Design, synthesis and biological evaluation of imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives as Mycobacterium tuberculosis pantothenate synthetase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.01.059

In the present study, we have designed imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives from earlier reported imidazo[1,2-a]pyridine based Mycobacterium tuberculosis (MTB) pantothenate synthetase (PS) inhibitors. We synthesized thirty compounds and they were evaluated for MTB PS inhibition study, in vitro anti-TB activities against replicative and non-replicative MTB, in vivo activity using Mycobacterium marinum infected Zebra fish and cytotoxicity against RAW 264.7 cell line. Among them compound 2-methyl-N′-(4-phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bc) emerged as potent compound active against MTB PS with IC₅₀ of 0.53 ± 0.13 μM, MIC of 3.53 μM, 2.1 log reduction against nutrient starved MTB, with 33% cytotoxicity at 50 μM. It also showed 1.5 log reduction of M. marinum load in Zebra fish at 10 mg/kg.

Full text of DOI:10.1016/j.bmc.2016.01.059

Accepted Manuscript
Design, synthesis and biological evaluation of imidazo[2,1-b]thiazole and ben-
zo[d]imidazo[2,1-b]thiazole derivatives as Mycobacterium tuberculosis pan-
tothenate synthetase inhibitors
Ganesh Samala, Parthiban Brindha Devi, Shalini Saxena, Nikhila Meda,
Perumal Yogeeswari, Dharmarajan Sriram
PII:
S0968-0896(16)30072-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.059
BMC 12802
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 December 2015
29 January 2016
30 January 2016
Please cite this article as: Samala, G., Devi, P.B., Saxena, S., Meda, N., Yogeeswari, P., Sriram, D., Design, synthesis
and biological evaluation of imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives as
Mycobacterium tuberculosis pantothenate synthetase inhibitors, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.01.059
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1
Design, synthesis and biological evaluation of imidazo[2,1-b]thiazole and
benzo[d]imidazo[2,1-b]thiazole derivatives as Mycobacterium tuberculosis pantothenate
synthetase inhibitors
Ganesh Samala, Parthiban Brindha Devi, Shalini Saxena, Nikhila Meda, Perumal
Yogeeswari, Dharmarajan Sriram*
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
Campus, Shameerpet, Hyderabad-500078, India.
ABSTRACT
In the present study, we have designed imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-
b]thiazole derivatives from earlier reported imidazo[1,2-a]pyridine based Mycobacterium
tuberculosis (MTB) pantothenate synthetase (PS) inhibitors. We synthesized thirty
compounds and they were evaluated for MTB PS inhibition study, in vitro anti-TB activities
against replicative and non-replicative MTB, in vivo activity using M. marinum infected
Zebra fish and cytotoxicity against RAW 264.7 cell line. Among them compound 2-methyl-
N'-(4-phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bc) emerged as
potent compound active against MTB PS with IC50 of 0.53±0.13 µM, MIC of 3.53 µM, 2.1
log reduction against nutrient starved MTB, with 33% cytotoxicity at 50 M. It also showed
1.5 log reduction of M. marinum load in Zebra fish at 10mg/kg.
Key
words:
Tuberculosis,
Pantothenate
synthetase,
imidazo[2,1-b]thiazole,
benzo[d]imidazo[2,1-b]thiazole.
*For Correspondence: Email- dsriram@hyderabad.bits-pilani.ac.in
Telephone: +91-4066303506; Fax: +91-4066303998
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1. Introduction
The global threat to human health posed by the Mycobacterium tuberculosis (MTB) was
recognized by the WHO, and it is estimated that one-third of the world’s population is
infected with the MTB (1). Synergy with the HIV, patient non-compliance, and the
consequences of the development of drug and multidrug-resistant strains of MTB has made
the situation ever more risk and it is widely acknowledged that novel intervention strategies
are needed (2). Pantothenate synthetase (PS; EC 6.3.2.1) enzyme catalyses the last step of
pantothenate biosynthesis, an ATP-dependent condensation of D-pantoate, and ß-alanine to
form pantothenate (3). Pantothenic acid (vitamin B5) is an essential molecule required for the
synthesis of coenzyme A and acyl carrier protein (ACP), that play important roles as acyl-
group carriers in fatty-acid metabolism, cell signalling, the tricarboxylic-acid cycle,
biosynthesis of polyketides, non-ribosomal peptides and several other reactions associated
with intermediary metabolism (4). PS is not essential for in vitro growth but is essential for
persistence of MTB in chronically infected mice. A strain of MTB with PS knock-out is
severely attenuated in mice (5), thus making this enzyme an attractive target; moreover
microorganisms need to synthesize pantothenate, while mammals obtain it from their diet. In
the recent years many MTB PS inhibitors are reported which includes 2-methylimidazo[1,2-
a]pyridine-3-carboxamides (6), thiazolidine derivatives (7), salicylanilide diethyl phosphates
(8), tetrahydrothieno[2,3-c]pyridine-3-carboxamide (9), intermediate bi-substrate analogue
(10), 3-biphenyl-4-cyanopyrrole-2-carboxylic acids (11), pyrazoles (12), 3-phenyl-4,5,6,7-
tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives (13)and actinomycin D inhibitors (14). In
the present study, we have designed, synthesized thirty imidazo[2,1-b]thiazole and
benzo[d]imidazo[2,1-b]thiazole derivatives and evaluated for MTB PS enzyme inhibition
study, in vitro anti-TB activities against replicative and nutrient starved non-replicative MTB,
in vivo activity in Zebra fish using M. marinum and cytotoxicity against RAW 264.7 cell line.
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2. Results and discussion
2.1. Designing of the compounds
In our previous work (6), we have used the crystal structure of the mycobacterial
pantothenate synthetase (PS) in complex with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-
methoxy-1H-indol-1-yl)acetic acid inhibitor (PDB: 3IVX) having resolution of 1.73A° as a
framework for virtual screening of known antitubercular compound database to identify lead
compounds against this enzyme. Based on the hit identified, we undertook synthesis of
analogues and evaluated for its biological activity. One of the lead had MTB PS activity of
1.90±0.12 μM and MIC of 4.53 μM. Based on these result and input from protein-ligand
interactions observed in the structure of MTB PS with lead molecules, further modifications
were explored as a ligand expansion step. Earlier we have reported imidazo[1,2-a]pyridine
derivatives as MTB PS inhibitors with IC50 ranging from 1.90±0.12 to 9.20±0.96 µM and
MTB with MIC ranging from 4.53 to 98.81 µM (6). In the present study, we have designed
novel imidazo[2,1-b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives by replacing
pyridyl ring of imidazo[1,2-a]pyridine with thiazole and benzothiazole nucleus (Figure 1).
Figure 1: Designing strategy of the compounds
2.2. Synthesis of designed compounds
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The target molecules were synthesized by three-step synthetic protocol (Scheme 1), wherein
the first step in the synthesis was the reaction between 2-aminothiazole (1a)/2-
aminobenzothiazole (1b) and 2-chloroethylacetoacetate in 1,2-dimethoxyethane at 90 °C to
yield the bi/tricyclic compounds 2a/2b. In the next step, two types of reactions were carried
out on ester group, one was the conversion of ester group into carboxylic acid (4a/4b) using
LiOH in Ethanol/Water (1:1), and the other was the direct conversion of ester into acid
hydrazide (3a/3b) using 35% aqueous solution of hydrazine hydrate in ethanol under reflux
conditions. Reaction of compound 3a/3b with various substituted aromatic carboxylic acids
in presence of coupling agents EDCI, and HOBt produced the compounds (5aa-5ae/5ba-
5be). In the third step, compound 3a/3b on reaction with various substituted aldehydes in
ethanol reflux conditions produced the acid hydrazones (6aa-6ae/6ba-6be) respectively in
excellent yields. During the reaction we observed the formation of desired product as a solid
then reaction mixture was filtered directly and washed with distilled water, cold ethanol and
hexane to obtain pure products without further purification steps. In the case of simple
amides, compound 4a/4b was treated with substituted aromatic/aliphatic primary amines in
presence of peptide coupling agent EDCI to produce final compounds (7aa-7ae/7ba-7be).
The purity of the synthesized compounds was checked by HPLC and elemental analyses and
the structures were identified by spectral data. In the nuclear magnetic resonance spectra (1H
13
NMR and C NMR), the signals of the respective protons of the prepared derivatives were
verified on the basis of their chemical shifts, multiplicities, and coupling constants. The
elemental analysis results were within ±0.4% of the theoretical values.
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Scheme 1: Synthetic protocol of designed compounds
2.3. Pantothenate synthetase enzyme inhibition studies
All the synthesized compounds were assayed for MTB PS inhibition study, that couples the
AMP produced in the condensation of β-alanine and pantoate with the oxidation of NADH to
NAD+ through myokinase, pyruvate kinase and lactate dehydrogenase (3). The NAD+
produced can be monitored spectrophotometrically at 340 nm. In the initial screening at 25
µM, all compounds showed more than 50% inhibition against MTB PS and were further
studied for IC50 measurements. All the compounds showed good IC50 in the range of
0.52±0.24 to 5.83±0.24 µM (Table 1); eight compounds (5ad, 5bc, 6ab, 6ac, 7aa, 7ab, 7ba,
7bd). inhibited MTB PS with nano molar concentration. Compounds N-(4-bromophenyl)-2-
methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide
(7ba)
and
2-Methyl-N'-(4-
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6
phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bc) emerged as the
most active compounds with an IC50 of 0.52±0.24 and 0.53±0.13 µM respectively.
Subsequently, all the molecule were docked into the crystal structure of MTB PS protein
(PDB Id: 3IVX) to establish the structure activity relationship. All the compounds showed
good binding energy in the range of -7.15 to -8.50 kcal/mol and showing good fitness with
the MTB PS protein. Most of the compounds are showing important hydrogen bonding
interaction with HIE44, Ser196, Ser197, Asp161, Gln72, HIE47 amino acid residues. Most of
the compounds were further stabilized by pi-pi interaction with HIE44 analogous to that
observed with the crystal ligand. One of the most active ligand N-(4-bromophenyl)-4-methyl-
7-thia-2,5-diazatricyclo pentaene-3-carboxamide (7ba) having IC50 of 0.52±0.24 µM
showing docking score of -7.66 kcal/mol. The ligand had two hydrogen bonding’s with
Asp161 and HIE44 also it showed a pi-pi interaction with HIE44 with a well fitted pose in
the active site in the hydrophobic pocket within the vicinity of Leu50, Val187, Met195,
Tyr82, Met40, and Pro38 and few polar amino acid residues HIE47, Ser196, Ser197, Gln72,
Gln164, Thr39 and Thr186 respectively. The binding pattern within the active site pocket of
the crystal ligand and reference ligand was quite similar and it is well fitted in the active site
of the protein, additionally the van der Waal’s forces were also observed which constituted
for a stable binding profile of the molecule as shown in the Figure 2.
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Figure 2: Binding pose and the interaction pattern of the compound 7aa (In 2D ligplot, pink line
represented hydrogen bond; green line represented pi-pi interaction, in surface representation the
blue colour indicate the hydrophobic nature; gray colour indicate the hydrophilic nature of the
active site).
Table 1: Biological activities of synthesized compounds
MP
( °C )
Com
poun
d
Yield
(%)
PanC IC50
in µM
MTB
MIC in
µM
Cytotoxicity at
50 µM (RAW
264.7 cells) %
inhibition
10.34
R
Phenyl
4-Tolyl
81 166-167 5.31±0.12
79 171-172 4.99±0.21
88 160-161
69 162-163 0.64±0.10
76 177-178
80 260-261
87 270-271 5.83±0.24
74 214-215 0.53±0.13
69 260-261 1.39±0.08
89 241-242 2.91±0.11
84 269-270 1.20±0.16
90 153-154 0.67±0.18
88 151-152 0.58±0.19
40.12
5.96
7.96
5aa
5ab
5ac
5ad
5ae
5ba
5bb
5bc
5bd
5be
6aa
6ab
6ac
39.25
2.98
27.47
31.19
29.71
53.01
10.40
32.03
45.41
22.23
12.84
23.12
4-Phenoxyphenyl
1-Naphthyl
Cyclohexyl
1.23±0.3
17.83
20.40
35.67
17.15
3.53
15.60
17.53
34.40
17.74
23.20
5.38±0.9
1.10±04
Phenyl
4-Tolyl
4-Phenoxyphenyl
1-Naphthyl
Cyclohexyl
4-Bromophenyl
4-Trifluromethylphenyl
Phenyl
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3,4,5-Trimethoxyphenyl
4-N,N-dimethylphenyl
4-Bromophenyl
4-Trifluromethylphenyl
Phenyl
3,4,5-Trimethoxyphenyl
4-N,N-dimethylphenyl
4-Bromophenyl
Phenyl
4-Ethoxyphenyl
Benzyl
Cyclohexyl
4-Bromophenyl
Phenyl
4-Ethoxyphenyl
Benzyl
76 218-219 5.61±0.27
33.38
38.18
15.12
16.53
9.35
29.45
4.13
2.32
12.14
20.74
11.52
11.85
16.18
40.67
41.95
19.44
19.94
0.72
1.17
6.75
6ad
6ae
6ba
6bb
6bc
6bd
6be
7aa
7ab
7ac
7ad
7ae
7ba
7bb
7bc
7bd
7be
92 119-120
2.50±0.3
87 252-253 1.02±0.13
93 271-272 5.31±0.11
10.06
16.11
34.57
16.18
12.06
32.32
14.07
58.69
12.67
32.10
20.87
33.21
52.73
6.62
90 246-247
2.15±0.8
91 249-250 2.07±0.20
82 256-257 1.46±0.12
78 213-214 0.69±0.04
82 109-110 0.74±0.21
84 120-121 5.83±0.11
76 141-142 1.06±0.14
69 146-147 2.00±0.16
63 255-256 0.52±0.04
81 200-201 1.03±0.11
83 245-246 2.10±0.09
72 216-217
0.84±0.1
Cyclohexyl
81 243-244 1.02±0.11
13.39
NT
NT
>25
>25
Isoniazid
Ethambutol
7.64
2.4. In vitro MTB screening
All the synthesized compounds were also screened for their in vitro anti-TB activity against
log phase culture of MTB H37Rv (ATCC27294) using MABA assay method (15) with drug
concentrations from 50 µg/mL to 0.78µg/mL (double dilution) in duplicates. The minimum
inhibitory concentration (MIC) was determined for each compound which was measured as
the minimum concentration of compound required to completely inhibit the bacterial growth.
Isoniazid (INH) and ethambutol were used as reference compounds for comparison. The MIC
values of the synthesized compounds along with the standard drugs for comparison are
presented in Table 1. All the synthesized compounds showed activity against MTB with MIC
ranging from 2.32 to 41.95 µM. Six compounds (5ab, 5ac, 5bc, 6bc, 6be and 7aa) inhibited
MTB with MIC of <10 µM; four compounds (5ab, 5bc, 6be and 7aa) were found to be more
potent than standard first line anti-TB drug ethambutol (MIC of 7.64 µM) and none were
potent as INH. Compound 7aa was found to be the most active compound in vitro with a
MIC of 2.32 μM against log-phase culture of MTB. When compared to enzyme PS activity;
MIC results were found to be many folds high; the reason might be due to MTB cell wall
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penetration problem with these compounds or involvement of MTB efflux pumps. With
respect to structure-MTB activity relationship; in the case of imidazo[2,1-b]thiazole
derivatives, we have prepared five N,N'-diacylhydrazines (5aa-5ae), five N-acylhydrazones
(6aa-6ae) and five amides (7aa-7ae). In general amides were more potent followed by N,N'-
diacylhydrazines and then N-acylhydrazones. Within N,N'-diacylhydrazines substitution on
phenyl ring increases MTB activity ~5-8 times (5aa vs 5ab and 5ac), similarly converting
phenyl ring to cyclohexyl (5ae) and naphthyl (5ad) ring leads twice potent compounds. In the
case of N-acylhydrazones substitution on phenyl ring (6ac vs others) in general is not
favoured for MTB activity. In the case of amides, when compared to phenyl (7ab)
introduction of electron withdrawing bromo group (7aa) makes six times more potent MTB
activity and introduction of electron donating ethoxy group (7ac) makes less effective;
whereas replacement of phenyl with cyclohexyl (7ae) or benzyl (7ad) does not alter any
appreciable activity. In general imidazo[2,1-b]thiazoles were more potent than
benzo[d]imidazo[2,1-b]thiazole derivatives.
Further we also tested two most active compounds (5bc and 7aa) in nutrient starved MTB
culture (16). In this model nutrient starvation caused MTB to arrest growth, minimized
aerobic metabolism and became resistant to currently available anti-TB drugs while
maintaining viability. Nutrient starvation may therefore mimic some of the features of MTB
during the persistent state. Owing to its simplicity, reproducibility and ease of handling, we
utilized this model for testing our few of the potent enzyme/MTB inhibitors aimed at non-
replicative bacteria. In this model, cultures of MTB bacteria were subjected to nutrient
depletion by growing the culture in PBS (Phosphate buffer saline) for 6 weeks. Nutrient
starvation using this method triggered a dormancy response in the bacilli that was termed
non-replicating persistence (NRP), a physiological state thought to mimic the one exhibited
by MTB during various stages of persistent infection. After 6 weeks, the culture was treated
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with the synthesized and standard drugs at a concentration of 10 µg/ml in a tube and
incubated at 37 °C for 7 days exposure. The treated cell suspensions were diluted 10-fold up
to 10-6 using Middlebrook 7H9 medium supplemented with OADC and were plated in 48
well plates in triplicates. The plates were incubated at 37 °C for 4 weeks and the wells with
visible bacterial growth were counted as positive and maximum probable number (MPN)
values were calculated using standard statistical methods (17). At 10 µg/ml concentration,
standard first line anti-TB drugs INH and rifampicin (Rif) reduced ~1.1 and 2.1 log bacterial
reduction respectively whereas moxifloxacin (Moxi) showed very good activity with ~2.5 log
bacterial reduction (Figure 3). MIC of INH: 0.05 µg/ml, Rif: 0.1 µg/ml and moxifloxacin:
0.78 µg/ml against active MTB. Compounds 5ab and 7 aa reduced 2.1 and 2.0 log bacterial
reduction respectively and were more potent than INH and almost equipotent as Rif. MTB PS
is essential for biosynthesis of both CoA and ACP; which were in turn essential in fatty acid
biosynthesis that plays a key role in persistent growth and pathogenicity of MTB (18); so
these MTB PS inhibitors might be useful for treating both active and persistent TB infection.
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
Control
Isoniazid
10μg/ml
Rifampicin Moxifloxacin 7aa 10μg/ml 5bc 10μg/ml
10μg/ml 10μg/ml
Figure 3: Inhibitors effect of tested compounds against nutrient starved MTB
2.5. Antimycobacterial screening using M. marinum induced adult zebra fish model (19)
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In this model we used M. marinum, which is genetically close to MTB. It causes systemic
tuberculosis-like granulomatous infection, superficial lesions which are examined similar to
the MTB lesions clinically and pathologically (20) and also has less generation time (4-6 hr)
when compared to MTB (20 hr). Adult Zebra fish model (Danio rerio) is infected with M.
marinum which is known to produce normal pathogenesis similar to MTB; we observed
granulomas-like lesions on the dorsal side and around the fins as sign of TB during the
infection stage. Compound which showed good in vitro anti-TB activity (5bc) was tested in
this model (after 7 days post infection) at 10mg/kg body weight along with INH (10mg/kg),
Rif (5mg/kg), Moxi (5mg/kg), and negative control amoxicillin (10mg/kg). After 7 days of
oral treatment fishes were sacrificed and bacterial count was done using MPN assay method.
INH reduces ~1.8 log reduction of M. marinum load in Zebra fish at 10 mg/kg dose; whereas
compound 5bc showed ~1.5 log reduction of bacteria at same dose level. At the dose level of
5 mg/kg dose Rif and Moxi reduces 1.9 and 2.9 log reduction respectively (Figure 4).
Negative control drug amoxicillin does not show any activity till 10 mg/kg dose.
Figure 4: Antimycobacterial screening using M. marinum induced adult zebra fish model
2.6. In vitro cytotoxicity studies
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All the compounds were also tested for in vitro cytotoxicity against RAW 264.7 cells (mouse
macrophage cell line) at 50 μM concentration using (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. As MTB survives inside macrophages any new
molecules should not show any toxicity to macrophages; so we carried out this study in vitro.
Percentage inhibitions of cells were reported in Table 1. The most promising anti-TB
compound 5bc showed only 10.4% cytotoxicity at 50μM.
3. Conclusion
We designed and synthesized novel compounds from our earlier reported MTB PS lead
inhibitors and evaluated for their biological evaluation. Among them seventeen compounds
(5ac, 5ad, 5ba, 5bc, 5bd, 6aa, 6ab, 6ac, 6ba, 6be, 7aa, 7ab, 7ad, 7ba, 7bb, 7bd and 7be)
showed more activity than lead against MTB PS and three compounds (5bc, 6be and 7aa)
were found to be more potent than lead against MTB MIC. Most active compounds (5bc and
7aa) also showed appreciable activity in nutrient starved MTB and M. marinum infected
Zebra fish model. As we mentioned earlier PS is not essential for in vitro survival of MTB;
further these molecules to be tested in vivo to prove their specificity towards MTB PS.
4. Experimental Section
4.1. Chemistry
Reagents and solvents obtained from commercial sources were used without further
purification. All the reactions were monitored by thin layer chromatography (TLC) on silica
gel 40 F254 (Merck, Darmstadt, Germany) coated on aluminium plates. All 1H and 13C NMR
spectra were recorded on a Bruker AM-400 and 100 MHz spectrometer, Bruker BioSpin
Corp., Germany. Chemical shifts are reported in parts per million (ppm) using tetramethyl
silane (TMS) as an internal standard. Temperatures are reported in degrees Celsius and are
uncorrected. Compounds were analysed for C, H, N using Elementar vario EL III analyser
and analytical results obtained were within ±0.4% of the calculated values for the formula
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shown. Purity of the final compounds were >95%; checked by HPLC (Agilent technologies –
Reverse phase C-18 column; run time 25 min; 0.1 TFA in CH3CN, 0.1 TFA in H2O as
eluent). Molecular weights of the synthesised compounds were checked by (Shimadzu,
LCMS-2020) ESI-MS method.
4.1.1. Preparation of ethyl 6-methylimidazo[2,1-b]thiazole-5-carboxylate (2a)
2-aminothiazole (3.00 g, 29.99 mmol) and 2-chloroethylacetoacetate (4.96 mL, 35.99 mmol)
were taken in 1,2-dimethoxyethane (30 mL) and heated at 90 °C for 6 h. The reaction mixture
was concentrated under reduced pressure, diluted with EtOAc (80 mL), washed the organic
layer with H2O (3 × 30 mL). The separated organic layer was dried over anhy Na2SO4 and
concentrated under vacuo to get crude compound. The crude compound was purified by
column chromatography using 20% EtOAc in Hexanes as eluent to get ethyl 6-
methylimidazo[2,1-b]thiazole-5-carboxylate (2a) (5.20 g, 82%) as an Off-white solid. ESI-
MS showed 211 [M+H]+ and carried to next step.
4.1.2. Preparation of ethyl 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylate (2b)
2-aminobenzothiazole (3.00 g, 19.97 mmol) and 2-chloroethylacetoacetate (3.30 mL, 23.96
mmol) were taken in 1,2-dimethoxyethane (30 mL) and heated at 90 °C for 6 h. The reaction
mixture was concentrated under reduced pressure, diluted with EtOAc (80 mL), washed the
organic layer with H2O (3 × 30 mL). The separated organic layer was dried over anhy
Na2SO4 and concentrated under vacuo to get crude compound. The crude compound was
purified by column chromatography using 25% EtOAc in Hexanes as eluent to get ethyl 2-
methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylate (2b) (4.32 g, 83%) as an Off-white
solid. ESI-MS showed 261 [M+H]+ and carried to next step.
4.1.3. Preparation of 6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (3a)
To the stirred solution of ethyl 6-methylimidazo[2,1-b]thiazole-5-carboxylate (2a) (5.20 g) in
Ethanol (40 mL) was added 35% aqueous solution of N2H4.H2O (40 mL) and refluxed for 3
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h. The reaction mixture was concentrated to half volume and cooled on ice bath, the solids
formed were filtered and dried in vacuum oven to get 6-methylimidazo[2,1-b]thiazole-5-
carbohydrazide (3a) (4.30 g, 89%) as an Off-white solid. ESI-MS showed 197 [M+H]+.
4.1.4. Preparation of 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (3b)
To the stirred solution of ethyl 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylate (2b)
(4.32 g) in Ethanol (40 mL) was added 35% aqueous solution of N2H4.H2O (35 mL) and
refluxed for 3 h. The reaction mixture was concentrated to half volume and cooled on ice
bath, the solids formed were filtered and dried in vacuum oven to get 2-
methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (3b) (3.69 g, 90%) as an Off-white
solid. ESI-MS showed 247 [M+H]+.
4.1.5. Preparation of 6-methylimidazo[2,1-b]thiazole-5-carboxylic acid (4a)
To the stirred solution of ethyl 6-methylimidazo[2,1-b]thiazole-5-carboxylate (2a) (3.00 g) in
ethanol/water (1:1) (30 mL) was added LiOH (4.00 g) and stirred at room temperature for 4
h. The reaction mixture was concentrated to half volume, and added 6N HCl at 0 °C till the
reaction mixture turned to H ~ 6, the solids formed were filtered and dried in vacuum oven
P
to get 6-methylimidazo[2,1-b]thiazole-5-carboxylic acid (4a) (2.10 g, 80%) as an Off-white
solid. ESI-MS showed 183 [M+H]+.
4.1.6. Preparation of 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylic acid (4b)
To the stirred solution of ethyl 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylate (2b)
(3.00 g) in ethanol/water (1:1) (30 mL) was added LiOH (4.00 g) and stirred at room
temperature for 4 h. The reaction mixture was concentrated to half volume, and added 6N
HCl at 0 °C till the reaction mixture turned to H ~ 6, the solids formed were filtered and
P
dried in vacuum oven to get 2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylic acid (4b)
(2.05 g, 76%) as an Off-white solid. ESI-MS showed 233 [M+H]+.
General procedure for the synthesis of final molecules (5aa-ae and 5ba-be)
14

15
To the stirred solution of R-COOH (1.0 equiv), in CH2Cl2 at 0 °C was added EDCI (1.2
equiv), HOBt (1.2 equiv) and Et3N (2.0 equiv) stirred for few minutes then was added 6-
methylimidazo[2,1-b]thiazole-5-carbohydrazide (for 5aa-ae)/ 2-methylbenzo[d]imidazo[2,1-
b]thiazole-3-carbohydrazide (for 5ba-be) (1.2 equiv), and allowed stir at rt for 3 h, The
reaction mixture was diluted with CH2Cl2 and washed with H2O and the separated organic
layer was concentrated under reduced pressure, purified by column chromatography.
4.1.8. N'-Benzoyl-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (5aa): To the stirred
solution of Benzoic acid (0.4 g, 3.27 mmol), in CH2Cl2 at 0 °C was added EDCI (0.76 g, 3.92
mmol), HOBt (0.53 g, 3.92 mmol), and Et3N (1.02 mL, 7.19 mmol) stirred for few minutes
then was added 6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (0.71 g, 3.60 mmol), and
allowed stir at rt for 3 h, The reaction mixture was diluted with CH2Cl2 (20 mL) and washed
with H2O (3 × 20 mL) and the separated organic layer was concentrated under reduced
pressure, purified by column chromatography using 25% EtOAc/Hexanes as eluent. MS(ESI)
1
m/z 301 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.48 (s, 1H), 8.49 (d, J = 8.0 Hz, 1H),
7.92–7.78 (m, 3H), 7.54–7.46 (m, 3H), 7.30 (d, J = 8.0 Hz, 1H), 2.60 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 168.3, 161.8, 153.4, 145.6, 142.2, 139.7, 137.9, 133.1, 127.6 (2C),
126.3(2C), 119.2, 17.8. Anal. calcd for C14H12N4O2S: C, 55.99; H, 4.03; N, 18.65 % Found
C, 56.03; H, 4.12; N, 18.71%.
4.1.9.
6-Methyl-N'-(4-methylbenzoyl)imidazo[2,1-b]thiazole-5-carbohydrazide
(5ab)
1
MS(ESI) m/z 315 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.36 (s, 1H), 8.50 (d, J = 8.0
Hz, 1H), 7.99–7.81 (m, 3H), 7.56 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 2.61 (s, 3H),
13
2.39 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 169.9, 162.3, 154.6, 146.2, 141.3, 140.4,
138.5, 133.6, 128.4 (2C), 126.9(2C), 120.6, 20.9, 18.2. Anal. calcd for C15H14N4O2S: C,
57.31; H, 4.49; N, 17.82 % Found C, 57.43; H, 4.52; N, 17.91%.
4.1.10. 6-Methyl-N'-(4-phenoxybenzoyl)imidazo[2,1-b]thiazole-5-carbohydrazide (5ac)
15

16
1
MS(ESI) m/z 393 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.63 (s, 1H), 8.51 (d, J = 8.0
Hz, 1H), 7.91 (d, J = 7.6 Hz, 2H), 7.76–7.63 (m, 3H), 7.56–7.45 (m, 3H), 7.42–7.30 (m, 3H),
13
2.58 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 166.9, 164.4, 160.3, 154.6, 151.7, 144.0,
139.6, 136.3, 135.1, 129.4 (2C), 127.1(2C), 124.9, 124.2 (2C), 122.4(2C), 119.1, 18.0. Anal.
calcd for C20H16N4O3S: C, 61.21; H, 4.11; N, 14.28 % Found C, 61.33; H, 4.20; N, 14.31%.
4.1.11. N'-(1-Naphthoyl)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (5ad)
MS(ESI) m/z 351 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.63 (s, 1H), 9.31 (d, J = 8.0
Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.81–7.72 (m, 2H), 7.68–7.54 (m, 3H), 7.47–7.32 (m, 3H),
13
2.61 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 169.0, 166.2, 162.0, 152.4, 149.7, 139.0,
137.3, 134.2, 133.9, 133.0, 129.4, 128.3, 126.4, 125.7, 125.1, 124.4, 119.3, 17.2. Anal. calcd
for C18H14N4O2S: C, 61.70; H, 4.03; N, 15.99 % Found C, 61.73; H, 4.09; N, 16.09%.
4.1.12. N'-(Cyclohexanecarbonyl)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (5ae)
1
MS(ESI) m/z 307 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.54 (s, 1H), 8.61 (d, J = 8.0
Hz, 1H), 7.92 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 2.56 (s, 3H), 2.18–2.14 (m, 1H), 1.78–1.50
(m, 10H); 13C NMR (100 MHz, DMSO-d6) δ 174.2, 164.8, 160.2, 148.3, 144.8, 138.4, 118.2,
47.9, 31.3(2C), 26.6(3C), 16.9. Anal. calcd for C14H18N4O2S: C, 54.88; H, 5.92; N, 18.29 %
Found C, 54.93; H, 5.98; N, 18.39%.
4.1.13. N'-Benzoyl-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5ba):
1
MS(ESI) m/z 351 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.44 (s, 1H), 8.09–7.81 (m,
3H), 7.74–7.63 (m, 4H), 7.56–7.44 (m, 3H), 2.63 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
167.2, 163.5, 158.6, 149.3, 143.6, 142.7, 138.6, 136.7, 134.2, 129.6, 128.0 127.8 (2C),
126.4(2C), 124.6, 120.4, 17.8. Anal. calcd for C18H14N4O2S: C, 61.70; H, 4.03; N, 15.99 %
Found C, 61.73; H, 4.12; N, 16.11%.
4.1.14.
(5bb)
2-Methyl-N'-(4-methylbenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide
16

17
MS(ESI) m/z 365 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.08–7.92 (m, 3H), 7.86 (d, J =
8.0 Hz, 2H), 7.72–7.64 (m, 2H), 7.54–7.39 (m, 3H), 2.60 (s, 3H), 2.42 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 168.6, 163.2, 155.8, 146.9, 140.4, 138.4, 137.3, 133.8, 129.6 (2C), 128.3,
127.4(2C), 126.5, 124.7, 124.0, 119.7, 22.3, 17.8. Anal. calcd for C19H16N4O2S: C, 62.62; H,
4.43; N, 15.37 % Found C, 62.63; H, 4.52; N, 15.41%.
4.1.15. 2-Methyl-N'-(4-phenoxybenzoyl)benzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide
(5bc)
MS(ESI) m/z 443 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 8.10–7.96 (m, 4H), 7.90–7.74 (m,
3H), 7.63 (d, J = 8.0 Hz, 2H), 7.60 (s, 1H), 7.56–7.45 (m, 5H), 2.62 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 167.4, 165.6, 162.5, 157.4, 153.0, 145.5, 140.3, 137.4, 136.2, 133.2, 128.9
(2C), 127.4(2C), 126.6, 125.7, 124.9(2C), 124.2, 123.9(2C), 121.4, 117.9, 17.1. Anal. calcd
for C24H18N4O3S: C, 65.14; H, 4.10; N, 12.66 % Found C, 65.23; H, 4.20; N, 12.71%.
4.1.16. N'-(1-Naphthoyl)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (5bd)
1
MS(ESI) m/z 401 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.28 (d, J = 8.0 Hz, 1H),
8.04–7.87 (m, 4H), 7.81–7.74 (m, 4H), 7.66–7.53 (m, 4H), 2.59 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 168.3, 167.1, 163.5, 155.7, 150.4, 139.5, 138.2, 136.4, 135.6, 133.6, 129.4,
129.2, 128.6, 127.4, 126.4, 126.2, 125.8, 125.0, 124.8, 124.3, 117.8, 17.2. Anal. calcd for
C22H16N4O2S: C, 65.98; H, 4.03; N, 13.99 % Found C, 66.03; H, 4.09; N, 14.09%.
4.1.17. N'-(Cyclohexanecarbonyl)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-
carbohydrazide (5be)
MS(ESI) m/z 357 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.07–7.96 (m, 3H), 7.69–7.58
(m, 3H), 2.58 (s, 3H), 2.16–2.11 (m, 1H), 1.76–1.51 (m, 10H); 13C NMR (100 MHz, DMSO-
d6) δ 172.9, 163.3, 162.7, 152.2, 139.4, 137.6, 126.8, 125.4, 124.7, 124.1, 119.4, 47.2,
31.6(2C), 25.9(3C), 16.7. Anal. calcd for C18H20N4O2S: C, 60.65; H, 5.66; N, 15.72 % Found
C, 60.73; H, 5.68; N, 15.89%.
17

18
General procedure for the synthesis of final molecules (6aa-ae and 6ba-be)
To the stirred solution of 6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (3a) (for 6aa-ae)/
2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (3b) (for 6ba-be) (1.0 equiv),
aldehyde (1.1 equiv), conc. H2SO4 (cat) were taken in Ethanol and refluxed for 1 h. The
formed solids were filtered, dried and triturated with CH2Cl2/hexanes to get pure products.
4.1.18. N'-(4-Bromobenzylidene)-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (6aa):
6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (0.4 g, 2.03 mmol), 4-bromobenzaldehyde
(0.22 mL, 2.23 mmol), conc. H2SO4 (3 drops) were taken in ethanol (7 mL) and refluxed for
30 min. The solids in the reaction mixture were filtered, washed with Water, cold Ethanol,
Hexanes and dried in vacuum oven to get (0.54 g, 72%) title compound.
1
MS(ESI) m/z 364 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.61 (s, 1H), 8.32 (d, J = 7.2
Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.68–7.49 (m, 3H), 7.29 (d, J = 7.6 Hz, 1H), 2.62 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 166.3, 162.1, 149.8, 146.1, 141.4, 130.6, 128.7,
127.4(2C), 125.4(2C), 124.1, 118.6, 17.3. Anal. calcd for C14H11BrN4OS: C, 46.29; H, 3.05;
N, 15.42 % Found C, 46.33; H, 3.09; N, 15.51%.
4.1.19.
6-Methyl-N'-(4-(trifluoromethyl)benzylidene)imidazo[2,1-b]thiazole-5-
carbohydrazide (6ab)
1
MS(ESI) m/z 353 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.63 (s, 1H), 8.49 (d, J =7.6
Hz, 1H), 7.74 (d, J =7.6 Hz, 2H), 7.63 (d, J =7.6 Hz, 2H), 7.48 (s, 1H), 7.30 (d, J =7.6 Hz,
1H), 2.60 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 164.4, 162.1, 148.9, 146.5, 137.6,
133.3, 130.6, 129.1, 127.3(2C), 126.2(2C), 124.4, 118.6, 17.4. Anal. calcd for
C15H11F3N4OS: C, 51.13; H, 3.15; N, 15.90 % Found C, 51.23; H, 3.19; N, 15.96%.
4.1.20. N'-Benzylidene-6-methylimidazo[2,1-b]thiazole-5-carbohydrazide (6ac)
1
MS(ESI) m/z 285 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.34 (s, 1H), 8.52 (d, J = 8.1
13
Hz, 2H), 8.12 (s, 1H), 7.69–7.36 (m, 5H), 2.64 (s, 3H); C NMR (100 MHz, DMSO-d6) δ
18

19
168.4, 164.9, 152.9, 148.3, 139.4, 129.8, 128.3, 124.9(2C), 124.0(2C), 123.7, 119.1, 18.9.
Anal. calcd for C14H12N4OS: C, 59.14; H, 4.25; N, 19.70 % Found C, 59.33; H, 4.29; N,
19.91%.
4.1.21. 6-Methyl-N'-(3,4,5-trimethoxybenzylidene)imidazo[2,1-b]thiazole-5-carbohydrazide
(6ad)
MS(ESI) m/z 375 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.45 (s, 1H), 8.53 (s, 1H), 8.39
13
(d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.27 (s, 2H), 3.94 (s, 9H), 2.61 (s, 3H); C
NMR (100 MHz, DMSO-d6) δ 162.5, 161.9, 156.3(2C), 144.8, 143.1, 142.3, 140.6, 139.5,
137.4, 121.2(2C), 120.1, 63.4, 60.9(2C), 17.1. Anal. calcd for C17H18N4O4S: C, 54.53; H,
4.85; N, 14.96 % Found C, 54.63; H, 4.92; N, 14.98%.
C15H11F3N4OS: C, 51.13; H, 3.15; N, 15.90 % Found C, 51.23; H, 3.19; N, 15.96%.
4.1.22.N'-(4-(Dimethylamino)benzylidene)-6-methylimidazo[2,1-b]thiazole-5-
carbohydrazide (6ae)
1
MS(ESI) m/z 328 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.55 (s, 1H), 8.46 (d, J = 8.0
Hz, 1H), 7.56–7.47 (m, 3H), 7.29 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 2H), 3.15 (s, 6H),
13
2.62 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 163.4, 162.5, 156.3, 146.2, 143.8, 141.4,
136.3, 129.3(2C), 125.1, 120.4, 115.2(2C), 44.1(2C), 17.3. Anal. calcd for C16H17N5OS: C,
58.70; H, 5.23; N, 21.39 % Found C, 58.73; H, 5.29; N, 21.51%.
4.1.23.
N'-(4-Bromobenzylidene)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-
carbohydrazide (6ba)
1
MS(ESI) m/z 414 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.58 (s, 1H), 8.04–7.81 (m,
3H), 7.71 (d, J = 7.6 Hz, 2H), 7.64–7.51 (m, 4H), 2.60 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 164.7, 163.3, 160.8, 144.0, 139.5, 137.3, 136.8, 135.4, 133.0, 130.7(2C), 129.0,
128.6(2C), 126.7, 123.4, 120.4, 17.4. Anal. calcd for C18H13BrN4OS: C, 52.31; H, 3.17; N,
13.56 % Found C, 52.33; H, 3.29; N, 13.71%.
19

20
4.1.24.
2-Methyl-N'-(4-(trifluoromethyl)benzylidene)benzo[d]imidazo[2,1-b]thiazole-3-
carbohydrazide (6bb)
1
MS(ESI) m/z 403 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.58 (s, 1H), 8.09–7.94 (m,
3H), 7.76 (d, J =8.0 Hz, 2H), 7.65–7.48 (m, 4H), 2.62 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 163.6, 163.0, 156.6, 140.1, 138.4, 137.1, 134.3, 131.8, 129.5, 128.2, 128.9(2C), 128.3,
127.5(2C), 126.6, 123.9, 119.1, 16.9. Anal. calcd for C19H13F3N4OS: C, 56.71; H, 3.26; N,
13.92 % Found C, 56.83; H, 3.29; N, 14.06%.
4.1.25. N'-Benzylidene-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carbohydrazide (6bc)
1
MS(ESI) m/z 335 [M+H]+. H NMR (400 MHz, CDCl3): δ 8.55 (d, J = 8.0 Hz, 1H), 8.02–
7.91 (m, 3H), 7.78 (d, J = 8.0 Hz, 2H), 7.65–7.51 (m, 5H), 2.62 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 163.3, 162.6, 158.9, 146.2, 139.0, 137.5, 136.8, 134.5, 133.0, 128.4(2C), 126.2,
125.7(2C), 124.2, 123.4, 119.6, 17.4. Anal. calcd for C18H14N4OS: C, 64.65; H, 4.22; N,
16.75 % Found C, 64.73; H, 4.29; N, 16.91%.
4.1.26.
2-Methyl-N'-(3,4,5-trimethoxybenzylidene)benzo[d]imidazo[2,1-b]thiazole-3-
carbohydrazide (6bd)
1
MS(ESI) m/z 425 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 8.56 (s, 1H), 7.99–7.90 (m,
2H), 7.54–7.42 (m, 3H), 7.21 (s, 2H), 3.96 (s, 9H), 2.60 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 163.6, 162.9, 160.2, 150.3(2C), 146.4, 142.6, 139.3, 136.2, 135.1, 134.6, 129.8,
126.3, 124.6, 121.4, 114.6(2C), 62.1, 60.6(2C), 17.3. Anal. calcd for C21H20N4O4S: C, 59.42;
H, 4.75; N, 13.20 % Found C, 59.63; H, 4.82; N, 13.38%.
C15H11F3N4OS: C, 51.13; H, 3.15; N, 15.90 % Found C, 51.23; H, 3.19; N, 15.96%.
4.1.27.
N'-(4-(Dimethylamino)benzylidene)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-
carbohydrazide (6be)
1
MS(ESI) m/z 328 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.79 (s, 1H), 8.61 (s, 1H),
8.01–7.90 (m, 2H), 7.69–7.54 (m, 4H), 7.15 (d, J = 8.0 Hz, 2H), 3.13 (s, 6H), 2.61 (s, 3H);
20

21
13C NMR (100 MHz, DMSO-d6) δ 164.3, 161.1, 158.4, 153.6, 146.5, 137.8, 135.1, 133.8,
130.4, 128.2(2C), 126.0, 125.3, 124.6, 122.4(2C), 118.1, 43.2(2C), 16.9. Anal. calcd for
C20H19N5OS: C, 63.64; H, 5.07; N, 18.55 % Found C, 63.73; H, 5.19; N, 18.71%.
General procedure for the synthesis of final molecules (7aa-ae and 7ba-be)
To the stirred solution of 6-methylimidazo[2,1-b]thiazole-5-carboxylic acid (for 7aa-ae)/ 2-
methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxylic acid (for 7ba-be) (1.0 equiv), in CH2Cl2
at 0 °C was added EDCI (1.2 equiv), HOBt (1.2 equiv) and Et3N (2.0 equiv) stirred for few
minutes then was added R-NH2 (1.2 equiv), and allowed stir at rt for 3 h, the reaction mixture
was diluted with CH2Cl2 and washed with H2O and the separated organic layer was
concentrated under reduced pressure, purified by column chromatography.
4.1.28. N-(4-Bromophenyl)-6-methylimidazo[2,1-b]thiazole-5-carboxamide (7aa): To the
stirred solution of 6-methylimidazo[2,1-b]thiazole-5-carboxylic acid (0.4 g, 2.19 mmol), in
CH2Cl2 at 0 °C was added EDCI (0.50 g, 2.64 mmol), HOBt (0.36 g, 2.64 mmol), and Et3N
(0.62 mL, 4.38 mmol) stirred for few minutes then was added 4-bromoaniline (0.45 g, 2.64
mmol), and allowed stir at rt for 3 h, The reaction mixture was diluted with CH2Cl2 (25 mL)
and washed with H2O (3 × 20 mL) and the separated organic layer was concentrated under
reduced pressure, purified by column chromatography using 40% EtOAc/hexanes as eluent.
1
MS(ESI) m/z 335 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 9.70 (s, 1H), 8.57 (d, J = 8.0
Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 7.6 Hz, 1H), 2.61 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 164.5, 162.3, 143.2, 141.7, 133.9, 132.8, 130.2,
128.3, 126.5(2C), 121.4, 118.4, 16.9. Anal. calcd for C13H10BrN3OS: C, 46.44; H, 3.00; N,
12.50 % Found C, 46.53; H, 3.12; N, 12.71%.
4.1.29. 6-Methyl-N-phenylimidazo[2,1-b]thiazole-5-carboxamide (7ab)
MS(ESI) m/z 258 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.21 (s, 1H), 8.52 (d, J = 8.0
13
Hz, 1H), 7.74–7.56 (m, 5H), 7.24 (d, J = 8.0 Hz, 1H), 2.64 (s, 3H); C NMR (100 MHz,
21

22
DMSO-d6) δ 166.9, 156.3, 146.5, 138.0, 132.2, 129.4, 127.7 (2C), 126.2, 124.4(2C), 119.4,
16.9. Anal. calcd for C13H11N3OS: C, 60.68; H, 4.31; N, 16.33 % Found C, 60.73; H, 4.42; N,
16.51%.
4.1.30. N-(4-Methoxyphenyl)-6-methylimidazo[2,1-b]thiazole-5-carboxamide (7ac)
MS(ESI) m/z 302 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 10.34 (s, 1H), 8.49 (d, J = 8.0 Hz,
1H), 7.62 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 8.0 Hz, 2H), 4.12 (q, J =
13
7.2 Hz, 2H), 2.63 (s, 3H), 1.38 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl3) δ 165.2,
160.2, 158.4, 145.3, 137.4, 134.6, 126.3(2C), 123.2, 119.4(2C), 118.1, 71.3, 17.2, 15.8. Anal.
calcd for C15H15N3O2S: C, 59.78; H, 5.02; N, 13.94 % Found C, 59.93; H, 5.12; N, 14.11%.
4.1.31. N-Benzyl-6-methylimidazo[2,1-b]thiazole-5-carboxamide (7ad)
1
MS(ESI) m/z 272 [M+H]+. H NMR (400 MHz, CDCl3): δ 9.45 (s, 1H), 8.52 (d, J = 8.0 Hz,
1H), 7.51–7.38 (m, 5H), 7.36 (d, J = 8.0 Hz, 1H), 4.23 (s, 2H), 2.62 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 163.4, 160.6, 144.4, 142.3, 137.3, 136.2, 127.4(2C), 126.5(2C), 125.9, 119.4,
51.3, 16.8. Anal. calcd for C14H13N3OS: C, 61.97; H, 4.83; N, 15.49 % Found C, 62.03; H,
4.92; N, 15.61%.
4.1.32. N-Cyclohexyl-6-methylimidazo[2,1-b]thiazole-5-carboxamide (7ae)
1
MS(ESI) m/z 264 [M+H]+. H NMR (400 MHz, CDCl3): δ 9.27 (s, 1H), 8.49 (d, J = 7.6 Hz,
1H), 7.31 (d, J = 7.6 Hz, 1H), 3.34–3.28 (m, 1H), 2.61 (s, 3H), 1.71–1.63 (m, 4H), 1.48–1.24
(m, 6H); 13C NMR (100 MHz, CDCl3) δ 162.7, 158.3, 146.2, 142.3, 136.2, 119.4, 61.3,
34.2(2C), 27.3, 25.2(2C), 16.2. Anal. calcd for C13H17N3OS: C, 59.29; H, 6.51; N, 15.96 %
Found C, 59.43; H, 6.64; N, 16.11%.
4.1.33. N-(4-Bromophenyl)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide (7ba):
1
MS(ESI) m/z 386 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 10.71 (s, 1H), 8.03–7.90 (m,
2H), 7.83 (d, J = 7.2 Hz, 2H), 7.69–7.54 (m, 4H), 2.59 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 162.7, 160.2, 156.4, 138.4, 136.9, 133.6, 132.2, 131.5, 130.3(2C), 128.2, 126.5, 126.1,
22

23
123.3(2C), 119.5, 15.6. Anal. calcd for C17H12BrN3OS: C, 52.86; H, 3.13; N, 10.88 % Found
C, 52.93; H, 3.22; N, 10.99%.
4.1.34. 2-Methyl-N-phenylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide (7bb)
MS(ESI) m/z 308 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.35 (s, 1H), 8.02 (d, J = 7.2
13
Hz, 1H), 7.72–7.63 (m, 3H), 7.58 (d, J = 7.6 Hz, 2H), 7.47–7.35 (m, 3H), 2.62 (s, 3H); C
NMR (100 MHz, DMSO-d6) δ 163.4, 159.5, 156.2, 139.4, 137.6, 136.1, 135.6, 134.2,
128.8(2C), 127.4, 127.0, 126.5, 125.3(2C), 120.4, 16.4. Anal. calcd for C17H13N3OS: C,
66.43; H, 4.26; N, 13.67 % Found C, 66.70; H, 4.42; N, 13.81%.
4.1.35. N-(4-Ethoxyphenyl)-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide (7bc)
1
MS(ESI) m/z 352 [M+H]+. H NMR (400 MHz, CDCl3): δ 9.34 (s, 1H), 8.09 (d, J = 6.8 Hz,
1H), 8.04 (d, J = 6.8 Hz, 1H), 7.77–7.63 (m, 4H), 7.22 (d, J = 7.6 Hz, 2H), 4.14 (q, J = 7.2
Hz, 2H), 2.61 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 164.6, 161.3,
156.5, 152.4, 139.3, 136.1, 134.2, 133.6, 130.3, 127.2, 124.4, 122.4(2C), 119.9, 119.4(2C),
69.4, 16.6, 15.4. Anal. calcd for C19H17N3O2S: C, 64.94; H, 4.88; N, 11.96 % Found C,
64.99; H, 4.92; N, 12.09%.
4.1.36. N-Benzyl-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide (7bd)
MS(ESI) m/z 322 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 10.33 (s, 1H), 8.10–8.03 (m, 2H),
13
7.69–7.42 (m, 7H), 4.19 (s, 2H), 2.63 (s, 3H); C NMR (100 MHz, CDCl3) δ 162.5, 160.5,
158.9, 140.1, 138.6, 136.8, 135.2, 129.1(2C), 127.4, 126.5(2C), 125.1, 123.5, 123.0, 119.4,
52.2, 16.9. Anal. calcd for C18H15N3OS: C, 67.27; H, 4.70; N, 13.07 % Found C, 67.33; H,
4.82; N, 13.21%.
4.1.37. N-Cyclohexyl-2-methylbenzo[d]imidazo[2,1-b]thiazole-3-carboxamide (7be)
MS(ESI) m/z 314 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 8.07 (d, J = 6.8 Hz, 1H), 8.01 (d, J
= 6.8 Hz, 1H), 7.67–7.55 (m, 3H), 3.33–3.28 (m, 1H), 2.63 (s, 3H), 1.74–1.66 (m, 4H), 1.51–
1.27 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 164.5, 162.8, 158.4, 137.2, 135.9, 135.2, 127.5,
23

24
126.3, 126.0, 122.2, 60.3, 34.6(2C), 27.8, 26.0(2C), 16.9. Anal. calcd for C17H19N3OS: C,
65.15; H, 6.11; N, 13.41 % Found C, 65.23; H, 6.14; N, 13.61%
4.2. Biological activity
4.2.1. MTB PS screening
The MTB panC gene (Rv3602c) encoding the pantothenate synthetase was cloned and
transformed into BL21 (DE3) cells and the expression of the protein was performed. For the
assay, to each well of a 96-well plate, 60μl of PS reaction mixture consisting of 0.4 mM
NADH, 5 mM pantoic acid, 10 mM MgCl2, 5mM β-alanine, 10 mM ATP, 1 mM potassium
phosphoenolpyruvate, and 20 μl of enzyme mixture consists of 18 units/ml each of chicken
muscle myokinase, rabbit muscle pyruvate kinase, and rabbit muscle lactate dehydrogenase
diluted in 100 mM HEPES buffer were added. The reaction mixture and enzyme mixture
were added to the plate to a final volume of 100 μl with 100 mM HEPES buffer (pH 7.8).
Concentration of enzyme was determined based on the range finding experiments by varying
the concentration of enzymes. Compounds were then added to the plates (from 25 µM to
lower concentration) and the reaction was initiated with the addition of 10 μL of 4.32 pM of
MTB PS, diluted in buffer. The test plate was immediately transferred to a microplate reader
and the depletion of NADH was measured at 340 nm. The reaction components except MTB
PS were mixed in the well and the background reaction was measured; MTB PS was then
added and the reaction kinetics was monitored. Reactions were carried out at 37°C in a heat-
controlled PerkinElmer Victor X3 Spectrophotometer. % Inhibitions were calculated using
following formula: 100 x [(1 - compound rate - background rate) / (full reaction rate -
background rate)]
4.2.2. In vitro MTB screening
The compounds were further screened for their in vitro antimycobacterial activity against M.
tuberculosis H37Rv by microplate Alamar blue assay method (15). Briefly, the inoculum was
24

25
prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone,
0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted
to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock
solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested.
Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter
plate using 100 µl 7H9-S. A growth control containing no antibiotic and a sterile control were
also prepared on each plate. Sterile water was added to all perimetre wells to avoid
evaporation during the incubation. The plate was covered, sealed in plastic bags and
incubated at 37oC in normal atmosphere. After 7 days incubation, 30 ml of alamar blue
solution was added to each well, and the plate was re-incubated overnight. A change in
colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the
MIC was defined as the lowest concentration of drug that prevented this change in colour.
4.2.3. In vitro cytotoxicity screening
Some compounds were further examined for toxicity in a RAW 264.7 cell line at the
concentration of 50 µM. After 72 h of exposure, viability was assessed on the basis of
cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 non-
radioactive cell proliferation assay.
4.2.4. In vitro nutrient starved MTB screening
Culture of Mycobacterium tuberculosis H37RV were grown in Middlebrook 7H9 medium
supplemented with OADC (nutrient rich medium) was pelleted and washed twice with PBS
(Phosphate buffer saline, HiMedia Laboratories). The pellet was re-suspended in PBS in
sealed bottles and is incubated at 37 °C for 6 weeks. Six week starved cultures were treated
with standard drugs like Isoniazid, Rifampicin and Moxifloxacin along with synthesized
drugs for 7 days at a concentration of 10ug/ml. The treated cell suspensions were diluted 10-
fold up to 10-6 using Middlebrook 7H9 medium supplemented with OADC and 100 µl of
25

26
each dilutions were plated in 48 well plates in triplicates along with 900 µl of Midllebrook
7H9 medium (HiMedia Laboratories) supplemented with OADC (HiMedia Laboratories).
The microplates were incubated at 37 °C for 28 days without agitation. Wells with visible
bacterial growth were counted as positive, and MPN values were calculated using standard
statistical methods.
4.2.5. Antimycobacterial screening using Mycobacterium marinum induced adult zebra
fish screening
One of the most active compounds was further evaluated for its in-vivo activity using adult
Zebra fish model. We used Mycobacterium marinum strain (ATCC BAA-535) grown at 30
°C in Middlebrook 7H9 broth. Fish were initially weighed and monitored for its locomotor
activities and were grouped into control and treatment groups (n=6). All the fish were
infected by intraperitoneal injection with 20 µl of thawed bacterial stocks (around 0.75
million bacteria). They were observed for lesions, reduction in swimming activities and
squamous eruptions in the initial 7 day infection stage which was followed by treatment
stage. The drug solutions were prepared based on the fish’s body weight and oral dosing
amount of 5 µL. Fish were then administered drug orally using micropipette for 7 days. After
treatment they were allowed to swim in 1.5mg/mL solution of kanamycin sulphate before
proceeding for sacrifice at the end of study i.e., 14th day. Finally, all of them were sacrificed
using homogenization technique and the tissue sample was prepared in Middlebrook 7H9
broth. The collected homogenate was serially diluted to 10-6 times and plated into 48-well
plates, incubated at 30 °C for 24 hours. The plates were checked for the bacterial counts
using MPN assay method.
Acknowledgements
Authors acknowledge DST, India for its financial support to carry out this work. Ganesh
Samala (SRF) is thankful to CSIR, India for fellowship; DS acknowledges University Grants
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27
Commission, New Delhi, India for UGC Research Award. We also acknowledge Dr David
Eisenberg and Dr Shuishu Wang, Department of Biochemistry & Molecular biology,
USUHS, Maryland for providing MTB PS clone.
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Design, synthesis and biological evaluation of imidazo[2,1-
b]thiazole and benzo[d]imidazo[2,1-b]thiazole derivatives as
Mycobacterium tuberculosis pantothenate synthetase
inhibitors
Ganesh Samala, Parthiban Brindha Devi, Shalini Saxena, Nikhila Meda Perumal Yogeeswari,
Dharmarajan Sriram*
imidazo[2,1-b]thiazole
O
NH
imidazo[1,2-a]pyridine
O
N
O
CH3
NHNH
CH3
S
N
Replacement
of pyridyl ring
with thiazole
N
O
N
NH
and benzthiazole
N
PanC IC50 = 1.90±0.12 µM
MTB MIC = 4.53 µM
[EARLIER REPORTED COMPOUND]
CH3
S
N
benzo[d]imidazo
[2,1-b]thiazole
Compound 5bc emerged as potent compound active against MTB PS with IC50 of 0.53±0.13 µM, MIC
of 3.53 µM, 2.1 log reduction against nutrient starved MTB. It also showed 1.5 log reduction of M.
marinum load in Zebra fish at 10mg/kg.
29