e in Figure 1) have shown the inhibition
of replicating Mtb with MIC value of 52.12 µM and 60.38 µM respectively. It is suggested
that the time-dependent anti-TB effect of NTZ [37] can be enhanced with the modification of
head groups [38]. Similarly, by considering the structural features of other compounds shown
in Figure 1, the rational design of the prototypic modified analogues are proposed as shown
in Figure 1 (f). The designed hybrid 2-(2-hydrazinyl)thiazole scaffold derivatives are
retained with core 2-aminothiazole scaffold and additionally both lipophilic and hydrophilic
inducing groups are introduced proportionally to maintain the cell wall permeability and the
solubility.
2.1. Lipinski rule analysis
Before the synthesis of designed 2-(2-hydrazinyl)thiazole derivatives, the suitability
of these compounds as drugs has been evaluated using Lipinski rule of five. The Lipinski rule
of five, while designing the drug, takes the account of the correlation between the physico –
chemical properties of the chemical entities and the drug pharmacokinetics in the human
body, including their absorption, distribution, metabolism and excretion (ADME) [36].
According to this rule, the chemical molecule is more likely to exhibit poor absorption or
permeation when the molecular weight is more than 500, LogP (Octanol/Water partition
coefficient), a measure of lipophilicity, is over 5, H-bond donors exceeds 5, H-bond acceptors
exceed 5 and the sum of N atoms and O atoms is over 10. Regardless of some exceptions to
this rule, it is a simple and widely accepted method to screen all patterns of drugs.
Molinspiration server is used to deduce the data based on Lipinski rule of five for all the 2-(2-
hydrazinyl)thiazole derivatives and the results are summarized in Table 1.
<Insert Table 1>
The data reveal that the molecular weights of the designed compounds are below 500,
and are in the range of 249.295 to 379.438. The LogP values are found to be in the range of
1.901 to 4.465. These values are found to be in the range recommended by Lipinski rule.
Similarly, all the compounds have shown 4 to 7 hydrogen bond acceptors except compound
2r which has shown the highest (8) hydrogen bond acceptors. Similarly, all the compounds
are possessing less than 2 hydrogen bond donors which are well below the value
recommended by Lipinski. Hence, all these compounds have shown no violations of Lipinski
rule and found to be possessing drug like molecule (DLM) features.
4

ACCEPTED MANUSCRIPT
2.2. Synthesis of 2-(2-hydrazinyl)thiazole derivatives
As all the molecules have DLM features, synthesis of targeted 2-(2-
hydrazinyl)thiazole derivatives has been carried out according to the synthetic pathways
described in Scheme 1. To synthesize 2-(2-hydrazinyl)thiazole derivatives, as shown in
Scheme 1 (a), first thiosemicarbazones (1 a-w and 3a-f) are synthesized from
thiosemicarbazide and wide range of substituted aromatic and heteroatom containing
1
carbonyl compounds using the literature procedure [39-42]. The H Nuclear Magnetic
Resonance (NMR) spectra of these thiosemicarbazones show the characteristic peak due to
imine methyl protons between 2.5 ppm and 3.0 ppm. Proton present in the Schiff base
resonates at 7.0 ppm and the aromatic phenyl ring protons resonate between 7.00 ppm and 8.6
ppm. The characteristic peak due to =NH proton appears between 8.5 ppm and 9.0 ppm as a
singlet and the peak corresponding to -NH proton resonate between 10 ppm and 11.5 ppm.
Due to the exchange of H between the terminal NH and S, the characteristic peak due to this
proton appears approximately at 11.5 ppm. The carbon present in C=N group resonates
around 150 ppm in 13C NMR spectra of thiosemicarbazones. These results confirm that the
condensation between carbonyl group and thiosemicarbazide is successful. In addition,
disappearance of the broad peak due to carbonyl (C=O) functional group at 1600-1700 cm-1
and the appearance of the characteristic peak of N=CH group in the range of 1542–1579 cm–
1
in the Infrared (IR) spectra of compounds further confirm the absence of carbonyl reactant
and formation of desired thiosemicarbazones. Apart from the synthesis of reported
thiosemicarbazones, three new thiosemicarbazones have also been synthesized and the
structures of these three compounds are also confirmed using the same way used for the
reported compounds.
<Insert Scheme 1>
In the next step, the desired 2-(2-hydrazinyl)thiazole derivatives with aryl (2a-z) and
hetero aryl (4a-f, 5a-d and 6a-f) substitutions at 2nd position have been synthesized from
corresponding thiosemicarbazones and aliphatic α-halo ketones as shown in Scheme 1(b).
All the synthesized compounds have been characterized using NMR, IR and mass
spectroscopic techniques. Except compound 6a and 6b, R3 in all 2-(2-hydrazinyl)thiazole
derivatives is CH3. The protons present in this CH3 group are observed at 2.25 ppm for α-
chloro ketones. After cyclization, these CH3 protons are shifted downfield at 2.40-2.60 ppm
5

ACCEPTED MANUSCRIPT
due to the aromatic nature of the newly formed thiazole ring. This result confirms the
formation of thiazole ring. It is interesting to note that the NH protons present in
thiosemicarbazones appear at 9.0-9.5 ppm whereas, due to the formation of aromatic thiazole
ring, these protons appear downfield at 10.5-11.5 ppm in 2-(2-hydrazinyl)thiazole
derivatives. Similarly, appearance of peaks corresponding to aliphatic protons in the region
13 - 65 ppm and three characteristic peaks of the thiazole ring at approximately 90-110, 135-
150 and 165-175 ppm confirm the formation of the 2-(2-hydrazinyl)thiazole derivatives. The
precise matching of the experimental mass and CHNS values with the calculated values
confirms the formation of analytically pure desired products. All the synthesized 2-(2-
hydrazinyl)thiazole derivatives with structural variations at 2 –, 4 – and 5 – positions are
listed in Table 2.
<Insert Table 2>
2.3. Single crystal X-ray study
The 2-aminothiazole ring and aromatic rings attached to C=N group may exist in
either E configuration or Z configuration. As the configuration of these compounds may
decide the drug properties, it is important to understand the configuration of newly
synthesized 2-(2-hydrazinyl)thiazole derivatives. For this, two compounds, 2i and 2l have
been selected as the representative of 2-(2-hydrazinyl)thiazole derivatives and single crystal
of these two compounds have been grown in acetonitrile by slow evaporation at 25-30 °C.
The solved single crystal structures of 2i and 2l are shown in Figure 2. The results reveal that
the molecules are planar and 2-aminothiazole ring and aromatic ring at C=N are in E-
configuration. The crystal structure of 2i preferred to stabilize with strong O-H···N intra
molecular hydrogen bonds (2.01 Å, 137.73°) between N of C=N and ortho-O-H of the phenyl
ring. In addition to this, weak C-H···O hydrogen bond interaction (2.13 Å, 129.10°) [43] also
stabilizes this compound. The molecules are packed in head to head dimer fashion with N-
H···O (1.91 Å, 169.56°) intermolecular hydrogen bond interactions leading to the stabilization
of the three dimensional assembly. Similarly, 2l stabilizes by the weak intra molecular C-
H···O interactions (2.29 Å, 129.10°) and two strong inter molecular N-H···N (2.17 Å,
171.43°) hydrogen bond interactions between N-H at 2-position of thiazole ring and N of
thiazole ring.
<Insert Figure 2>
6

ACCEPTED MANUSCRIPT
2.4. Antimycobacterial activity
After the successful design, synthesis and structural confirmation, the antimicrobial
activities of the newly synthesized 2-(2-hydrazinyl)thiazole derivatives (2a- 6b) have been
tested against Mtb, H37Rv. In this study, the minimum inhibitory concentration (MIC) of 2-
(2-hydrazinyl)thiazole derivatives that yield ≥ 80% inhibition using Resazurin microtiter
assay (REMA) [44] is considered as positive. Isoniazide, the first line TB drug in clinical
practice is used as a reference compound. The MIC values of in vitro test results are given in
Table 1 along with Lipinski rule results. It is interesting to note that anti-TB activity results
are varied based on the substitutions at 4th and 5th positions of thiazole ring and substitution at
hydrazinyl group.
Compound 2a, the primary molecule of thiozole derivatives 2a-2w, shows no activity
with MIC value of more than 200 µM. Compound 2b, imine form of 2a also shows similar
activity. When 2b is substituted with fluorine at 4th position, it shows the moderate activity
with MIC value of 200 µM. This indicates that Schiff base and imine systems are not
influencing the activity significantly, but it is the functional groups substituted on the phenyl
ring decide the anti-TB activity. To further explore, single and multiple substitutions of
chlorine at 2nd, 3rd and 4th positions (2d-2g) are changed. But, none of them show MIC value
less than 200 µM. Similarly, when Br is introduced at 3rd position (2h), the MIC value is
more than 200 µM. In addition, the effect of OH and alkoxy group on the phenyl ring has
been studied extensively. In this series, the OH group is attached at 2nd, 3rd and 4th positions.
The 2-hydroxyphenyl system (2i) shows significant activity with MIC value of 25 µM
whereas its counterparts (2j, 2k) do not show MIC values less than 200 µM. To further
explore the activity of OH and its methyl and ethyl derivatives, the OH is replaced with
OCH3 at 4th position (2l), new OCH3 and OC2H5 groups are introduced at 3rd positions by
keeping OH groups constant at 4th positions (2m, 2n). The OH and OCH3 groups are also
exchanged (2o). Apart from this, di and tri OCH3 (2p, 2r) and 1, 3-benzodioxo systems (2q)
are also studied. Unfortunately, all of these compounds are not active as the MIC values are
more than 200 µM. To further explore, compounds with alkyl substitutions such as CH3 and -
C(CH3)2 groups at 4th position (2s, 2t), NO2 substitutions at 2nd and 4th position (2w, 2v) and -
N(CH3)2 substitution at 4th position (2u) are also studied. In this series, -NO2 and -N(CH3)2
substitutions at 4th position show moderate activity with MIC value of 200 µM. Efforts have
also been made to introduce and identify the effect of -COCH3 group in place of -COOC2H5
7

ACCEPTED MANUSCRIPT
at 5th position of the thiazole ring (2x, 2y, 2z) as these compounds have keto-enol
tautomerism to influence activity [45, 46]. But, there is no significant difference in the
activity.
In continuation of this study, aromatic heterocyclic systems which include both five
membered and six membered rings are introduced in place of phenyl ring. The five
membered heterocyclic systems such as thiophene (4b, 5b), furan (4a, 5a) and 3-indole (4c,
5c) are introduced at 2nd position of thiazole ring and 5th position of thiazole ring is varied
with COOC2H5 and COCH3 groups. It is interesting to note that thiophene with COOC2H5 at
5th position (4b) shows good inhibition against Mtb with MIC value of 100 µM. Replacement
of COOC2H5 with COCH3 (5b) decreases the activity to a MIC value of 200 µM. In contrast,
furan (4a, 5a) and 3-indole (4c, 5c) systems show moderate activity with MIC value of 200
µM. Similarly, 2-, 3- and 4-pyridyl systems with variations at 4th and 5th positions using
aliphatic ketone and ester groups have also been tested. In this series, 2-pyridyl system with
COOC2H5 group at 5th position (4d) shows significant activity with MIC value of 12.50 µM
whereas replacement of -COOC2H5 group with -COCH3 group at 5th position (5d) decreases
the activity to more than six folds with MIC value of 100 µM. Similarly, a compound with H
atom at 5th position and CH2COOC2H5 group at 4th position (6b) shows moderate activity
with MIC value of 200 µM. 3-Pyridyl and 4-pyridyl systems with CH3 at 4th position and
COOC2H5 at 5th position (4e, 4f) show moderate activity with MIC value of 200 µM. Further
variations of 3-pyridyl ring system with H at 5th position and CH2COOC2H5 at 4th position
(6a) show no difference in the activity. In conclusion, 2-pyridyl and 2-hydroxyphenyl
hydrazinyl substitutions at the 2nd position of thiazole ring and CH3 at 4th and COOC2H5 at 5th
positions of thiazole ring (2i, 4d) show significant inhibition of Mtb with MIC value of 12.50
µM and 25.50 µM respectively which is comparable with the literature [33, 34] results. The
structural similarity between these two compounds is four membered and five membered
coordination systems. This could be the possible structural feature to enhance the activity. It
may also be concluded that the COOC2H5 at 5th position of thiazole ring is an activity
enhancing group.
2.5. Cytotoxicity studies
The in vitro cytotoxicity was tested against human lung carcinoma type II epithelial
cells (A549) [47]. A549 cells were treated with 2b, 4d, 5d and 6b at different concentration
in a 96 wells plate for 48 h and the cell growths were analyzed by MTT assay. The
8

ACCEPTED MANUSCRIPT
cytotoxicity of the compounds were calculated from their IC50 values and the results are
summarized in Table 3. The cytotoxicity order of these compounds is 5d>4d>2b>6b with
IC50 values of 50, 140, 150 and 200 respectively. The effect of 2b, 4d, 5d and 6b on the
proliferation of cells with concentration variations is depicted in the Figure 3. All these results
suggest that the compounds with 2-pyridyl substitution with an ester group attached to the
thiazole ring at 4th and 5th position, 6b and 4d, have shown the low cytotoxicity with IC50
values 200 and 140. Whereas 2-pyridyl substitution with the keto group at the 5th position,
5d, has shown high cytotoxicity with IC50 value of 50. The aromatic ring system, 2b has
shown IC50 value of 150. The low toxicity values and the high antimycobacterial values of
the 2-pyridyl system at 2-position suggest that these compounds could be efficient therapeutic
candidates to treat TB.
<Insert Table 3 and Figure 3>
2.6.
Docking studies with KasA protein
After successful finding of DLMs, it is important to understand how these DLMs
interact with Mtb. Docking studies give a fair idea related to drug-receptor interactions. For
this study, KasA protein has been chosen as it is assumed that the interaction of drug with this
protein is the important step during the deactivation of Mtb. Two representative compounds,
2a and 4d, have been chosen for this study. The compound 2a has been selected as it is the
basic structure of all compounds presented in this study and 4d has been selected as it shows
the highest activity among the compounds reported in this report.
Compounds 2a and 4d show the inhibition constant (Ki) values of 3.29 µM and 1.46
µM with binding energy of -7.48 Kcal/mol and -7.96 Kcal/mol respectively. The inhibition of
the protein by 4d is through hydrogen bonding interactions of NH in 4d with a COO - group
of THR – 315, C=N of 4d with a COO- group of VAL – 278 and C=O in 4d with a NH group
present in HIS -311, and HIS – 345 protein residues as shown in Figure 4. Similarly, in case
of 2a, C=N and C=O functional groups form hydrogen bonding interactions with COO- group
of VAL – 278 and NH group of HIS -311, and HIS – 345 respectively as shown in Figure 4.
The hydrogen bonding interactions of these compounds with KasA protein residues are
similar to that of TLM, the known TB drug which inhibits KasA protein. These results
suggest that the present molecules could be the possible candidates to inhibit mycolic acid
synthesis, an essential step in cell wall biosynthesis, by inhibiting the KasA protein of
mtFabH. The differences in Ki and binding energy values of 2a and 4d and their patterns of
9

ACCEPTED MANUSCRIPT
binding interactions with the KasA protein suggests that 4d could be better candidate for
inhibition of KasA protein of mtFabH.
<Insert Figure 4>
3. Conclusion
2-(2-Hydrazinyl)thiazole derivatives have been synthesized from corresponding
thiosemicarbazones with aliphatic α-halo ketones. Among the compounds, 4d and 2i are
found to be potential molecules against Mtb, H37Rv with MIC values 12.5 µM and 25 µM
respectively. The activity result suggests that molecules with structural features of either
hydrogen bond acceptor or hydrogen bond donor at 2-position of the aromatic substituents
induce activity. Among these, the 2-pydidyl system proves to be the promising substituent. In
the cytotoxicity studies against human lung carcinoma type II epithelial cells, compound 6b
exhibits low cytotoxicity. The protein – ligand interaction studies reveal that the hydrogen
bonding interactions of 2a and 4d with KasA protein are similar to that of TLM. The aim to
explore the 2-(2-hydrazinyl)thiazole derivatives with special focus on the present lead
compounds to achieve improved antimycobacterial activities and further validation of them to
use as potential drugs are under progress in our laboratory.
4. Experimental
4.1. Materials and methods
Thiosemicarbazide and carbonyl compounds were purchased either from Sigma–
Aldrich, USA or Himedia Biosciences or Spectrochem Pvt. Ltd (Mumbai, India). α-
Haloketones were purchased from Himedia Biosciences. All the chemicals were used without
further purification. 1H NMR and 13C NMR spectra were recorded with 400 MHz and 101
MHz respectively using Bruker Avance-II NMR instrument. IR spectra were recorded on a
Thermo Nicolet 6700 FT-IR spectrometer and only major peaks are reported in cm-1.
Elemental analysis of all the compounds was performed on Elementar Vario EL-II CHNS
analyzer. Mass spectra (MS) were recorded on a Thermo Scientific High Resolution
Magnetic Sector MS DFS by chemical ionization (CI) or negative-ion electrospray ionization
(ESI) method. The single crystals were analyzed using Oxford Diffractometer and data
collection, cell refinement and data reduction were carried out using CrysAlis PRO, Oxford
Diffraction Ltd., Version 1.171.34.44 (release 25-10-2010 CrysAlis171. NET) software. The
programs used to solve the structures and to refine structures are olex2.solve (compiled Oct
10

ACCEPTED MANUSCRIPT
25 2010,18:11:34) and SHELXL respectively. All the IUPAC names for the synthesized
compounds were deduced using MarvinSketch software version 5.11.5.
4.2. General procedures for the synthesis of thiosemicarbazone derivatives
The appropriate thiosemicarbazide (1.0 mmol), aldehyde or ketone (1.1 mmol) were
dissolved in ethanol (10 ml) and acetic acid (0.2 mmol) was added to the above solution. The
reaction mixture was refluxed for 5-6 h and then cooled to room temperature. The resulting
precipitate was filtered, washed by ether and recrystallized from ethanol to obtain the
corresponding thiosemicarbazones, 1a-w and 3a-f. All the compounds were characterized by
spectral techniques and were found to be identical with the reported data in the literature [39-
42]. The unreported data for the thiosemicarbazones are given below.
4.2.1. [(E)-[(3-Bromophenyl)methylidene]amino]thiourea (1h)
White solid from EtOH (81% yield). m.p. 224-226 °C. 1H NMR (400 MHz, δ, ppm,
DMSO-d6): δ 11.52 (s, 1H), 8.27 (s, 1H), 8.21 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 8.02 (s, 1H),
7.70 (d, J = 7.8 Hz, 1H), 7.57 (m, 1H), 7.36 (t, J = 7.9 Hz, 1H). 13C NMR (101 MHz, δ, ppm,
DMSO-d6): δ 178.19, 140.46, 136.68, 132.27, 130.68, 128.88, 126.95, 122.34.
4.2.2. [(E)-[(4-Methylphenyl)methylidene]amino]thiourea (1s)
White solid from EtOH (77% yield). m.p. 194-196 °C. 1H NMR (400 MHz, δ, ppm,
CDCl3): δ 10.22 (s, 1H), 7.92 (s, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.23 (s, 1H), 7.19 (d, J = 7.9
Hz, 2H), 6.60 (s, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, δ, ppm, DMSO-d6): δ 178.27,
144.51, 141.39, 130.40, 129.72, 127.62, 77.48, 77.16, 76.85, 21.67.
4.2.3. [(E)-{[4-(Propan-2-yl)phenyl]methylidene}amino]thiourea (1t)
White solid from EtOH (85% yield). m.p. 245-246 °C. 1H NMR (400 MHz, δ, ppm,
DMSO-d6): δ 11.38 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.92 (s, 1H), 7.70 (d, J = 8.3 Hz, 1H),
7.26 (d, J = 8.2 Hz, 1H), 2.89 (hept, J = 6.9 Hz, 1H), 1.19 (d, J = 6.9 Hz, 1H).13C NMR (101
MHz, δ, ppm, CDCl3): δ 178.10 (s), 152.20 (s), 144.60 (s), 130.78 (s), 127.73 (s), 127.07 (s),
77.48 (s), 77.16 (s), 76.84 (s), 34.23 (s), 23.83 (s).
11

ACCEPTED MANUSCRIPT
4.3. General procedures for the synthesis of 2-(2-hydrazinyl)thiazole derivatives
The appropriate thiosemicarbazone (1.0 mmol) and aliphatic α-halo ketones (1.1
mmol) were dissolved in ethanol (10 ml). The reaction mixture was refluxed for 1-3 h and
then was cooled to room temperature. The resulting precipitate was filtered, washed with cold
ethanol and recrystallized from ethanol to obtain the corresponding 2-(2-hydrazinyl)thiazole
derivatives, 2a-z, 4a-4f, 5a-5f and 6a-6b in analytically pure form. All the compounds were
characterized by spectral techniques and the data confirm the proposed structures.
4.3.1. Ethyl 4-methyl-2-[(E)-2-(phenylmethylidene)hydrazin-1-yl]-1,3-thiazole-5-carboxylate
(2a)
Light brown solid from EtOH; (78 % yield). m.p. 196-197 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.33 (t, J = 6.8 Hz, 3H), 2.58(3H, s), 4.23(q, J = 6.8, 2H), 7.38 - 7.40 (m, 3H),
7.63 -7.65 (m, 2H), 7.87(s, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3): 14.44, 17.04, 60.61,
111.45, 127.02, 128.80, 130.05, 133.89, 143.84, 157.17, 162.47, 170.96; IR (KBr, νmax, cm-1):
3199, 3084, 2997, 2932, 1670, 1575, 1370, 1320, 1280, 1087; Anal. Calcd. for C14H15N3O2S:
C, 58.11; H, 5.23; N, 14.52; S, 11.08; Found: C, 58.09; H, 5.28; N, 14.51; S, 10.97; HR-MS
(ESI+): Calcd. m/z for C14H15N3O2S: 289.08850; Found m/z [M + H]+: 289.08854.
4.3.2. Ethyl 4-methyl-2-[(E)-2-(1-phenylethylidene)hydrazin-1-yl]-1,3-thiazole-5-carboxylate
(2b)
White solid from EtOH; (75 % yield). m.p. 182-183 °C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.37 (t, J = 6.8 Hz, 3H), 2.40 (s, 3H), 2.78(3H, s), 4.33(q, J = 6.8, 2H), 7.24 - 7.40
(m, 3H), 7.63 -7.65 (m, 2H), 7.87(s, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3): 14.52, 15.42,
17.08, 60.77, 112.15, 126.72, 129.40, 131.05, 133.79, 144.14, 158.17, 162.67, 171.06; IR
(KBr, νmax, cm-1): 3199, 3084, 2997, 2932, 1670, 1575, 1370, 1320, 1280, 1087; Anal. Calcd.
for C15H17N3O2S: C, 59.38; H, 5.65; N, 13.85; S, 10.57; Found: C, 59.37; H, 5.63; N, 13.88;
S, 10.56; HR-MS (ESI+): Calcd. m/z for C15H17N3O2S: 303.10414; Found m/z [M + H]+:
303.10426.
4.3.3. Ethyl 2-[(E)-2-[1-(4-fluorophenyl)ethylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2c)
12

ACCEPTED MANUSCRIPT
Brown solid from EtOH (80% yield). m.p. 177 -178 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.24 (t, J = 7.20 Hz, 3H), 2.30 (s, 3H), 2.46 (s, 3H), 4.18 (q, J = 7.20 Hz, 2H),
7.23 (d, J = 8.50 Hz, 2H), 7.81 (d,J = 8.53, 2H), 11.82 (s, 1H); 13C NMR (101 MHz, δ, ppm,
DMSO-d6): 14.23, 14.33, 16.53, 21.04, 60.10, 115.21, 128.03, 128.11, 134.16, 161.94,
163.978, 169.82, 172.02; IR (KBr, νmax, cm-1): 3200, 3089, 2983, 1681, 1556, 1423, 1372,
1271, 1090; Anal. Calcd. for C15H16FN3O2S: C, 56.06; H, 5.02; N, 13.08; S, 9.98; Found: C,
56.10; H, 5.05; N, 13.12; S, 9.94; HR-MS (ESI+): Calcd. m/z for C15H16FN3O2S: 323.04952;
Found m/z [M + H]+: 323.04958.
4.3.4. Ethyl 2-[(E)-2-[(2-chlorophenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2d)
Antique white solid from EtOH (83% yield). m.p. 172 -180 °C; 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 1.26 (t, J = 7.1, 3H) , 2.48 (s, 3H), 4.20 (q, J = 7.1, 2H), 7.45 – 7.39 (m,
2H), 7.53 – 7.48 (m, 1H), 7.98 – 7.91 (m, 1H), 8.43 (s, 1H); 13C NMR (101 MHz, δ, ppm,
DMSO-d6): 14.27, 16.74, 60.17, 116.48, 126.55, 127.66, 129.92, 131.13, 132.56,134.05,
144.24, 156.53, 162.15, 169.09; IR (KBr, νmax, cm-1): 3142, 3038, 2985, 2936, 1706, 1558,
1426, 1372, 1313, 1271, 1215, 1088; Anal. Calcd. for C14H14ClN3O2S: C, 51.93; H, 4.36; N,
12.98; S, 9.90; Found: C, 51.96; H, 4.31; N, 13.01; S, 9.95; HR-MS (ESI+): Calcd. m/z for
C14H14ClN3O2S: 323.04953; Found m/z [M + H]+: 323.04958.
4.3.5. Ethyl 2-[(E)-2-[1-(3-chlorophenyl)ethylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2e)
Antique white solid from EtOH (80% yield). m.p. 192 -194 °C; 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 1.26 (t, J = 7.2 Hz, 3H), 2.47 (s, 3H,), 4.20 (q, J =7.2 Hz, 2H), 7.45 (t, J
= 3.5 Hz, 2H), 7.64 (t, J = 3.5 Hz, 1H), 7.70 (s, 1H), 8.06 (s,1H), 12.53 (s, 1H); 13C NMR
(101 MHz, δ, ppm, DMSO-d6): 14.29, 17.11, 18.50, 60.17, 116.88, 125.26, 125.95, 129.37,
130.73, 133.64, 136.24, 154.79, 161.81, 167.10, 169.19; IR (KBr, νmax, cm-1): 3143, 3038,
2986, 2905, 1705, 1573, 1470, 1432, 1373, 1317, 1275, 1214, 1128, 1091, 973; Anal. Calcd.
for C15H16ClN3O2S: C, 53.33; H, 4.77; N, 12.44; S, 9.49; Found: C, 53.36; H, 4.82; N, 12.48;
S, 9.50; HR-MS (ESI+): Calcd. m/z for C15H16ClN3O2S: 337.06518; Found m/z [M + H]+:
337.06522.
13

ACCEPTED MANUSCRIPT
4.3.6. Ethyl 2-[(E)-2-[(4-chlorophenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2f)
Antique white solid from EtOH (77% yield). m.p. 206-208 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.34 (t, J = 7.14 Hz, 3H), 2.57 (s, 3H), 4.24 (q, J = 7.14 Hz, 2H), 7.36 (d, J =
8.53 Hz, 2H), 7.58 (d, J = 8.53 Hz, 2H), 7.80 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-
d6): 14.26, 16.98, 60.08, 109.22, 128.18, 128.85, 132.95, 134.15, 143.00, 158.00, 161.83,
169.11; IR (KBr, max, cm-1): 3200, 3089, 2983, 1681, 1556, 1423, 1372, 1271, 1090; Anal.
Calcd. for C14H14ClN3O2S: C, 51.93; H, 4.36; N, 12.98; S, 9.91; Found: C, 52.01; H, 4.31; N,
12.91; S, 9.89; HR-MS (ESI+): Calcd. m/z for C14H14ClN3O2S: 323.04952; Found m/z [M +
H]+: 323.04958.
4.3.7. Ethyl 2-[(E)-2-[(2,4-dichlorophenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-
5-carboxylate (2g)
Antique white solid from EtOH (75% yield). m.p. 223-224 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.36 (t, J = 7.1, 1H), 2.61 (s, 1H), 4.27 (q, J = 7.1, 1H), 4.78 (s, 1H), 7.28 (dd,
J = 8.6, 1.7, 1H), 7.39 (d, J = 1.9, 1H), 7.99 (d, J = 8.6, 1H), 8.24 (s, 1H); 13C NMR (101
MHz, δ, ppm, DMSO-d6): 14.36. 17.19, 60.04, 110.96, 115.76, 120.97, 125.39, 145.08,
147.10, 149.07, 161.98, 169.02; IR (KBr, νmax, cm-1): 3459, 3221, 3092, 2976, 2933, 1676,
1591, 1565, 1422, 1472, 1334, 1100, 876; Anal. Calcd. for C14H13Cl2N3O2S: C, 46.94; H,
3.66; N, 11.73; S, 8.95; Found: C, 46.97; H, 3.68; N, 11.76; S, 8.99; HR-MS (ESI+): Calcd.
m/z for C14H13Cl2N3O2S: 357.0105; Found m/z [M + H]+: 357.0117.
4.3.8. Ethyl 2-[(E)-2-[(3-bromophenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2h)
Yellow solid from EtOH (82% yield). m.p. 209-210 °C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.37 (t, J = 7.13 Hz, 3H), 2.60 (s, 3H), 4.31 (q, J = 7.13 Hz, 2H), 7.27 (t, J = 8.00
Hz, 2H), 7.51 (d, J = 8.00 Hz, 1H), 7.55 (d, J = 7.70 Hz, 1H), 7.83 (s, 1H), 7.78 (s, 1H); 13C
NMR (101 MHz, δ, ppm, CDCl3): 14.56, 16.66, 60.24,117.48, 123.09, 128.37, 130.47,
133.27, 134.68, 137.51, 143.46, 156.82, 160.99, 170.51; IR (KBr, νmax, cm-1): 3141, 3037,
2985, 2904, 1701, 1558, 1433, 1274, 1091; Anal. Calcd. for C14H14BrN3O2S: C, 45.66; H,
14

ACCEPTED MANUSCRIPT
3.83; N, 11.41; S, 8.71 Found: C, 45.62; H, 3.79; N, 11.49; S, 8.65; HR-MS (ESI+): Calcd.
m/z for C14H14BrN3O2S: 366.99901; Found m/z [M + H]+: 366.99907.
4.3.9. Ethyl 2-[(E)-2-[(2-hydroxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (2i)
Antique white solid from EtOH; (88 % yield). m.p. 197 -200 °C; 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 1.25 (t, J = 6.47 Hz, 3H), 2.45 (s, 3H), 4.16 (q, J = 6.47 Hz, 3H), 6.98 –
6.79 (m, 2H), 7.24 (d, J = 8.01 Hz, 1H), 7.62 (d, J = 8.01 Hz, 1H), 10.31 (s, 1H), 8.40 (s, 1H),
12.36 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.12, 16.54, 60.17, 116.20, 119.51,
119.65, 127.30,131.09, 144.15, 156.62, 161.77, 168.39; IR (KBr, νmax, cm-1): 3307, 3154,
3027, 2960, 2825, 1678, 1611, 1513, 1372, 1223, 1165, 1093, 968; Anal. Calcd. for
C14H15N3O3S: C, 55.07; H, 4.95; N, 13.76; S, 10.5; Found: C, 55.10; H, 4.96; N, 13.78; S,
10.56; HR-MS (ESI+): Calcd. m/z for C14H15N3O3S: 305.08341; Found m/z [M + H]+:
305.08346.
4.3.10. Ethyl 2-[(E) -2-[(3-hydroxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-
5-carboxylate (2j)
1
White solid from EtOH (72% yield). mp 199 -201°C; H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.34 (t, J = 7.2, 3H), 2.38 (s, 3H), 4.11 (q, J = 7.2, 2H), 6.85 (dd, J =6.0 Hz, 3.2
Hz, 2H,), 7.58 (dd , J =6.0, 3.2 Hz, 2H), 7.88 (d, J =6.8, 1H,), 9.92 (s, 1H,), 12.12 (s, 1H);
13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.75, 17.33, 61.20, 116.45, 12.03, 128.48, 143.94,
156.93, 160.31, 162.09, 168.16; IR (KBr, νmax, cm-1): 3307, 3154, 3027, 2960, 2825, 1678,
1611, 1513, 1372, 1223, 1165, 1093, 968; Anal. Calcd. for C14H15N3O3S: C, 55.07; H, 4.95;
N, 13.76; S, 10.50; Found: C, 55.13; H, 4.94; N, 13.78; S, 10.54; HR-MS (ESI+): Calcd. m/z
for C14H15N3O3S: 305.08341; Found m/z [M + H]+: 305.08353.
4.3.11. Ethyl 2-[(E) -2-[(4-hydroxyphenyl) methylidene] hydrazin-1-yl]-4-methyl-1,3-
thiazole-5-carboxylate (2k)
15

ACCEPTED MANUSCRIPT
Antique white from EtOH (76% yield). mp 179 -180 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.24 (t, J = 7.2, 3H), 2.44 (s, 3H), 4.16 (q, J = 7.2, 2H), 6.80 (dd, J =6.0 Hz, 3.2
Hz, 2H,), 7.48 (dd , J =6.0, 3.2 Hz, 2H), 7.97 (d, J =6.8, 1H,), 9.88 (s, 1H,), 12.17 (s, 1H);
13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.35, 17.16, 60.04, 115.79, 125.03, 128.48,
143.94, 156.93, 159.31, 161.99, 169.16; IR (KBr, νmax, cm-1): 3307, 3154, 3027, 2960, 2825,
1678, 1611, 1513, 1372, 1223, 1165, 1093, 968; Anal. Calcd. for C14H15N3O3S: C, 55.07; H,
4.95; N, 13.76; S, 10.50; Found: C, 55.11; H, 4.96; N, 13.80; S, 10.56; HR-MS (ESI+):
Calcd. m/z for C14H15N3O3S: 305.08341; Found m/z [M + H]+: 305.08346.
4.3.12. Ethyl 2-[(E)-2-[(4-methoxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazole-
5-carboxylate (2l)
Antique white solid (81% yield). m.p.180 - 182°C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.33 (t, J = 7.08 Hz, 3H), 3.84 (s, 3H), 2.57 (s, 3H), 4.23 (q, J = 7.08 Hz, 2H), 5.57
(s, 1H), 6.91 (d, J = 8.76 Hz, 2H), 7.58 (d, J = 8.76 Hz, 2H), 7.82 (s, 1H); 13C NMR (101
MHz, δ, ppm, CDCl3): 14.15, 16.82, 55.16, 60.24, 110.73, 114.00, 126.39, 128.28, 143.58,
156.94, 160.99, 162.31, 170.51; IR (KBr, νmax, cm-1): 3168, 3056,298, 2845, 1700,
1606,1581, 1512,1432, 1370, 1314, 1253, 1166 ; Anal. Calcd. for C15H17N3O3S:C, 56.41; H,
5.37; N, 13.16; S, 10.04; Found: C, 56.37; H, 5.39; N, 13.19; S, 10.08; HR-MS (ESI+):
Calcd. m/z for C15H17N3O3S: 319.37878; Found m/z [M + H]+: 319.37890.
4.3.13. Ethyl 2-[(E)-2-[(3-hydroxy-4-methoxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-
1,3-thiazole-5-carboxylate (2m)
Light yellow from EtOH (88% yield). m.p. 226 - 229 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.25 (t, J = 7.2, 3H), 2.46 (S, 3H), 3.82 (s, 3H), 4.18 (q, J = 7.2, 2H), 6.82 (d, J
= 8.0, 1H),7.09 (d, J = 8.0, 1H,), 7.23 (s, 1H), 9.55 (s, 1H,), 12.25 (s, 1H,); 13C NMR (101
MHz, δ, ppm, DMSO-d6): 14.35, 17.17, 55.59, 60.03, 109.62, 115.68, 121.01, 125.40,
145.09, 147.95, 148.82, 161.98, 169.01; IR (KBr, νmax, cm-1): 3235, 3069, 2933, 2834, 1666,
1584, 1516, 1422, 1372, 1289, 1164, 1091; Anal. Calcd. for C15H17N3O4S: C, 53.72; H, 5.11;
N, 12.53; S, 9.56 Found: C, 53.76; H, 5.15; N, 12.56; S, 9.60; HR-MS (ESI+): Calcd. m/z for
C15H17N3O4S: 335.09398; Found m/z [M + H]+: 335.09311.
16

ACCEPTED MANUSCRIPT
4.3.14. Ethyl 2-[(E)-2-[(3-ethoxy-4-hydroxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-
thiazole-5-carboxylate (2n)
Antique white solid from EtOH (76% yield). m.p. 204-206 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 1.25 (t, J = 7.09 Hz, 3H), 1.36 (t, J = 6.98 Hz, 3H), 2.46 (s, 3H), 4.06 (q, J
= 6.93 Hz, 2H), 4.19 (q, J = 7.07 Hz, 2H), 6.84 (d, J = 8.13 Hz, 1H), 7.09 (d, J = 8.14 Hz,
1H), 7.21 (d, J = 1.92 Hz, 1H), 7.96 (s, 1H), 9.45 (s, 1H), 12.26 (s, 1H); 13C NMR (101 MHz,
δ, ppm, DMSO-d6): 14.36, 14.74, 17.19, 38.90, 39.10, 39.31, 39.52, 39.73, 39.94, 40.15,
60.04, 63.92, 110.96, 115.76, 120.97, 125.39, 145.08, 147.10, 149.07, 161.98, 169.02; IR
(KBr, νmax, cm-1): 3321, 3171, 3063, 2921, 1674, 1580, 1518, 1423, 1371, 1276, 1175, 1088;
Anal. Calcd. for C16H19N3O4S: C, 55.00; H, 5.48; N, 12.03; S, 9.18; Found: C, 55.03; H,
5.46; N, 11.96; S, 9.15; HR-MS (ESI+): Calcd. m/z for C16H19N3O4S: 349.10962; Found m/z
[M + H]+: 349.10975.
4.3.15. Ethyl 2-[(E)-2-[(3-hydroxy-4-methoxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-
1,3-thiazole-5-carboxylate (2o)
Sandy brown solid from EtOH (80% yield). m.p. 215 -217 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 1.26 (t, J = 7.12 Hz, 3H), 2.46 (s, 3H), 3.79 (s, 3H), 4.19 (q, J = 7.12 Hz,
2H), 6.94 (d, J = 8.36 Hz, 1H), 7.00 (dd, J = 1.94, 8.36 Hz, 1H), 7.23 (d, J = 1.90 Hz, 1H),
7.94 (s, 1H), 9.30 (s, 1H), 12.18 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.35,
17.16, 55.58, 60.06, 99.56, 111.67, 111.86, 120.12, 126.83, 144.20, 146.90, 149.72, 161.95,
169.15; IR (KBr, νmax, cm-1): 3235, 3069, 2933, 2834, 1666, 1584, 1516, 1422, 1372, 1289,
1164, 1091; Anal. Calcd. for C15H17N3O4S: C, 53.72; H, 5.11; N, 12.53; S, 9.56; Found: C,
53.79; H, 5.15; N, 12.55; S, 9.58; HR-MS (ESI+): Calcd. m/z for C15H17N3O4S: 335.09398;
Found m/z [M + H]+: 335.09409.
4.3.16. Ethyl 2-[(E)-2-[(3,4 -dimethoxyphenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-
thiazole-5-carboxylate (2p)
Antique white solid from EtOH (76% yield). m.p. 211-213 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.33 (t, J = 7.13 Hz, 3H), 3.91 (s, 3H), 2.58 (s, 3H), 3.96 (s, 3H), 4.25 (q, J =
17

ACCEPTED MANUSCRIPT
7.19 Hz, 2H), 6.85 (d, J = 8.30 Hz, 1H), 7.06 (d, J = 8.23 Hz, 1H), 7.82 (s, 1H), 7.31 (s, 1H);
13C NMR (101 MHz, δ, ppm, CDCl3): δ 14.50, 17.19, 56.09, 60.67, 108.12, 110.86, 111.18,
121.75, 126.94, 144.26, 149.51, 151.12, 157.09, 162.66, 170.59; IR (KBr, νmax, cm-1): 3211,
3106, 2986, 2935, 2831, 1664, 1510, 1424, 1268, 1088, 1024; Anal. Calcd. for C16H19N3O4S:
C, 55.00; H, 5.48; N, 12.03; S, 9.18; Found: C, 54.03; H, 5.49; N, 12.07; S, 9.19; HR-MS
(ESI+): Calcd. m/z for C16H19N3O4S: 349.10962; Found m/z [M + H]+: 349.10973.
4.3.17. Ethyl 2-[(E)-2-(2H-1,3-benzodioxol-4-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-
thiazole-5-carboxylate (2q)
Light green solid from EtOH (80% yield). m.p.242 -245 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 1.26 (t, J = 7.15 Hz, 3H), 2.46 (s, 3H), 4.19 (q, J = 7.15 Hz, 2H), 6.08 (s,
2H), 6.96 (d, J = 8.16 Hz, 1H), 7.13 (d, J = 8.16 Hz, 1H), 7.23 (s, 1H), 7.99 (s, 1H), 12.31 (s,
1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.30. 17.12, 60.05, 101.54, 104.92, 108.50,
121.10, 122.74, 128.42, 143.92, 147.97, 148.90, 155.86, 161.90, 169.09; IR (KBr, νmax, cm-1):
3156, 3039, 2986, 2907, 1707, 1583, 1497, 1430, 1374, 1256, 1095, 972, 931; Anal. Calcd.
for C15H15N3O4S: C, 54.04; H, 4.54; N, 12.60; S, 9.62; Found: C, 54.09; H, 4.55; N, 12.55; S,
9.68; HR-MS (ESI+): Calcd. m/z for C15H15N3O4S: 333.07833; Found m/z [M + H]+:
333.07838.
4.3.18. Ethyl 4-methyl-2-[(E)-2-[(3,4,5-trimethoxyphenyl)methylidene]hydrazin-1-yl]-1,3-
thiazole-5-carboxylate (2r)
Light green solid from EtOH (79% yield). m.p. 189-191°C. 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.34 (t, J = 7.10 Hz, 3H), 2.59 (s, 3H), 3.89 (d, J = 8.79 Hz, 9H), 4.27 (q, J =
7.10 Hz, 2H), 6.87 (s, 2H), 7.78 (s, 1H), 10.91 (s, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3):
14.50, 17.10, 56.36, 60.77, 61.05, 104.21, 111.43, 129.28, 140.14, 144.18, 153.65, 156.75,
162.57, 170.50; IR (KBr, νmax, cm-1): 3200, 3099, 2975, 2976, 2834, 1675, 1578, 1504, 1371,
1129, 1089, 1008; Anal. Calcd. for C17H21N3O5S: C, 53.81; H, 5.58; N, 11.07;S, 8.45; Found:
C, 53.89; H, 5.50; N, 11.11; S, 8.43; HR-MS (ESI+): Calcd. m/z for C17H21N3O5S:
379.12019; Found m/z [M + H]+: 379.12021.
18

ACCEPTED MANUSCRIPT
4.3.19. Ethyl 4-methyl-2-[(E)-2-[(4-methylphenyl)methylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (2s)
Antique white solid from EtOH (85% yield). m.p.189 - 191°C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.34 (t, J = 7.11 Hz, 3H), 2.59 (s, 3H), 2.38 (s, 3H), 4.25 (q, J = 7.11 Hz, 2H),
7.20 (d, J = 8.08 Hz, 2H), 7.55 (d, J = 8.08 Hz, 2H), 7.85 (s, 1H); 13C NMR (101 MHz, δ,
ppm, CDCl3): δ 14.50, 17.23, 21.62, 60.63, 111.36, 127.09, 129.59, 131.20, 140.45, 144.27,
157.34, 162.67, 170.81; IR (KBr, νmax, cm-1): 3459, 3194, 3101, 2979, 1665, 1570, 1428,
1371, 1274, 1123, 1091; Anal. Calcd. for C15H17N3O2S: C, 59.38; H, 5.65; N, 13.85; S,
10.57; Found: C, 59.36; H, 5.62; N, 13.87;S, 16.00; HR-MS (ESI+): Calcd. m/z for
C15H17N3O2S: 303.10414; Found m/z [M + H]+: 303.10416.
4.3.20. Ethyl 4-methyl-2-[(E)-2-{[3-(propan-2-yl)phenyl]methylidene}hydrazin-1-yl]-1,3-
thiazole-5-carboxylate (2t)
Pale green solid from EtOH (89% yield). m.p. 210 -212 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3) 1.25 (t, J = 6.8, 6H), 1.34 (t, J = 7.2, 3H), 2.59 (s, 3H), 2.93(m,1H), 4.25 (q, J
= 7.2,2H), 7.24 (d, J = 8.0, 2H),7.57 (d, J = 8.0, 2H), 7.86 (s, 1H,); 13C NMR (101 MHz, δ,
ppm, CDCl3): 14.53, 17.26, 23.92, 34.25, 60.63, 111.36, 126.99, 127.21, 131.55, 144.27,
151.41, 157.36, 162.69, 170.86; IR (KBr, νmax, cm-1): 3193, 3094, 2963, 1666, 1569, 1429,
1370, 1275, 1124, 1092; Anal. Calcd. for C17H21N3O2S: C, 61.61; H, 6.39; N, 12.68; S, 9.67;
Found: C, 61.64; H, 6.45; N, 12.63; S, 9.60; HR-MS (ESI+): Calcd. m/z for C17H21N3O2S:
331.13545; Found m/z [M + H]+: 331.13549.
4.3.21. Ethyl 2-[(E)-2-{[4-(dimethylamino)phenyl]methylidene}hydrazin-1-yl]-4-methyl-1,3-
thiazole-5-carboxylate (2u)
Light brown solid from EtOH (82% yield). m.p. 231 -232 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 1.36 (t, J = 7.17 Hz, 3H), 2.57 (s, 3H), 3.02 (s, 6H), 4.29 (q, J = 7.17 Hz, 2H),
6.69 (d, J = 8.85 Hz, 2H), 7.54 (d, J = 8.85 Hz, 2H), 7.80 (s, 1H); 13C NMR (400 MHz, δ,
ppm, CDCl3): 13.72, 16.58, 39.45, 59.69, 109.76, 111.11, 120.87, 127.73, 144.52, 150.93,
156.84, 162.10, 169.80; IR (KBr, νmax, cm-1): 3367, 3187, 3073, 2979, 2925, 1696, 1608,
1527, 1430, 1369, 1315, 1262, 1181, 1079; Anal. Calcd. for C16H20N4O2S: C, 57.81; H, 6.06;
19

ACCEPTED MANUSCRIPT
N, 16.85; S, 9.65; Found: C, 57.86; H, 6.10; N, 16.81; S, 9.71; HR-MS (ESI+): Calcd. m/z
for C16H20N4O2S: 332.13070; Found m/z [M + H]+: 332.13073.
4.3.22. Ethyl 4-methyl-2-[(E)-2-[(3-nitrophenyl)methylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (2v)
Yellow solid from EtOH (80% yield); m.p. 239 -243 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.28 (t, J = 7.2 Hz, 3H), 2.50 (s, 3H), 4.23 (q, J = 8.0 Hz, 2H), 7.89 (d, J = 8 Hz,
2H), 8.14 (s, 1H,), 8.24 (d, J = 8 Hz, 2H,), 9.88 (s, 1H), 12.67 (s, 1H); 13C NMR (101 MHz,
δ, ppm, DMSO-d6): 14.28, 16.87, 60.25, 124.05, 127.42, 140.33, 147.41, 161.72, 169.08; IR
(KBr, νmax, cm-1): 3140, 1704, 1566, 1513, 1432, 1342, 1321, 1274, 1088 cm-1; Anal. Calcd.
for C14H14N4O4S: C, 50.29; H, 4.22; N, 16.76; S, 9.59; Found: C, 50.27; H, 4.25; N, 16.75; S,
9.58; HR-MS (ESI+): Calcd. m/z for C14H14N4O4S: 334.07358; Found m/z [M + H]+:
334.07371.
4.3.23. Ethyl 4-methyl-2-[(E)-2-[(2-nitrophenyl)methylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (2w)
Yellow solid from EtOH (80% yield). m.p. 261 - 262 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.26 (t, J = 7.2 Hz, 3H), 2.48 (s,3H), 4.20 (q, J =7.2 Hz, 2H), 7.63 (t, J = 7.6 Hz,
1H,), 7.79 (t, J = 7.6 Hz, 1H), 8.04 (d, J = 7.6 Hz, 2H), 8.47 (s, 1H), 12.71 (s, 1H); 13C NMR
(101 MHz, δ, ppm, DMSO-d6): 14.24, 16.71, 60.20, 117.52, 124.68, 127.80, 130.23, 131.93,
133.60, 143.17, 147.74, 157.92, 161.75, 172.31; IR (KBr, νmax, cm-1): 3147, 3106, 3033,
2984, 1705, 1575, 1510, 1426, 1372, 1339, 1262, 1119, 1090, 970; Anal. Calcd. for
C14H14N4O4S: C, 50.29; H, 4.22; N, 16.76; S, 9.59; Found: C, 50.32; H, 4.24; N, 16.70; S,
9.61; HR-MS (ESI+): Calcd. m/z for C14H14N4O4S: 334.07358; Found m/z [M + H]+:
334.07362.
4.3.24. 1-{2-[(E)-2-[(2,4-dichlorophenyl)methylidene]hydrazin-1-yl]-4-methyl-1,3-thiazol-5-
yl}ethan-1-one (2x)
20

ACCEPTED MANUSCRIPT
Antique white solid from EtOH (88% yield). m.p. 240-242 °C; 1H NMR (400 MHz, δ,
ppm, CDCl3): 2.31 (s, 1H), 2.61 (s, 1H), 4.27 (q, J = 7.1, 1H), 4.78 (s, 1H), 7.28 (dd, J = 8.6,
1.7, 1H), 7.39 (d, J = 1.9, 1H), 7.99 (d, J = 8.6, 1H), 8.24 (s, 1H); 13C NMR (101 MHz, δ,
ppm, DMSO-d6): 14.36. 17.19, 115.76, 120.97, 125.39, 145.08, 147.10, 149.07, 161.98,
169.02; IR (KBr, νmax, cm-1): 3459, 3221, 3092, 2976, 2933, 1756, 1591, 1565, 1422, 1472,
1334, 1100, 876; Anal. Calcd. for C14H11Cl2N3OS: C, 47.57; H, 3.38; N, 12.08; S, 9.77;
Found: C, 47.59; H, 3.37; N, 12.07; S, 9.75; HR-MS (ESI+): Calcd. m/z for C14H11Cl2N3OS:
326.9999; Found m/z [M + H]+: 327.0004.
4.3.25. 1-{4-methyl-2-[(E)-2-[(3,4,5-trimethoxyphenyl)methylidene]hydrazin-1-yl]-1,3-
thiazol-5-yl}ethan-1-one (2y)
Light yellow solid from EtOH (88% yield). m.p. 177 -180 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 2.40 (s, 3H), 2.50 (s, 3H),3.68 (s, 3H),3.81 (s, 6H),7.00 (s, 1H),8.11 (s,
1H),8.60 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 16.06, 55.85, 62.07, 104.23,
128.46, 133.72, 140.68, 144.62, 154.47, 157.27, 170.96, 195.54; IR (KBr, νmax, cm-1): 3100,
2975, 2966, 2844, 1715, 1571, 1504, 1381, 1159, 1099, 1008; Anal. Calcd. for C16H19N3O4S:
C, 55.00; H, 5.48; N, 12.03; S, 9.18; Found: C, 55.05; H, 5.51; N, 12.07; S, 9.21; HR-MS
(ESI+): Calcd. m/z for C16H19N3O4S: 349.10963; Found m/z [M + H]+: 349.10966.
4.3.26. 1-{2-[(E)-2-(2H-1,3-benzodioxol-4-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-
thiazol-5-yl}ethan-1-one (2z)
Light yellow solid from EtOH (80% yield). m.p. 172-180 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 2.41 (s, 3H), 2.50 (s, 3H), 5.81 (s, 1H), 6.09 (s, 2H), 6.98 (d, J = 8.0, 1H),
7.15 (d, J = 8.0, 1H), 7.26 (s, 1H), 8.07 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6):
16.39, 26.23, 102.12, 115.80, 117.56, 119.66, 124.25, 133.36, 144.60,, 148.48, 153.40,
156.54, 173.05, 194.13; IR (KBr, νmax, cm-1): 3146, 3030, 2976, 2915, 1697, 1590, 1491,
1422, 1370, 1245, 1088, 975; Anal. Calcd. for C14H13N3O3S: C, 55.43; H, 4.32; N, 13.85; S,
10.57; Found: C, 55.45; H, 4.37; N, 13.88; S, 10.60; HR-MS (ESI+): Calcd. m/z for
C14H13N3O3S: 303.06776; Found m/z [M + H]+: 303.06780.
21

ACCEPTED MANUSCRIPT
4.3.27. Ethyl 2-[(E)-2-(furan-2-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (4a)
Brown solid from EtOH (71% yield). m.p. 194-196 °C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.34 (t, J = 7.12 Hz, 3H), 2.58 (s, 3H), 4.27 (q, J = 7.12 Hz, 2H), 6.48 (dd, J = 1.72,
3.42 Hz, 1H), 6.69 (d, J = 3.42 Hz, 1H), 7.53 (d, J = 1.72 Hz, 1H), 7.77 (s, 1H); 13C NMR
(101 MHz, δ, ppm, CDCl3): 14.41, 17.08, 60.27, 109.08, 112.36, 113.49, 134.77, 145.19,
149.27, 157.85, 162.02, 168.99; IR (KBr, νmax, cm-1): 3210, 3093, 2979, 1669, 1617, 1567,
1528, 1411, 1318, 1274, 1092; Anal. Calcd. for C12H13N3O3S: C, 51.60; H, 4.69; N, 15.04; S,
11.48; Found: C, 51.64; H, 4.68; N, 15.07, S, 11.42; HR-MS (ESI+): Calcd. for m/z
C12H13N3O3S: 279.06776; Found m/z [M + H]+ 279.06781.
4.3.28. Ethyl 4-methyl-2-[(E)-2-[1-(thiophen-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (4b)
Gray solid from EtOH (76% yield). m.p. 209-210 °C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.35 (t, J = 7.2, 3H), 2.24 (s, 3H), 2.57 (s, 3H), 4.29 (2H, q, J = 7.2 ), 7.01 (m,
1H),7.25 (d, J =3.6, 1H), 7.32 (m, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3): 13.98, 14.42,
17.11, 60.58, 111.47, 126.34, 127.26, 128.02, 143.01, 145.56, 156.76, 162.71, 170.28; IR
(KBr, νmax, cm-1): 3212, 3098, 2980, 1615, 1565, 1368, 1317, 1280, 1119; Anal. Calcd. for
C13H15N3O2S2: C, 50.46; H, 4.89; N, 13.58; S, 20.73; Found: C, 50.61; H, 4.85; N, 13.50; S,
22.00; HR-MS (ESI+): Calcd. m/z for C13H15N3O2S2: 309.06057; Found m/z [M + H]+:
309.06066.
4.3.29. Ethyl 2-[(E)-2-(1H-indol-3-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-thiazole-5-
carboxylate (4c)
Antique white solid from EtOH (86% yield). m.p. 282 -285 °C; 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 1.26 (t, J = 7.2 Hz, 3H), 2.50 (s, 3H), 4.23 (q, J=7.2 Hz, 2H,), 7.23 (dd,
J= 4 Hz,2.8 Hz, 2H,), 7.47 (dd, J= 4 Hz,2.8, 1H), 7.83 (d, J= 2.4 Hz, 1H), 8.21 (dd, J= 4
Hz,2.8, 1H) , 8.34 (s, 1H), 11.6(s, 1H), 12.17(s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-
22

ACCEPTED MANUSCRIPT
d6): 14.43, 17.37, 59.99, 108.15, 111.45, 112.02, 120.76, 121.58, 122.75, 124.10, 130.53,
137.18, 142.29, 158.61, 162.19, 168.99; IR (KBr, νmax, cm-1): 3306, 3052, 2898, 1686, 1613,
1550, 1419, 1119, 1093 cm-1; Anal. Calcd. for C16H16N4O2S: C, 58.52; H, 4.91; N, 17.06; S,
9.76; Found: C, 58.55; H, 4.99; N, 17.02; S, 9.72; HR-MS (ESI+): Calcd. m/z for
C16H16N4O2S: 328.09940; Found m/z [M + H]+: 328.09944.
4.3.30. Ethyl 4-methyl-2-[(E)-2-[1-(pyridin-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (4d)
Yellow solid from EtOH (81% yield). m.p. 206-208 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 1.28 (t, J = 7.0, 3H), 2.40 (s, 3H), 4.22 (q,J = 7.0, 2H), 2.51 (s, 3H), 7.42 – 7.37
(t, J = 7.3, 1H), 7.86 (t, J = 7.3, 1H), 8.05 (d, J = 7.8, 1H), 8.60 (d, J = 7.8, 1H), 11.91 (s,
1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 12.86, 14.25, 18.43, 60.11, 111.54, 119.83,
123.72,136.56, 143.86, 148.55, 154.74, 161.06, 170.55, 178.61; IR (KBr, νmax, cm-1): 3200,
3048, 2988, 2947, 1687, 1566, 1469, 1372, 1271, 1099; Anal. Calcd. for C14H16N4O2S: C ,
55.25; H, 5.30; N, 18.41; S, 10.53; Found: C, 55.24; H, 5.28; N, 18.44; S, 10.63; HR-MS
(ESI+): Calcd. m/z for C14H16N4O2S: 304.09519; Found m/z [M + H]+: 304.09523.
4.3.31. Ethyl 4-methyl-2-[(E)-2-[1-(pyridin-3-yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (4e)
Brown color solid from EtOH; (yield 78 %). m.p. 244 -245 °C; 1H NMR (400 MHz,
δ, ppm, DMSO-d6): 1.23 (t, J = 7.1, 3H), 2.44 (s, 3H), 2.32 (s, 3H), 4.17 (q, J = 7.1, 2H),
7.42 (dd, J = 8.0, 4.8, 1H), 8.10 (d, J = 8.0, 1H), 8.54 (d, J = 3.3, 1H), 8.92 (d, J = 3.3, 1H),
11.87 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6): 14.25, 16.35, 18.11, 60.12, 119.84,
123.73, 127.35, 136.56, 148.56, 151.94, 157.92, 162.13, 167.04, 169.86; IR (KBr, νmax, cm-1):
3123, 3045, 2978, 2923, 2796, 1671, 1618, 1556, 1474, 1367, 1321, 1097, 1037; Anal. Calcd.
for C14H16N4O2S: C, 55.25; H, 5.30; N, 18.41; S, 10.53; Found: C, 55.30; H, 5.32; N, 18.47;
S, 10.51; HR-MS (ESI+): Calcd. m/z for C14H16N4O2S: 304.09940; Found m/z [M + H]+:
304.09943.
23

ACCEPTED MANUSCRIPT
4.3.32. Ethyl 4-methyl-2-[(E)-2-[1-(pyridin-4-yl)ethylidene]hydrazin-1-yl]-1,3-thiazole-5-
carboxylate (4f)
Yellow solid from EtOH (80% yield). m.p. 217°C -218 °C. 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 1.27 (t, J = 7.1, 1H), 2.32 (s, 1H), 2.48 (s, 1H), 4.21 (q, J = 7.1, 1H), 7.71
(dd, J = 4.6, 1.6, 1H), 8.62 (dd, J = 4.6, 1.6, 1H),11.94 (s, 1H); 13C NMR (101 MHz, δ, ppm,
DMSO-d6): 15.00, 18.14, 22.02, 62.06, 120.03, 126.34, 139.00, 149.86, 154.80, 163.93,
168.48, 171.29; IR (KBr, νmax, cm-1): 3123, 2993, 2899, 1706, 1617, 1591, 1559, 1368, 1316,
1271, 1216, 1131, 1092; Anal. Calcd. for C14H16N4O2S: C, 55.25; H, 5.30; N, 18.41; S,
10.53; Found: C, 55.28; H, 5.37; N, 18.46; S, 10.58; HR-MS (ESI+): Calcd. m/z for
C14H16N4O2S: 304.09940; Found m/z [M + H]+: 304.09946.
4.3.33. 1-{2-[(E)-2-(furan-2-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-thiazol-5-yl}ethan-1-
one (5a)
Light green solid from EtOH (80% yield). m.p. 202 -203 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 2.37 (s, 3H), 2.46 (s, 3H), 6.61 – 6.58 (m, 1H), 6.86 (d, J = 3.46 Hz, 1H),
7.81 (d, J = 3.46 Hz, 1H), 7.96 (s, 1H), 12.39 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-
d6): 16.39, 26.23, 112.64, 119.32, 133.36, 142.86, 146.71, 150.57, 155.85, 171.29, 195.89; IR
(KBr, νmax, cm-1): 3212, 3073, 2999, 1690, 1617, 1577, 1528, 1411, 1328; Anal. Calcd. for
C11H11N3O2S: C, 53.00; H, 4.45; N, 16.86; S, 12.86; Found: C, 53.04; H, 4.46; N, 16.88; S,
12.89; HR-MS (ESI+): Calcd. m/z for C11H11N3O2S: 249.05720; Found m/z [M + H]+:
249.05723.
4.3.34. 1-{4-methyl-2-[(E)-2-[1-(thiophen-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazol-5-
yl}ethan-1-one (5b)
Yellow solid from EtOH (80% yield). m.p. 192 -193 °C; 1H NMR (400 MHz, δ, ppm,
DMSO-d6): 2.33 (s, 3H), 2.39 (s, 3H), 2.48 (s, 3H), 7.09 – 7.06 (m, 1H), 7.42 (d, J = 3.60 Hz,
1H), 7.54 (d, J = 4.99 Hz, 1H), 11.86 (s, 1H); 13C NMR (101 MHz, δ, ppm, DMSO-d6):
14.69, 18.44, 26.24, 125.41, 127.90, 129.47, 133.20, 155.66, 158.78, 170.62, 197.76; IR
(KBr, νmax, cm-1): 3210, 3072, 2975, 1692, 1545, 1364, 1327, 1285; Anal. Calcd. for
24

ACCEPTED MANUSCRIPT
C12H13N3OS2: C, 51.59; H, 4.69; N, 15.04; S, 22.95; Found: C, 51.63; H, 4.71; N, 15.08; S,
22.99; HR-MS (ESI+): Calcd. m/z for C12H13N3OS2: 279.05000; Found m/z [M + H]+:
279.05002.
4.3.35. 1-{2-[(E)-2-(1H-indol-3-ylmethylidene)hydrazin-1-yl]-4-methyl-1,3-thiazol-5-
yl}ethan-1-one (5c)
Light green solid from EtOH (81% yield). m.p. 182 -184 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 2.44 (s, 3H), 2.53 (s, 3H), 6.03 (s, 1H), 7.26 – 7.20 (m, 2H), 7.49 – 7.44 (m,
1H), 7.86 (d, J = 2.6, 1H), 8.17 (dd, J = 5.8, 3.1, 1H), 8.42 (s, 1H), 11.73 (s, 1H); 13C NMR
(101 MHz, δ, ppm, DMSO-d6): 16.39, 26.58, 52.23, 63.47, 121.07, 124.25, 128.08, 133.36,
135.83, 138.28, 140.39, 158.29, 170.61, 177.63, 195.89; IR (KBr, νmax, cm-1): 3316, 3042,
2890, 1696, 1620, 1570, 1439, 1109, 1087. Anal. Calcd. for C15H16N4OS: C, 59.98; H, 5.37;
N, 18.65; S, 10.67; Found: C, 59.95; H, 5.39; N, 18.69; S, 10.65; HR-MS (ESI+): Calcd. m/z
for C15H16N4OS: 300.10448; Found m/z [M + H]+: 300.10452.
4.3.36. 1-{4-methyl-2-[(E)-2-[1-(pyridin-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazol-5-
yl}ethan-1-one (5d)
Light red solid from EtOH (86% yield). m.p. 237-238 °C; 1H NMR (400 MHz, δ,
ppm, DMSO-d6): 2.45 (s, 3H), 2.62 (s, 3H), 2.80 (s, 3H), 7.44 – 7.35 (t, J = 7.3, 1H), 7.78 (t,
J = 7.3, 1H), 8.11 (d, J = 7.8, 1H), 8.65 (d, J = 7.8, 1H), 11.88 (s, 1H); 13C NMR (101 MHz,
δ, ppm, DMSO-d6): 12.74, 14.34, 18.63, 61.01, 110.89, 120.33, 124.12,137.50, 142.96,
149.55, 155.77, 162.16, 171.25, 177.61; IR (KBr, νmax, cm-1): 3200, 3048, 2988, 2947, 1687,
1636, 1459, 1376, 1267, 1105; Anal. Calcd. for C13H14N4OS: C, 56.91; H, 5.14; N, 20.42; S,
11.69; Found: C, 56.95; H, 5.18; N, 20.44; S, 11.67; HR-MS (ESI+): Calcd. m/z for
C13H14N4OS: 274.08883; Found m/z [M + H]+ : 274.08888.
4.3.37. Ethyl 2-{2-[(E)-2-[1-(pyridin-3-yl)ethylidene]hydrazin-1-yl]-1,3-thiazol-4-yl}acetate
(6a)
25

ACCEPTED MANUSCRIPT
1
Yellow green solid from EtOH (80% yield). m.p. 215-218 °C; H NMR (400 MHz, δ,
ppm, CDCl3): 1.28 (t, J = 7.1, 3H), 2.26 (s, 3H), 3.64 (s, 2H), 4.20 (q, J = 7.1, 2H), 6.55 (s,
1H), 7.33 (dd, J = 8.0, 4.8, 1H), 8.08 (dd, J = 8.0, 1.6, 1H), 8.59 (d, J = 4.7, 1H), 8.75 (s,
1H), 8.97 (s, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3): 14.2, 17.4, 35.4, 61.4, 104.3, 124.2,
126.5, 137.5, 150.9, 152.1, 153.5, 168.2, 169.0, 171.1; IR (KBr, νmax, cm-1): 3133, 3003,
2886, 1708, 1611, 1587, 1559, 1358, 1312, 1268, 1226, 1121, 1088; Anal. Calcd. for
C14H16N4O2S: C, 55.25; H, 5.30; N, 18.41; S, 10.53; Found: C, 55.29; H, 5.34; N, 18.47; S,
10.59; HR-MS (ESI+): Calcd. m/z for C14H16N4O2S: 304.09940; Found m/z [M + H]+:
304.09943.
4.3.38. Ethyl 2-{2-[(E)-2-[1-(pyridin-2-yl)ethylidene]hydrazin-1-yl]-1,3-thiazol-4-yl}acetate
(6b)
Brown solid from EtOH (80% yield). m.p. 197 -198 °C; 1H NMR (400 MHz, δ, ppm,
CDCl3): 1.28 (t, J = 7.1, 3H) 2.37 (s, 3H), 3.64 (s, 2H), 4.19 (q, J = 7.1, 2H), 6.55 (s, 1H),
7.25 – 7.19 (m, 1H), 7.69 (t, J = 7.8, 1H), 8.10 (d, J = 8. 1, 1H), 8.56 (d, J = 4.8, 1H), 8.78
(s, 1H); 13C NMR (101 MHz, δ, ppm, CDCl3): 11.2, 14.1, 15.2, 61.3, 104.3, 122.2, 126.2,
136.1, 145.6, 149.1, 150.8, 154.8, 169.2, 170.4; IR (KBr, νmax, cm-1): 3208, 3038, 2983, 2956,
1690, 1558, 1479, 1362, 1277, 1120; Anal. Calcd. for C14H16N4O2S: C , 55.25; H, 5.30; N,
18.41; S, 10.53; Found: C , 55.28; H, 5.32; N, 18.46; S, 10.57; HR-MS (ESI+): Calcd. m/z
for C14H16N4O2S: 304.09940; Found m/z [M + H]+: 304.09947.
4.4. In vitro assay for evaluation of antimycobacterial assay
All test compound stocks as well as dilutions were prepared in DMSO. M.
tuberculosis MICs of test compounds were determined in 7H9 broth by a standard
microdilution method [48] with some modifications. Briefly, 1µl of serial twofold dilutions
of test compound made in DMSO were added to wells of a 384 well plate, with the final
concentrations ranging from 100 mM-0.19 mM. Two sets of control wells were prepared: a
media control with only medium (Middlebrook 7H9 medium supplemented with 0.2%
glycerol, 0.05% Tween 80 (Sigma), and 10% albumin dextrose catalase (Difco Laboratories,
Detroit, Mich.) and a culture control with a bacterial suspension but no compound. Then,
26

ACCEPTED MANUSCRIPT
40µl (3-7 x 105 CFU/ml) of the bacterial culture was added to all the wells except the media
control wells. The plates were packed in gas permeable polyethylene bags and incubated at
37°C for 5 days. Following this incubation period, 8 µl of a freshly prepared 1:1 mixture of
Resazurin (0.02% in water) and 10% Tween 80 was added to all the wells. The plates were
re-incubated for an additional 24 h at 37°C and the colour conversion of all wells were
recorded. A blue colour in the well indicated no growth and a pink colour indicated the
growth. Absorbance at 575 nm and 610 nm was monitored and the ratio (A575/A610
)
calculated. The least concentration which yielded 80% inhibition was considered as MIC; the
media control is considered 100% inhibition and the culture control as 0% inhibition.
Isoniazid was used as a reference drug for the assay.
4.5. In vitro assay for evaluation of cytotoxicity assay
4.5.1. Cell culture
The human lung carcinoma type II epithelial cells (A549) cells were grown in RPMI-
1640 medium containing 5% (v/v) fetal bovine serum (FBS). Cultures of A549 cells were
maintained in medium containing 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-
glutamine in 75 cm2 culture flasks at 37 oC in a humidified atmosphere with a supply of 5%
CO2. The cells were sub cultured twice per week by seeding at a density of about 3x105
cells/ml.
4.5.2. MTT Assay
The cell viability assay was carried out using MTT as per the protocol described
earlier with slight modifications [47]. In brief, A549 cells (3 × 103 cells/well) in RPMI-1640
medium with a final volume of 200 µl were seeded into 96 wells culture plate and incubated
overnight at 37 oC with the supply of 5 % CO2. The cells were treated with various
synthesized drugs and incubated for 48 hrs. Further, following a wash with Phosphate
Buffered Saline (PBS), the cells were treated with 20 µL of MTT (5 mg/ml) alone and
incubated for 4 h at 37 °C in CO2 incubator. The blue Formosan products formed in cells
were dissolved in DMSO (200 µL) and spectrophotometrically measured at 540 nm. The
concentration of the compound that inhibited cell growth by 50% (IC50) was determined from
cell survival plots and the percentage of inhibition results were expressed as mean ±SE (n=5)
and percentage of growth in the treated samples was calculated with respect to control. The
27