Design and synthesis of imidazo[2,1-b]thiazole linked triazole conjugates: Microtubule-destabilizing agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.060

A series of imidazo[2,1-b]thiazole linked triazole conjugates were synthesized by using Huisgen 1,3-dipolar cyclo-addition reaction (click chemistry approach) and evaluated for their antiproliferative activity against some human cancer cell lines like, HeLa (cervical), DU-145 (prostate), A549 (lung), MCF-7 (breast) and HepG2 (liver). Among them, Conjugates 4g and 4h demonstrated a significant antiproliferative effect against human lung cancer cells (A549) with IC₅₀values of 0.92 and 0.78 μM respectively. Flow cytometric analysis revealed that these conjugates induced cell cycle arrest in G2/M phase in A549 lung cancer cells. The tubulin polymerization assay and immunofluorescence analysis showed that these conjugates effectively inhibit microtubule assembly in cell free and cell based (A549) experiment respectively. Moreover, the apoptosis inducing properties were evaluated by Hoechst staining, mitochondrial membrane potential and Annexin V-FITC assay. Further, western blot analysis was performed for proapoptotic protein Bax and antiapoptotic protein Bcl-2 and the results demonstrated that there was up regulation of Bax and down regulation of Bcl-2 suggesting that these compounds induced apoptosis in human lung cancer cells, A549.

Full text of DOI:10.1016/j.ejmech.2016.09.060

Accepted Manuscript
Design and synthesis of imidazo[2,1-b]thiazole linked triazole conjugates:
Microtubule-destabilizing agents
Siddiq Pasha Shaik, V. Lakshma Nayak, Faria Sultana, A.V. Subba Rao, Anver
Basha Shaik, Korrapati Suresh Babu, Ahmed Kamal
PII:
S0223-5234(16)30796-6
10.1016/j.ejmech.2016.09.060
EJMECH 8926
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 June 2016
Revised Date: 17 September 2016
Accepted Date: 19 September 2016
Please cite this article as: S.P. Shaik, V.L. Nayak, F. Sultana, A.V.S. Rao, A.B. Shaik, K.S.
Babu, A. Kamal, Design and synthesis of imidazo[2,1-b]thiazole linked triazole conjugates:
Microtubule-destabilizing agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.060.
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ACCEPTED MANUSCRIPT
Design and Synthesis of Imidazo[2,1-b]thiazole linked Triazole Conjugates:
Microtubule-Destabilizing Agents
Siddiq Pasha Shaik, V. Lakshma Nayak, Faria Sultana, A. V. Subba Rao, Anver Basha Shaik,
Korrapati Suresh Babu and Ahmed Kamal

ACCEPTED MANUSCRIPT
Design and Synthesis of Imidazo[2,1-b]thiazole linked Triazole Conjugates:
Microtubule-Destabilizing Agents
Siddiq Pasha Shaik,a,b V. Lakshma Nayak,a Faria Sultana,a A. V. Subba Rao,a,b Anver Basha
Shaik,a Korrapati Suresh Babua and Ahmed Kamal*a,b,c
b
aMedicinal Chemistry and Pharmacology Division, Academy of Scientific and Innovative
Research, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad-500007, India
cChemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
Abstract
A series of imidazo[2,1-b]thiazole linked triazole conjugates were synthesized by using Huisgen
1,3-dipolar cyclo-addition reaction (click chemistry approach) and evaluated for their
antiproliferative activity against some human cancer cell lines like, HeLa (cervical), DU-145
(prostate), A549 (lung), MCF-7 (breast) and HepG2 (liver). Among them, Conjugates 4g and 4h
demonstrated a significant antiproliferative effect against human lung cancer cells (A549) with
IC50 values of 0.92 and 0.78 µM respectively. Flow cytometric analysis revealed that these
conjugates induced cell cycle arrest in G2/M phase in A549 lung cancer cells. The tubulin
polymerization assay and immunofluorescence analysis showed that these conjugates effectively
inhibit microtubule assembly in cell free and cell based (A549) experiment respectively.
Moreover, the apoptosis inducing properties were evaluated by Hoechst staining, mitochondrial
membrane potential and Annexin V-FITC assay. Further, western blot analysis was performed
for proapoptotic protein Bax and antiapoptotic protein Bcl-2 and the results demonstrated that
there was up regulation of Bax and down regulation of Bcl-2 suggesting that these compounds
induced apoptosis in human lung cancer cells, A549.
Keywords:
Imidazo[2,1-b]thiazoles; Triazole conjugates; Cytotoxicity; Tubulin depolymerization;
Apoptosis

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1. Introduction
Microtubules are one of the key structural components of the cytoskeleton in eukaryotic
cells comprising of α and β-tubulin heterodimers. They play an important role in the fundamental
cellular processes such as structural and regulatory functions, including the maintenance of cell
shape and intracellular transport machinery as well as cell growth and mitosis and have emerged
as an attractive and viable target in the development of anticancer drugs [1-4]. Based on their
mechanism of action and four different binding sites on tubulin these antimitotic agents are
further classified into: the taxane site and the laulimalide/peloruside A site, both for microtubule-
stabilizing agents, and the vinca site and the colchicine site for microtubule destabilizing agents
[5, 6]. Based on the previous studies it is observed that taxanes and vinca alkaloids binding sites
in tubulin are usually more successful in clinical oncology, however none of the colchicine site
inhibitors have been approved by FDA as clinically useful antitumor agents [7-9]. Recently
developed small molecules showed attractive target for tubulin inhibitors [10-13]. Therefore,
there is growing interest in identifying and developing newer molecules that could inhibit tubulin
polymerization.
Imidazo[2,1-b]thiazole scaffolds have drawn considerable interest because of their broad
spectrum of pharmacological activities, particularly antitumor activity [14, 15]. Its derivative
imidazo[2,1-b]thiazole-guanylhydrazone (1) showed potent antiproliferative activity against a
number of cancer cell lines and is considered as a promising lead [16]. These fused heterocycles
exhibit anticancer activity through different molecular mechanisms such as by inhibiting p90
ribosomal S6 kinase 2 (RSK2) and tubulin polymerization [17-21]. Recently, we reported
imidazo[2,1-b]thiazole-chalcone conjugates as tubulin polymerization inhibitors and binds at the
colchicine site [14]. Nocodazole (2) is a well-known tubulin polymerization inhibitor, which
inhibits cell proliferation and is largely used as a positive control [22, 23]. Whereas, 1,2,3-
triazole derivatives have attracted the medicinal chemists, due to their wide range of biological
activities, such as antitumor, anti-HIV, antifungal and antitubercular activities [24-27]. They
have also been used as building blocks for the synthesis of important bio-conjugates. Considering
the biological significance of this scaffold, researchers have used this moiety in the development
of various biologically active compounds. Earlier studies in this laboratory on triazole linked 2-
anilinomethylnicotinamides (3, Fig. 1) as potential microtubule polymerization inhibitors have
exhibited promising activity against certain cancer cell lines [28].

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In continuation to our search for synthetic small molecule tubulin inhibitors, herein we
designed and attached the “triazole” moiety to imidazo[2,1-b]thiazole motif that resulted in
imidazo[2,1-b]thiazole linked triazole conjugates (4a-x) as shown in Fig. 1. They have been
evaluated for their cytotoxicity, effect on tubulin polymerization and cell cycle to understand
their possible mechanism of action.
< Fig. 1 >
2. Results and Discussion
2.1. Chemistry
Imidazo[2,1-b]thiazole linked triazole conjugates (4a-x) were synthesized as shown in
Scheme 1. Ethyl-2-aminothiazole-4-carboxylate (7) was synthesized by the condensation of ethyl
bromopyruvate and thiourea in ethanol under reflux for 4 h. Cyclization of this precursor (7) with
various phenacyl bromides (8a-d) in ethanol at reflux temperature results in the formation of
intermediates (9a-d), which upon lithium hydroxide monohydrate (LiOH.H2O) mediated ester
hydrolysis produces carboxylic acids (10a-d). These upon coupling with propargylamine
hydrochloride under N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HOBt) in dry dimethylformamide (DMF) conditions provide the
alkyne precursors (11a-d). The other subunit, benzyl azides (14a-f) were prepared from benzyl
bromides (13a-f) with sodium azide (NaN3) in dimethyl sulfoxide (DMSO) [29]. Finally, we
obtained the desired 1,2,3-triazole linked imidazo[2,1-b]thiazoles (4a-x) in high regioselectively
by exposing imidazo[2,1-b]thiazole alkynes (11a-d) to benzyl azides (14a-f) in the presence of
catalytic copper(׀׀) sulfate pentahytrate (CuSO4·5H2O) and sodium ascorbate in a H2Oꢀt-butyl
alcohol t-BuOH) mixture in good to excellent yields. All the structures of these conjugates were
(
confirmed by spectral data (1H NMR, 13C NMR, MS and HRMS).
< Scheme 1 >
2.2. Biological activity
2.2.1. Antiproliferative activity
All the newly synthesized compounds (11a-d, 4a-x) were evaluated for their
antiproliferative activity in a panel of five human cancer cell lines, namely, HeLa (cervical), DU-
145 (prostate), A549 (lung), MCF-7 (breast) and HepG2 (liver) by employing MTT assay [30].

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The results are summarized in Table 1 and expressed as IC50 values. All the compounds (4a-x)
exhibited pronounced activity against A549 and MCF-7 cell lines compared to other tested cell
lines. Amongst these 4g and 4h showed significant cytotoxic activity against A549 (IC50 values
of 0.924 and 0.778 µM) and MCF-7 (IC50 values of 1.737 and 1.013 µM) cell lines.
In order to get the insight in to the structure-activity relationship (SAR), we have varied
the substitution pattern of the both aryl rings of these conjugates. The cytotoxic activities of
intermediates 11a-d revealed that electron-donating substituents like methoxy on the aryl ring
(11b) increases the activity on all tested cell lines, in contrast electronegative substituents like
fluorine (11c) and chlorine (11d) on this ring suppressed the activity.
Similarly, conjugates 4a-b and 4g-h with electron-donating substituents on the benzyl
ring of the triazole subunit showed significant cytotoxicity with IC50 values in micro to
submicromolar range, in contrast electronegative substituent like fluoro (4r and 4x) decreases the
cytotoxicity. Amongst them 3,4-disubstituted conjugates like 4g and 4h displayed promising
cytotoxicity (IC50 values of 0.924 and 0.778 µM), particularly against A549 cell lines, these are
superior to compound 3 (IC50 values of 1.57 µM). It was observed that mono, 3,5-disubstituted
as well as trisubstituted conjugates showed less activity when compared to 3,4-disubstituted
conjugates. These studies also suggest that electron donating substituents like methoxy on the
aryl ring of the imidazo[2,1-b]thiazole subunit and electron rich 3,4-disubstituted substituents on
the benzyl ring of the triazole subunit are required for the cytotoxic potential. Based on the
results obtained from the MTT assay, the most cytotoxic conjugates (4g and 4h) were examined
for detailed biological studies, such as tubulin polymerization inhibition, immunohistochemistry
and apoptosis induction. In addition, these conjugates were also investigated for a western blot
analysis as well as molecular docking studies.
< Table 1 >
< Fig. 2 >
2.2.2. Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint or by induction of apoptosis or a combined effect of both
cycle block and apoptosis [31, 32]. The in vitro screening results revealed that compounds, 4g
and 4h showed significant antiproliferative activity against lung cancer (A549). Therefore, it was

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considered of interest to understand whether this inhibition of cell growth was on account of cell
cycle arrest. In this study A549 cells were treated with these conjugates at concentrations of 0.5
and 1 µM for 48 h. The data obtained clearly indicated that these compounds show G2/M cell
cycle arrest in comparison with the untreated cells. These conjugates (4g and 4h) showed 20.4
and 21.7 % of cell accumulation in G2/M phase at 0.5 µM concentration, whereas they exhibited
26.2 and 28.5 % of cell accumulation at 1 ꢁM concentration, respectively (Fig. 3, Table 2).
< Fig. 3 >
< Table 2 >
2.2.3. Effect on tubulin polymerization
In general G2/M cell cycle arrest is strongly associated with the inhibition of tubulin
polymerization and since conjugates 4g and 4h cause cell cycle arrest at G2/M phase, it was
considered of interest to investigate their microtubule inhibitory function [33]. Tubulin subunits
are known to heterodimerize and self-assemble to form microtubules in a time dependent
manner. The progression of tubulin polymerization was thus examined by monitoring the
increase in fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate
for 1 h at 37 °C with and without the conjugates in comparison to nocodazole a reference
compound [34, 35]. The test compounds (4g and 4h) significantly inhibited tubulin
polymerization by 70.2 and 74.3 % respectively, whereas the reference compound (nocodazole)
exhibited 71.0 % inhibition (Fig. 4). This was followed by evaluation of IC50 values for these
conjugates and the results are shown in Table 3. The test compounds 4g and 4h showed tubulin-
assembly inhibition with IC50 values of 1.62 and 1.43 µM respectively, whereas the reference
compound nocodazole showed 1.59 µM.
< Fig. 4 >
< Table 3 >
2.2.4. Immunohistochemistry studies on tubulin
In addition to tubulin polymerization studies, we also investigated alterations in the
microtubule network in A549 cells induced by these conjugates (4g and 4h) using florescence
microscopy [36]. Therefore, A549 cells were treated with these conjugates for 48 h and the
results demonstrated a well-organized microtubular network in the control cells. However, cells

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treated with test compounds showed disrupted microtubule organization as seen in Fig. 5, thus
confirming the inhibition of tubulin polymerization.
< Fig. 5 >
2.2.5. Hoechst staining for apoptosis
Apoptosis is one of the major pathways that lead to the process of cell death, wherein
chromatin condensation and fragmented nuclei are known as the classic characteristics of
apoptosis. It was considered of interest to investigate the apoptotic inducing effect of these two
potent conjugates (4g and 4h) by Hoechst staining (H33258) method in A549 cancer cell line.
Therefore, A549 cells were treated with test compounds at 0.5 µM concentrations for 48 h.
Manual field quantification of apoptotic cells based on cytoplasmic condensation, presence of
apoptotic bodies, nuclear fragmentation and relative fluorescence of the test compounds (4g and
4h) revealed that there is significant increase in the percentage of apoptotic cells (Fig. 6).
< Fig. 6 >
2.2.6. Measurement of mitochondrial membrane potential (ꢀΨm)
The maintenance of mitochondrial membrane potential (∆Ψm) is important for
mitochondrial integrity and bioenergetic function [37]. Mitochondrial changes, including loss of
mitochondrial membrane potential (∆Ψm), are key events that take place during drug-induced
apoptosis. In order to study the apoptosis inducing effect of compounds 4g and 4h, mitochondrial
membrane potential (∆Ψm) changes were detected using the fluorescent probe JC 1 (5,5,6,6-
tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide-5µg/mL). The red fluorescence
and green fluorescence were detected at FL-2 and FL-1 channel, respectively. During apoptosis
mitochondrial membrane potiential (∆Ψm) decreases and is measured by decrease in the ratio of
red fluorescence (FL-2) to green fluorescence (FL-1). After 48 h of treatment, it was observed
that the mitochondrial membrane potential (ꢂΨm) of A549 cells is reduced significantly (Fig. 7).
< Fig. 7 >
2.2.7. Annexin V-FITC for apoptosis
The apoptotic effect of 4g and 4h was further evaluated by Annexin V FITC/PI (AV/PI)
dual staining assay to examine the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis [38]. In this study

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A549 cells were treated with these compounds for 48 h at 0.5 and 1 µM concentrations to
examine the apoptotic effect. It was observed that these compounds showed significant apoptosis
against A549 cells as shown in Fig. 8 and Table 4 and the results indicated that compounds 4g
and 4h exhibited 9.34 and 22.44 % apoptosis at 0.5 µM concentration, whereas they exhibited
30.64 and 31.88 % apoptosis at 1 µM concentration respectively for 48 h.
< Fig. 8 >
< Table 4 >
2.2.8. Western blot analysis of Bcl-2 and Bax proteins
The apoptotic regulation depends mainly on the internal balance between anti-apoptotic
protein Bcl-2 activity and proapoptotic protein Bax activity [39]. In this context, A549 cells were
treated with compounds 4g and 4h at 0.5 µM concentrations for 48 h. After 48 h of treatment, it
was observed that the antiapoptotic protein Bcl-2 was down regulated and the proapoptotic
protein Bax was up regulated (Fig. 9). This data revealed that these compounds induced cell
death by apoptosis in human lung cancer cells, A549.
< Fig. 9 >
2.3. Molecular modeling
Molecular docking studies were performed extensively for the potential ligands (4g and
4h) to support the discussion of the biological results obtained in the previous sections.
Autodock 4 was employed and these compounds were docked in the colchicine binding site of
the tubulin (PDB code: 3E22) [18, 40]. The colchicine site is a deep pocket generally located at
the α,β interface of tubulin heterodimers and it was identified by Ravelli and coworkers in 2004
[41]. The backbone of protein is visualized as cylindrical cartoon of wheat and pale yellow colors
that correspond to α- and β-chains respectively. However, the significant hydrogen bonding
interactions are shown as red dotted lines and the amino acid residues that interact with ligands
are indicated as magenta stick models. The promising conjugates were docked with similar
fashion at the colchicine binding site of tubulin and sandwiched between the amino acid residues
like αAsn101, αAsn102, αTyr224, αSer178, αVal181, αVal182, αGln11 of α-chain and
β
Lys254, βLeu255, βCys241, βAsn258, βThr239, βMet259 and βVal238 of β-chain. Conjugate
4g exhibits significant hydrogen bonding between hydroxyl hydrogen of α Tyr224 and methoxy
oxygen of phenyl ring that is directly linked to the imidazo[2,1-b]thiazole subunit (OH---O). In

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addition, two more strong hydrogen bonds were observed between hydroxyl hydrogen of benzyl
ring of the triazole subunit and carbonyl oxygen of both βThr238 as well as βVal238 (O---H---
O). Moreover, the carbonyl oxygen of conjugate 4g forms a weak hydrogen bonding with NH of
aAsn258 (O---HN). Interestingly the sulfur atom of core scaffold imidazo[2,1-b]thiazole also
forms a weak hydrogen bond with NH of
electrostatic interactions with 4g. Additionally, some of the hydrophobic interactions are also
noticed between the ligand and Asn101, αAsn102, αSer178, αVal181, αVal182 and Lys254.
βLeu255 (S---NH), however, βCys241 exhibits weak
β
Compound 4h is also docked in a similar fashion at the interface of two chains that displays
strong hydrogen bonding between methoxy oxygen of phenyl ring directly linked to the
imidazo[2,1-b]thiazole subunit and NH of the αGln11 (O---NH). In addition, some of the weak
hydrogen bonding interaction observed between carbonyl oxygen of 4h and NH of Asn258 (O---
NH) as well as sulfur of imidazo[2,1-b]thiazole subunit and NH of Leu255 (S---NH). The
Cys241 forms a weak electrostatic interaction with the methoxy oxygen of the dimethoxy benzyl
ring that is linked to the triazole subunit, while the other surrounded amino acid residues
experience hydrophobic contacts. However the triazole rings of both the conjugates are burried at
the hydrophobic region and plays a bridging role between the two chains that leads to a
successful docking at the taget binding site (Fig. 10). To gain further insight of our findings, we
domonstrated the superimposition of colchcine with the potential ligands and the respective
poses were visualized as seen in Fig. 10.
< Fig. 10 >
3. Conclusion
In conclusion, we have synthesized a series imidazo[2,1-b]thiazole linked triazole
conjugates by using Huisgen 1,3-dipolar cyclo-addition reaction and evaluated for their
antiproliferative activity. Among them, conjugates 4g and 4h showed significant cytotoxic
activity against the human lung cancer cell line (A549). The flow cytometric analysis revealed
that these conjugates cause cell cycle arrest at G2/M phase. Furthermore, they effectively
inhibited microtubule assembly and disrupt the microtubule organization in the lung cancer cells.
The molecular docking studies revealed that these conjugates bind at the colchicine site of the
tubulin. Further, Hoechst staining, mitochondrial membrane potential and Annexin V FITC assay
suggested that they induced cell death by apoptosis. Moreover, western blot analysis of

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proapoptotic protein Bax and antiapoptotic protein Bcl-2 results suggesting that these conjugates
induced apoptosis in human lung cancer cells, A549. The SAR provided a useful insight that
could be utilized in developing improved leads based on these conjugate scaffolds. Thus these
conjugates could be considered as potential scaffolds that are useful in the development of newer
leads for the treatment of lung cancer.
4. Experimental section
4.1. Chemistry
All the chemicals and reagents used in this study were obtained from Aldrich (Sigma-
Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill,
MA, USA), or Spectrochem Pvt. Ltd (Mumbai, India) and were used without further purification.
The reactions were monitored by TLC performed on silica gel glass plates containing 60 GF-
254, and visualized was done by using a UV light or iodine indicator. Column chromatography
1
was performed using Merck 60-120 mesh silica gel. H NMR spectra were recorded on Bruker
UXNMR/XWIN-NMR (300 MHz) or Inova Varian-VXRunity (400, 500 MHz) instruments. 13C
NMR spectra were recorded on a Bruker UXNMR/XWIN-NMR (75 MHz) instrument. Chemical
shifts (δ) were reported in ppm downfield from an internal standard TMS and coupling constants
are expressed in Hz. Signal multiplicities are represented by s (singlet), d (doublet), t (triplet),
quartet (q) ds (double singlet), dd (double doublet), m (multiplet) and br s (broad singlet). ESI
spectra were recorded on a Micro mass Quattro LC using ESI+ software with a capillary voltage
of 3.98 kV and an ESI mode positive ion trap detector. High-resolution mass spectra (HRMS)
were recorded on a QSTAR XL hybrid MS-MS mass spectrometer. Melting points were
determined with an electrothermal melting point apparatus, and are uncorrected.
4.1.1. Procedure for the synthesis of ethyl-2-aminothiazole-4-carboxylate (7)
To a solution of ethyl bromopyruvate (6, 15 g, 77.32 mmol) in ethanol (EtOH) (100 mL),
thiourea (5, 5.87 g, 77.32 mmol) was added and the reaction mixture was heated under reflux
temperature (80 °C) for 4 h in inert atmosphere.The reaction mixture was cooled to room
temperature and concentrated under reduced pressure to remove the EtOH. The resulting crude
mass was dissolved in ice water (100 mL) and neutralized with aqueous ammonia. The resulting
solid was filtered, washed with cold water and air dried. This gave 12.5 g (94 %) of a yellow
solid that was used in the next step without further purification.

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1
Mp: 179-181 °C; H NMR (500 MHz, DMSO-d6): δ 7.35 (s, 1H, Ar-H of thiazole), 6.82 (s, 2H,
NH2), 4.29 (q, J = 7.1 Hz, 2H, CH2), 1.35 (t, J = 7.1 Hz, 3H, CH3); MS (ESI): m/z 173 [M + H]+.
4.1.2.General method for the synthesis of ethyl 6-(aryl)imidazo[2,1-b]thiazole-3-carboxylates
(9a-d)
A mixture of ethyl-2-aminothiazole-4-carboxylate (7, 1.0 equiv) and phenacyl bromides
(8a-d; 1.2 equiv) were dissolved in EtOH (10 ml for 1g) was heated at reflux temperature (80
°C) for 8 h. The reaction mixture was cooled to room temperature and concentrated under
vacuum. The resulting crude mass was dissolved in ice water, neutralized with saturated sodium
bicarconate (NaHCO3) solution, and extracted with ethyl acetate (EtOAc). The organic layer was
dried over anhydrous sodium sulfate (Na2SO4), evaporated in a vacuum and purified by column
chromatography by using an ethyl acetate and hexane solvent system to afford the pure title
compounds (9a-d).
4.1.2.1 Ethyl 6-phenylimidazo[2,1-b]thiazole-3-carboxylate (9a)
1
Yellow solid; 81% yield; mp: 91-93 °C; H NMR (500 MHz, CDCl3): δ 8.33 (s, 1H, Ar-H of
thiazole), 7.84 (d, J = 8.2 Hz, 2H, Ar-H), 7.75 (s, 1H, Ar-H of imidazole), 7.40 (t, J = 7.4 Hz,
2H, Ar-H), 7.29 (t, J = 7.4 Hz, 1H, Ar-H), 4.44 (q, J = 14.1, 7.0 Hz, 2H, CH2), 1.43 (t, J = 7.0
Hz, 3H, CH3); MS (ESI): m/z 273 [M + H]+.
4.1.2.2. Ethyl 6-(4-methoxyphenyl)imidazo[2,1-b]thiazole-3-carboxylate (9b)
1
Reddish solid; 79% yield; mp: 95-97 °C; H NMR (400 MHz, CDCl3): δ 8.21 (s, 1H, Ar-H of
thiazole), 7.77 (d, J = 8.4 Hz, 2H, Ar-H), 7.68 (s, 1H, Ar-H of imidazole), 6.93 (d, J = 8.4 Hz,
2H, Ar-H), 4.40 (q, J = 13.8, 6.9 Hz, 2H, CH2), 3.82 (s, 3H, OMe), 1.41 (t, J = 6.9 Hz, 3H, CH3);
MS (ESI): m/z 303 [M + H]+.
4.1.2.3. Ethyl 6-(4-fluorophenyl)imidazo[2,1-b]thiazole-3-carboxylate (9c)
1
Yellow solid; 78% yield; mp: 94-96 °C; H NMR (500 MHz, CDCl3): δ 8.27 (s, 1H, Ar-H of
thiazole), 7.79 (d, J = 8.2 Hz, 2H, Ar-H), 7.74 (s, 1H, Ar-H of imidazole), 7.13 - 7.06 (m, 2H,
Ar-H), 4.44 (q, J = 13.8, 7.0 Hz, 2H, CH2), 1.43 (t, J = 7.0 Hz, 3H, CH3); MS (ESI): m/z 291 [M
+ H]+.
4.1.2.4. Ethyl 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-3-carboxylate (9d)
1
Yellow solid; 86% yield; mp: 97-99 °C; H NMR (500 MHz, CDCl3): δ 8.28 (s, 1H, Ar-H of
thiazole), 7.77 (d, J = 8.5 Hz, 2H, Ar-H), 7.71 (s, 1H, Ar-H of imidazole), 7.34 (d, J = 8.5 Hz,

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2H, Ar-H), 4.44 (q, J = 15.1, 7.1 Hz, 2H, CH2), 1.43 (t, J = 7.1 Hz, 3H, CH3); MS (ESI): m/z 307
[M + H]+.
4.1.3. General method for the synthesis of ethyl 6-(aryl)imidazo[2,1-b]thiazole-3-carboxylic
acids (10a–d)
To a solution of the corresponding ethyl 6-(aryl)imidazo[2,1-b]thiazole-3-carboxylate
(9a-d, 1 equiv) in tetrahydro furan (THF):Methanol:Water (1:1:1) system, was added equimolar
lithium hydroxide monohyrate (LiOH·H2O) at room temperature and continued stirring for 3 h.
After completely consumed the starting material, the solvent mixture was evaporated and an
appropriate amount of water was added. The crude compound was extracted by ethyl acetate.
The aqueous layer was acidified using dil. HCl. The precipitate was filtered, washed with cold
water, and dried under vacuo to afford the title compounds (10a-d), which were used in the next
step without further purification.
4.1.4. General method for the synthesis of 6-(aryl)-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-
carboxamides (11a-d)
Above obtained carboxylic acid (10a-d; 1.0 equiv) was dissolved in dry dimethyl
formamide (DMF), cooled to 0 °C (ice bath). EDCI (1.5 equiv), HOBt (1.2 equiv),
propargylamine hydrochloride (1.2 equiv) and triethylamine (1.2 equiv) were added slowly.
After 10 min removed the ice bath and the mixture was stirred at room temperature for 8 h until
the starting materials were consumed. Then ice cold water was added to the reaction mixture and
extracted with dichloro methane (DCM). The combined organic extracts were washed with aq
NaHCO3 and dried with Na2SO4 and concentrated in vacuo. The residue was purified by column
chromatography by using an ethyl acetate and hexane solvent system to afford the title
compounds (11a-d).
4.1.4.1. 6-Phenyl-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-carboxamide (11a)
Light yellow solid; 78% yield; mp: 189-191 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.10
(t, J = 5.1 Hz, 1H, NH), 8.56 (s, 1H, Ar-H of thiazole), 8.08 (s, 1H, Ar-H of imidazole), 7.85 (d,
J = 7.3 Hz, 2H, Ar-H), 7.41 (t, J = 7.5 Hz, 2H, Ar-H), 7.28 (t, J = 7.3 Hz, 1H, Ar-H), 4.20 (dd, J
= 5.4, 2.5 Hz, 2H, CH2), 2.51 (t, J = 2.4 Hz, 1H, acetylenic); MS (ESI): m/z 282 [M + H]+;
HRMS calcd for C15H12N3OS [M + H]+ 282.0696, found 282.0696.
4.1.4.2. 6-(4-Methoxyphenyl)-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-carboxamide(11b)

ACCEPTED MANUSCRIPT
Light yellow solid; 76% yield; mp: 189-191 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 8.98
(s, 1H, NH), 8.44 (s, 1H, Ar-H of thiazole), 7.98 (s, 1H, Ar-H of imidazole), 7.76 (d, J = 8.7 Hz,
2H, Ar-H), 6.93 (d, J = 8.7 Hz, 2H, Ar-H), 4.19 (dd, J = 5.3, 2.4 Hz, 2H, CH2), 3.84 (s, 3H,
OMe), 2.40 (t, J = 2.4 Hz, 1H, acetylenic); MS (ESI): m/z 312 [M + H]+; HRMS calcd for
C16H14N3O2S [M + H]+ 312.0801, found 312.0812.
4.1.4.3. 6-(4-Fluorophenyl)-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-carboxamide (11c)
Light yellow solid; 75% yield; mp: 189-191 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.15
(s, 1H, NH), 8.52 (s, 1H, Ar-H of thiazole), 8.17 (s, 1H, Ar-H of imidazole), 7.85 - 7.76 (m, 2H,
Ar-H), 7.10 (t, J = 8.3 Hz, 2H, Ar-H), 4.19 (s, 2H, CH2), 2.44 (s, 1H, acetylenic); MS (ESI): m/z
300 [M + H]+; HRMS calcd for C15H11FN3OS [M + H]+ 300.0601, found 300.0603.
4.1.4.4. 6-(4-Chlorophenyl)-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-carboxamide (11d)
White solid; 77% yield; mp: 189-191 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 8.95 (s, 1H,
NH), 8.54 (s, 1H, Ar-H of thiazole), 8.02 (s, 1H, Ar-H of imidazole), 7.78 (d, J = 7.8 Hz, 2H, Ar-
H), 7.36 (d, J = 7.9 Hz, 2H, Ar-H), 4.19 (s, 2H, CH2), 2.36 (s, 1H, acetylenic); MS (ESI): m/z
316 [M + H]+; HRMS calcd for C15H11ClN3OS [M + H]+ 316.0306, found 316.0309.
4.1.5. General method for the synthesis of imidazo[2,1-b]thiazole linked triazole conjugates (4a-
x)
To a solution of 6-(aryl)-N-(prop-2-yn-1-yl)imidazo[2,1-b]thiazole-3-carboxamide (11a-
d, 1.0 equiv ) and benzyl azides (14a-f, 1.10 equiv) in 2 : 1 mixture of water and tert-butyl
alcohol, sodium ascorbate (0.1 equiv) and copper(II) sulfate (0.05 equiv) were added
sequentially. The reaction was stirred at room temperature for 5 h and TLC analysis indicated
completion of reaction. The solvent was concentrated under vacuum and the resulting mixture
was extracted with dichloromethane (CH2Cl2), dried with Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography by using an ethyl acetate and hexane
solvent system to afford the pure title compounds (4a-x).
4.1.5.1. N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-phenylimidazo[2,1-
b]thiazole-3-carboxamide (4a)
White solid; 69% yield; mp: 189-191 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.17 (s, 1H,
NH), 8.53 (s, 1H, Ar-H of thiazole), 8.03 (s, 1H, Ar-H of imidazole), 7.82 (m, 2H, OH and –
C=CH of triazole), 7.68 (s, 2H, Ar-H), 7.49 - 7.30 (m, 2H, Ar-H), 7.27 (d, J = 6.7 Hz, 1H, Ar-H),
6.82 (d, J = 4.1 Hz, 3H, Ar-H), 5.38 (s, 2H, benzylic CH2), 4.63 (d, J = 4.2 Hz, 2H, CH2 -

ACCEPTED MANUSCRIPT
amide), 3.83 (s, 3H, OMe); 13C NMR (75 MHz, DMSO-d6) δ 157.89 (–CO), 148.02, 147.20,
146.64, 146.07, 144.44, 133.54, 128.22, 126.89, 126.74, 125.74, 124.73, 122.13, 119.51, 116.59,
114.56, 110.82, 110.51, 55.50 (OMe), 53.52 (benzylic CH2), 34.33 (CH2 – amide); MS (ESI):
m/z 461 [M + H]+; HRMS calcd for C23H21N6O3S [M + H]+ 461.1390, found 461.1394.
4.1.5.2.
N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-phenylimidazo[2,1-
b]thiazole-3-carboxamide (4b)
Light yellow solid; 76% yield; mp: 191-193 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.01
(s, 1H, NH), 8.44 (s, 1H, Ar-H of thiazole), 7.81 (d, J = 7.5 Hz, 2H, Ar-H), 7.76 (s, 1H, Ar-H of
imidazole), 7.67 (s, 1H, –C=CH of triazole), 7.37 (t, J = 7.5 Hz, 2H, Ar-H), 7.29 - 7.22 (m, 1H,
Ar-H), 6.88 - 6.82 (m, 2H, Ar-H), 6.80 (s, 1H, Ar-H), 5.40 (s, 2H, benzylic CH2), 4.63 (d, J = 5.2
Hz, 2H, CH2 - amide), 3.84 (s, 3H, OMe), 3.81 (s, 3H, OMe); 13C NMR (75 MHz,
CDCl3+DMSO-d6) δ 158.51(–CO), 149.70, 149.52, 148.49, 147.54, 144.83, 134.04, 128.74,
127.48, 126.41, 126.22, 125.27, 123.14, 121.18, 116.76, 111.50, 110.83, 56.02 (OMe),
54.45(benzylic CH2), 34.69 (CH2 – amide); MS (ESI): m/z 475 [M + H]+; HRMS calcd for
C24H23N6O3S [M + H]+ 475.1547, found 475.1552.
4.1.5.3.
N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-phenylimidazo[2,1-
b]thiazole-3-carboxamide (4c)
White solid; 74% yield; mp: 169-171 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.11 (t, J =
5.2 Hz, 1H, NH), 8.52 (s, 1H, Ar-H of thiazole), 7.97 (s, 1H, Ar-H of imidazole), 7.83 (d, J = 7.5
Hz, 2H, Ar-H), 7.70 (s, 1H, –C=CH of triazole), 7.39 (t, J = 7.5 Hz, 2H, Ar-H), 7.26 (t, J = 7.3
Hz, 1H, Ar-H), 6.41 (s, 3H, Ar-H), 5.43 (s, 2H, benzylic CH2), 4.65 (d, J = 5.2 Hz, 2H, CH2 -
amide), 3.75 (s, 6H, OMe); 13C NMR (100 MHz, CDCl3) δ 161.49 (–CO), 158.54, 148.56,
147.64, 144.89, 136.22, 134.16, 128.78, 127.50, 126.27, 125.34, 123.47, 116.70, 110.88, 106.46,
100.40, 55.55 (OMe), 54.61(benzylic CH2), 34.75 (CH2 – amide); MS (ESI): m/z 475 [M + H]+;
HRMS calcd for C24H23N6O3S [M + H]+ 475.1547, found 475.1553.
4.1.5.4.
6-Phenyl-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[2,1-
b]thiazole-3-carboxamide (4d)
Light yellow solid; 81% yield; mp: 192-194 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.26
(s, 1H, NH), 8.52 (s, 1H, Ar-H of thiazole), 8.06 (s, 1H, Ar-H of imidazole), 7.91 (s, 1H, –C=CH
of triazole), 7.82 (d, J = 7.4 Hz, 2H, Ar-H), 7.37 (t, J = 7.4 Hz, 2H, Ar-H), 7.25 (t, J = 7.2 Hz,

ACCEPTED MANUSCRIPT
1H, Ar-H), 6.60 (s, 2H, Ar-H), 5.46 (s, 2H, benzylic CH2), 4.62 (d, J = 4.5 Hz, 2H, CH2 -
amide), 3.78 (s, 6H, OMe), 3.73 (s, 3H, OMe); 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 157.60
(–CO), 152.85, 147.89, 146.35, 137.32, 133.78, 130.40, 128.18, 126.74, 125.72, 124.47, 122.55,
116.85, 110.32, 105.08, 59.94 (OMe), 55.60 (OMe), 53.36 (benzylic CH2), 34.37 (CH2 – amide);
MS (ESI): m/z 505 [M + H]+; HRMS calcd for C25H25N6O4S [M + H]+ 505.1653, found
505.1656.
4.1.5.5. N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-phenylimidazo[2,1-b]thiazole-
3-carboxamide (4e).
1
Light brown solid; 84% yield; mp: 166-168 °C; H NMR (500 MHz, CDCl3): δ 8.44 (s, 1H, Ar-
H of thiazole), δ 8.14 (s, 1H, NH), 7.83 (d, J = 8.2 Hz, 2H, Ar-H), 7.70 (dd, J = 5.6, 3.3 Hz, 1H,
Ar-H), 7.65 (s, 1H, Ar-H of imidazole), 7.59 (s, 1H, –C=CH of triazole), 7.52 (dd, J = 5.6, 3.3
Hz, 1H, Ar-H), 7.39 (t, J = 7.7 Hz, 2H, Ar-H), 7.35 - 7.30 (m, 3H, Ar-H), 7.11 (t, J = 7.4 Hz, 1H,
Ar-H), 6.99 - 6.96 (m, 3H, Ar-H), 6.92 (s, 1H, Ar-H), 5.48 (s, 2H, benzylic CH2), 4.67 (d, J = 5.7
Hz, 2H, CH2 - amide); 13C NMR (75 MHz, DMSO-d6) δ 166.94 (–CO), 157.63, 156.90, 156.17,
148.19, 146.26, 144.63, 138.21, 134.03, 131.54, 130.39, 130.05, 128.64, 127.13, 125.79, 124.81,
123.66, 123.31, 122.85, 118.76, 118.01, 110.60, 52.35 (benzylic CH2), 34.47 (CH2 – amide); MS
(ESI): m/z 507 [M + H]+; HRMS calcd for C28H23N6O2S [M + H]+ 507.1598, found 507.1602.
4.1.5.6. N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-phenylimidazo[2,1-b]thiazole-3-
carboxamide (4f)
Light yellow solid; 71% yield; mp: 209-211 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.16
(s, 1H, NH), 8.52 (s, 1H, Ar-H of thiazole), 8.00 (s, 1H, Ar-H of imidazole), 7.83 (d, J = 7.3 Hz,
2H, Ar-H), 7.61 (s, 1H, –C=CH of triazole), 7.41 - 7.23 (m, 5H, Ar-H), 7.06 (t, J = 8.6 Hz, 2H,
13
Ar-H), 5.51 (s, 2H, benzylic CH2), 4.64 (d, J = 5.7 Hz, 2H, CH2
̶
amide); C NMR (75 MHz,
CDCl3+DMSO-d6) δ 157.62 (–CO), 147.83, 146.86, 144.69, 133.71, 129.68, 129.57, 128.12,
126.70, 125.69, 124.44, 122.31, 116.71, 115.39, 115.11, 110.31, 52.43 (benzylic CH2), 34.29
(CH2 – amide); MS (ESI): m/z 433 [M + H]+; HRMS calcd for C22H18FN6OS [M + H]+
433.1241, found 433.1245.
4.1.5.7.
N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-
methoxyphenyl)imidazo[2,1-b]thiazole-3-carboxamide (4g)

ACCEPTED MANUSCRIPT
White solid; 68% yield; mp: 191-193 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.11 (t, J =
5.4 Hz, 1H, NH), 8.57 (Brs, 1H, OH), 8.42 (s, 1H, Ar-H of thiazole), 7.97 (s, 1H, Ar-H of
imidazole), 7.75 (d, J = 8.7 Hz, 2H, Ar-H), 7.66 (s, 1H, –C=CH of triazole), 6.93 (d, J = 8.8 Hz,
2H, Ar-H), 6.83-6.79 (m, 3H, Ar-H), 5.38 (s, 2H, benzylic CH2), 4.63 (d, J = 5.6 Hz, 2H, CH2 -
amide), 3.84 (s, 3H, OMe), 3.83 (s, 3H, OMe); 13C NMR (100 MHz, DMSO-d6) δ 158.60 (–CO),
157.65, 147.93, 147.64, 146.57, 146.21, 144.52, 128.45, 126.65, 126.15, 125.81, 122.89, 119.13,
117.59, 115.42, 114.09, 112.18, 109.54, 55.62 (OMe), 55.09 (OMe), 52.56 (benzylic CH2), 34.47
(CH2 – amide); MS (ESI): m/z 491 [M + H]+; HRMS calcd for C24H23N6O4S [M + H]+ 491.1496,
found 491.1504.
4.1.5.8.
N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-
methoxyphenyl)imidazo[2,1-b]thiazole-3-carboxamide (4h)
White solid; 78% yield; mp: 244-246 °C; 1H NMR (500 MHz, CDCl3): δ 8.57 (s, 1H, NH), 8.34
(s, 1H, Ar-H of thiazole), 7.761 (d, J = 8.39, 2H, Ar-H), 7.66-7.62 (m, 2H, Ar-H of imidazole
and –C=CH of triazole), 6.93 (d, J = 8.5 Hz, 2H, Ar-H), 6.87 (q, J = 9.00, J = 17.09, 2H, Ar-H),
6.81 (s, 1H, Ar-H), 5.43 (s, 2H, benzylic CH2), 4.64 (d, J = 5.0 Hz, 2H, CH2
̶ amide), 3.86 (s,
6H, OMe), 3.83 (s, 6H, OMe); 13C NMR (75 MHz, DMSO−d6): δ 158.58 (–CO), 157.65, 148.72,
147.92, 146.29, 144.53, 128.22, 126.71, 126.12, 125.79, 122.90, 120.69, 117.51, 114.08, 112.13,
111.85, 109.47, 55.50 (OMe), 55.08 (OMe), 52.69 (benzylic CH2), 34.48 (CH2 – amide); MS
(ESI): m/z 505 [M + H]+; HRMS calcd for C25H25N6O4S [M + H]+ 505.1653, found 505.1657.
4.1.5.9.
N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-
methoxyphenyl)imidazo[2,1-b]thiazole-3-carboxamide (4i)
1
Light brown solid; 73% yield; mp: 189-191 °C; H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.21
(s, 1H, NH), 8.41 (s, 1H, Ar-H of thiazole), 8.01 (s, 1H, Ar-H of imidazole), 7.82 (s, 1H, –C=CH
of triazole), 7.74 (d, J = 8.6 Hz, 2H, Ar-H), 6.92 (d, J = 8.6 Hz, 2H, Ar-H), 6.43 (s, 2H, Ar-H),
6.39 (s, 1H, Ar-H), 5.45 (s, 2H, benzylic CH2), 4.63 (d, J = 5.5 Hz, 2H, CH2 - amide), 3.82 (s,
3H, OMe), 3.74 (s, 6H, OMe); 13C NMR (75 MHz, DMSO-d6) δ 160.60 (–CO), 158.54, 157.64,
147.91, 146.23, 144.57, 138.13, 126.66, 126.09, 125.75, 123.25, 117.55, 114.04, 109.45, 106.07,
99.45, 55.17 (OMe), 55.04 (OMe), 52.71 (benzylic CH2), 34.42 (CH2 – amide); MS (ESI): m/z
505 [M + H]+; HRMS calcd for C25H25N6O4S [M + H]+ 505.1653, found 505.1652.

ACCEPTED MANUSCRIPT
4.1.5.10.
6-(4-Methoxyphenyl)-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4j)
White solid; 80% yield; mp: 194-196 °C; 1H NMR (400 MHz, CDCl3): δ 8.38 (br, 1H, NH), 8.33
(s, 1H, Ar-H of thiazole), 7.76 (d, J = 8.8 Hz, 2H, Ar-H), 7.67 (s, 1H, Ar-H of imidazole), 7.62
(s, 1H, –C=CH of triazole), 6.93 (d, J = 8.8 Hz, 2H, Ar-H), 6.51 (s, 2H, Ar-H), 5.42 (s, 2H,
benzylic CH2), 4.66 (d, J = 5.7 Hz, 2H, CH2 - amide), 3.83 (s, 6H, OMe), 3.82 (s, 6H, OMe); 13C
NMR (75 MHz, DMSO-d6) δ 158.54 (–CO), 157.63, 152.93, 147.92, 144.56, 137.22, 131.40,
126.65, 126.08, 125.74, 123.10, 117.55, 114.05, 109.42, 105.67, 59.92 (OMe), 55.83 (OMe),
55.04 (OMe), 52.99 (benzylic CH2), 34.44 (CH2 – amide); MS (ESI): m/z 535 [M + H]+; HRMS
calcd for C26H27N6O5S [M + H]+ 535.1758, found 535.1764.
4.1.5.11.
6-(4-Methoxyphenyl)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4k)
White solid; 80% yield; mp: 234-236 °C; 1H NMR (400 MHz, CDCl3): δ 8.72 (t, J = 5.6 Hz, 1H,
NH), 8.34 (s, 1H, Ar-H of thiazole), 7.74 (d, J = 8.8 Hz, 2H, Ar-H), 7.68 (d, J = 5.1 Hz, 2H, Ar-
H of imidazole and –C=CH of triazole), 7.33-7.28 (m, 3H, Ar-H), 7.10 (t, J = 7.4 Hz, 1H, Ar-
H), 7.00 - 6.90 (m, 7H, Ar-H), 5.46 (s, 2H, benzylic CH2), 4.64 (d, J = 5.7 Hz, 2H, CH2 - amide),
3.82 (s, 3H, OMe); 13C NMR (75 MHz, CDCl3): δ 159.11(– CO), 158.54, 158.16, 156.32,
148.29, 147.47, 144.95, 135.93, 130.62, 129.95, 126.87, 126.49, 126.13, 123.94, 123.44, 122.55,
119.28, 118.72, 118.17, 116.30, 114.10, 109.77, 55.31 (OMe), 54.10 (benzylic CH2), 34.59 (CH2
– amide); MS (ESI): m/z 537 [M + H]+; HRMS calcd for C29H25N6O3S [M + H]+ 537.1703,
found 537.1708.
4.1.5.12. N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-methoxyphenyl)imidazo[2,1-
b]thiazole-3-carboxamide (4l)
Light brown solid; 70% yield; mp: 214-216 °C; 1H NMR (500 MHz, CDCl3): δ 8.58 (s, 1H, NH),
8.32 (s, 1H, Ar-H of thiazole), 7.73 (d, J = 8.7 Hz, 2H, Ar-H), 7.67 (s, 1H, Ar-H of imidazole),
7.64 (s, 1H, –C=CH of triazole), 7.29 - 7.24 (m, 3H, Ar-H), 7.05 (t, J = 8.6 Hz, 2H, Ar-H), 6.91
(d, J = 8.8 Hz, 2H, Ar-H), 5.45 (s, 2H, benzylic CH2), 4.63 (d, J = 5.6 Hz, 2H, CH2 - amide),
3.82 (s, 3H, OMe); 13C NMR (125 MHz, CDCl3) δ 164.07 (–CO), 162.09, 159.33, 158.52,
148.34, 148.26, 147.45, 144.91, 130.31, 130.24, 130.08, 126.66, 126.26, 123.08, 116.48, 116.30,
114.27, 109.86, 55.44 (OMe), 53.82 (benzylic CH2), 34.78 (CH2 – amide); MS (ESI): m/z 463
[M + H]+; HRMS calcd for C23H20FN6O2S [M + H]+ 463.1347, found 463.1360.

ACCEPTED MANUSCRIPT
4.1.5.13.
6-(4-Fluorophenyl)-N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4m)
White solid; 71% yield; mp: 204-206 °C; 1H NMR (300 MHz, DMSO-d6): δ 9.26 (t, J = 5.5 Hz,
1H, NH), 8.98 (Brs, 1H, OH), 8.50 (s, 1H, Ar-H of thiazole), 8.07 (s, 1H, Ar-H of imidazole),
7.90 - 7.79 (m, 3H, Ar-H and –C=CH of triazole), 7.11 (t, J = 8.7 Hz, 2H, Ar-H), 6.78 – 6.69
(m, 3H, Ar-H), 5.39 (s, 2H, benzylic CH2), 4.59 (d, J = 5.5 Hz, 2H, CH2 - amide), 3.79 (s, 3H,
OMe); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 162.98 (–CO), 159.72, 157.50, 147.88, 147.48,
146.42, 145.38, 144.32, 130.22, 127.49, 126.30, 126.20, 125.73, 122.20, 118.95, 118.64, 116.90,
115.10, 114.83, 111.40, 110.16, 55.36 (OMe), 52.90 (benzylic CH2), 34.33 (CH2 – amide); MS
(ESI): m/z 479 [M + H]+; HRMS calcd for C23H20FN6O3S [M + H]+ 479.1296, found 479.1297.
4.1.5.14.
N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-
fluorophenyl)imidazo[2,1-b]thiazole-3-carboxamide (4n)
1
White solid; 73% yield; mp: 209-211 °C; H NMR (300 MHz, DMSO-d6): δ 9.10 (s, 1H, NH),
8.39 (s, 1H, Ar-H of thiazole), 7.94 (s, 1H, Ar-H of imidazole), 7.76 - 7.63 (m, 3H, Ar-H and –
C=CH of triazole), 7.00 (t, J = 8.49 Hz, 2H, Ar-H), 6.81 (s, 3H, Ar-H), 5.37 (s, 2H, benzylic
CH2), 4.54 (s, 2H, CH2 - amide), 3.78 – 3.68 (m, 6H, OMe); 13C NMR (75 MHz,
CDCl3+DMSO-d6): δ 163.05 (–CO), 159.79, 159.63, 157.63, 148.65, 147.88, 145.53, 144.41,
130.03, 126.82, 126.20, 126.09, 125.69, 122.04, 120.41, 116.59, 115.05, 114.77, 111.04, 110.93,
110.06, 55.37 (OMe), 53.24 (benzylic CH2), 34.31 (CH2 – amide); MS (ESI): m/z 493 [M + H]+;
HRMS calcd for C24H22FN6O3S [M + H]+ 493.1453, found 493.1458.
4.1.5.15.
N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-
fluorophenyl)imidazo[2,1-b]thiazole-3-carboxamide (4o)
White solid; 71% yield; mp: 233-235 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.25 (t, J =
5.4 Hz, 1H, NH), 8.49 (s, 1H, Ar-H of thiazole), 8.07 (s, 1H, Ar-H of imidazole), 7.90 (s, 1H, –
C=CH of triazole), 7.82 (dd, J = 8.6, 5.5 Hz, 2H, Ar-H), 7.10 (t, J = 8.7 Hz, 2H, Ar-H), 6.44 (d, J
= 2.0 Hz, 2H, Ar-H), 6.39 (d, J = 2.1 Hz, 1H, Ar-H), 5.46 (s, 2H, benzylic CH2), 4.61 (d, J = 5.5
Hz, 2H, CH2 - amide), 3.74 (s, 6H, OMe); 13C NMR (75 MHz, DMSO-d6) δ 163.05, 160.64,
157.60 (– CO), 148.22, 145.33, 144.57, 138.14, 130.58, 126.80, 126.69, 125.80, 123.26, 118.02,
115.61, 115.33, 110.51, 106.11, 99.50, 55.20 (OMe), 52.76 (benzylic CH2), 34.47 (CH2 –
amide); MS (ESI): m/z 493 [M + H]+; HRMS calcd for C24H22FN6O3S [M + H]+ 493.1453,
found 493.1453.

ACCEPTED MANUSCRIPT
4.1.5.16.
6-(4-Fluorophenyl)-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4p)
White solid; 82% yield; mp: 225-227 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.24 (s, 1H,
NH), 8.48 (s, 1H, Ar-H of thiazole), 8.05 (s, 1H, Ar-H of imidazole), 7.85 - 7.79 (m, 3H, Ar-H
and –C=CH of triazole), 7.09 (t, J = 8.5 Hz, 2H, Ar-H), 6.60 (s, 2H, Ar-H), 5.46 (s, 2H, benzylic
13
CH2), 4.62 (d, J = 5.1 Hz, 2H, CH2 - amide), 3.79 (s, 6H, OMe), 3.74 (s, 3H, OMe); C NMR
(75 MHz, CDCl3+DMSO-d6): δ 163.88 (–CO), 162.98, 159.74, 157.56, 152.83, 147.86, 145.44,
144.39, 137.31, 130.25, 126.21, 126.10, 125.69, 122.40, 116.74, 115.07, 114.79, 104.99, 59.94,
55.57 (OMe), 53.39 (benzylic CH2), 34.32 (CH2 – amide); MS (ESI): m/z 523 [M + H]+; HRMS
calcd for C25H24FN6O4S [M + H]+ 523.1558, found 523.1563.
4.1.5.17. 6-(4-Fluorophenyl)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[2,1-
b]thiazole-3-carboxamide (4q)
1
White solid; 81% yield; mp: 180-182 °C; H NMR (400 MHz, CDCl3): δ 8.38 (s, 1H, Ar-H of
thiazole), 8.28 (t, J = 5.6 Hz, 1H, NH), 7.84 - 7.75 (m, 2H, Ar-H), 7.67 (s, 1H, Ar-H of
imidazole), 7.62 (s, 1H, –C=CH of triazole), 7.33 (td, J = 5.3, 2.0 Hz, 3H, Ar-H), 7.14 - 7.04 (m,
3H, Ar-H), 7.01 - 6.96 (m, 3H, Ar-H), 6.94 (dd, J = 9.9, 1.6 Hz, 2H, Ar-H), 5.49 (s, 2H, benzylic
13
CH2), 4.67 (d, J = 5.8 Hz, 2H, CH2 - amide); C NMR (75 MHz, CDCl3+DMSO-d6): δ 157.67
(–CO), 157.14, 155.87, 147.87, 145.58, 144.55, 136.30, 130.04, 129.83, 129.31, 126.19, 126.08,
125.68, 123.15, 122.30, 122.10, 118.41, 117.89, 117.61, 116.55, 115.03, 114.74, 110.06, 52.98
(benzylic CH2), 34.28 (CH2 – amide); MS (ESI): m/z 525 [M + H]+; HRMS calcd for
C28H22FN6O2S [M + H]+ 525.1503, found 525.1508.
4.1.5.18.
N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6-(4-fluorophenyl)imidazo[2,1-
b]thiazole-3-carboxamide (4r)
White solid; 72% yield; mp: 203-205 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.23 (s, 1H,
NH), 8.48 (s, 1H, Ar-H of thiazole), 8.05 (s, 1H, Ar-H of imidazole), 7.91 - 7.75 (m, 4H, Ar-H
and – C=CH of triazole), 7.41 - 7.29 (m, 2H, Ar-H), 7.09 (q, J = 7.9, 16.2 Hz, 4H, Ar-H), 5.53
(s, 2H, benzylic CH2), 4.61 (d, J = 5.0 Hz, 2H, CH2 - amide); 13C NMR (75 MHz, DMSO-d6) δ
163.49 (–CO), 163.05, 159.82, 157.60, 148.23, 145.34, 144.63, 132.31, 130.58, 130.37, 130.27,
126.80, 125.79, 123.12, 118.03, 115.68, 115.40, 110.51, 51.97 (benzylic CH2), 34.46 (CH2 –
amide); MS (ESI): m/z 451 [M + H]+; HRMS calcd for C22H17F2N6OS [M + H]+ 451.1147,
found 451.1153.

ACCEPTED MANUSCRIPT
4.1.5.19.
6-(4-Chlorophenyl)-N-((1-(3-hydroxy-4-methoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4s)
White solid; 69% yield; mp: 185-187 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.23 (s, 1H,
NH), 8.90 (Brs, 1H, OH), 8.54 (s, 1H, Ar-H of thiazole), 8.07 (s, 1H, Ar-H of imidazole), 7.85 -
7.76 (m, 3H, Ar-H and –C=CH of triazole), 7.35 (d, J = 8.2 Hz, 2H, Ar-H), 6.89 - 6.71 (m, 3H,
Ar-H), 5.38 (s, 2H, benzylic CH2), 4.60 (d, J = 4.8 Hz, 2H, CH2 - amide), 3.80 (s, 3H, OMe); 13C
NMR (75 MHz, DMSO-d6): δ 157.49 (–CO), 148.87, 148.02, 147.48, 146.40, 145.13, 132.55,
131.77, 128.21, 127.38, 125.91, 125.70, 122.18, 118.98, 117.12, 115.07, 111.33, 110.70, 55.36
(OMe), 52.96 (benzylic CH2), 34.34 (CH2 – amide); MS (ESI): m/z 495 [M + H]+; HRMS calcd
for C23H20ClN6O3S [M + H]+ 495.1001, found 495.1002.
4.1.5.20.
6-(4-Chlorophenyl)-N-((1-(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4t)
White solid; 76% yield; mp: 164-166 °C; 1H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H, NH), 8.41
(s, 1H, Ar-H of thiazole), 7.76 (s, 1H, Ar-H of imidazole), 7.73 (s, 2H, Ar-H), 7.67 (s, 1H, –
C=CH of triazole), 7.34 (d, J = 8.5 Hz, 2H, Ar-H), 6.89 - 6.80 (m, 3H, Ar-H), 5.44 (s, 2H,
benzylic CH2), 4.64 (d, J = 5.6 Hz, 2H, CH2 - amide), 3.87 (s, 3H, OMe), 3.84 (s, 3H, OMe); 13C
NMR (125 MHz, CDCl3): δ 158.41 (–CO), 149.85, 149.64, 148.63, 146.59, 144.74, 133.15,
132.64, 128.95, 126.57, 126.36, 126.25, 123.12, 121.27, 116.81, 111.56, 110.98, 56.16 (OMe),
56.10 (OMe), 54.60 (benzylic CH2), 34.74 (CH2 – amide); MS (ESI): m/z 509 [M + H]+; HRMS
calcd for C24H22ClN6O3S [M + H]+ 509.1157, found 509.1156.
4.1.5.21.
6-(4-Chlorophenyl)-N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo[2,1-b]thiazole-3-carboxamide (4u)
White solid; 73% yield; mp: 211-213 °C; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.25 (s, 1H,
NH), 8.53 (s, 1H, Ar-H of thiazole), 8.07 (s, 1H, Ar-H of imidazole), 7.89 (s, 1H, –C=CH of
triazole), 7.80 (d, J = 8.4 Hz, 2H, Ar-H), 7.36 (d, J = 8.4 Hz, 2H, Ar-H), 6.44 (d, J = 1.8 Hz, 2H,
Ar-H), 6.39 (d, J = 1.9 Hz, 1H, Ar-H), 5.46 (s, 2H, benzylic CH2), 4.62 (d, J = 5.4 Hz, 2H, CH2 -
amide), 3.73 (s, 6H, OMe); 13C NMR (75 MHz, DMSO-d6): δ 160.53 (–CO), 157.52, 148.00,
145.13, 144.34, 136.94, 132.51, 131.79, 128.18, 125.85, 125.69, 122.57, 117.03, 110.64, 105.64,
99.36, 54.81 (OMe), 53.15 (benzylic CH2), 34.33 (CH2 – amide); MS (ESI): m/z 509 [M + H]+;
HRMS calcd for C24H22ClN6O3S [M + H]+ 509.1157, found 509.1162.

ACCEPTED MANUSCRIPT
4.1.5.22.
6-(4-Chlorophenyl)-N-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)imidazo [2,1-b]thiazole-3-carboxamide (4v)
1
White solid; 83% yield; mp: 212-214 °C; H NMR (500 MHz, CDCl3): δ 8.41 (s, 1H, Ar-H of
thiazole), 8.29 (s, 1H, NH), 7.76 (d, J = 8.4 Hz, 2H, Ar-H), 7.67 (s, 1H, Ar-H of imidazole), 7.64
(s, 1H, –C=CH of triazole), 7.36 (d, J = 8.4 Hz, 2H, Ar-H), 6.52 (s, 2H,Ar-H), 5.43 (s, 2H,
benzylic CH2), 4.68 (d, J = 5.5 Hz, 2H, CH2 - amide), 3.84 (s, 3H, OMe), 3.83 (s, 6H, OMe); 13C
NMR (75 MHz, CDCl3+DMSO-d6): δ 160.57 (–CO), 157.62, 148.01, 145.29, 144.45, 136.54,
132.40, 131.92, 128.15, 125.79, 122.33, 116.80, 110.58, 105.59, 99.46, 54.80 (OMe), 53.35
(benzylic CH2), 34.32 (CH2 – amide); MS (ESI): m/z 539 [M + H]+; HRMS calcd for
C25H24ClN6O4S [M + H]+ 539.1263, found 539.1263.
4.1.5.23. 6-(4-Chlorophenyl)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[2,1-
b]thiazole-3-carboxamide (4w)
White solid; 85% yield; mp: 181-183 °C; 1H NMR (500 MHz, CDCl3): δ 8.75 (s, 1H, NH), 8.42
(s, 1H, Ar-H of thiazole), 7.73 (dd, J = 16.2, 8.2 Hz, 4H, Ar-H and Ar-H of imidazole), 7.37 -
7.29 (m, 5H, Ar-H and –C=CH of triazole ), 7.11 (t, J = 7.4 Hz, 1H, Ar-H), 6.9 - 6.92 (m, 5H,
Ar-H), 5.48 (s, 2H, benzylic CH2), 4.65 (d, J = 5.5 Hz, 2H, CH2 - amide); 13C NMR (100 MHz,
CDCl3): δ 158.46 (–CO), 158.34, 156.43, 148.69, 146.69, 144.88, 135.99, 133.10, 132.77,
130.76, 130.08, 128.94, 126.58, 126.23, 124.10, 123.41, 122.66, 119.41, 118.89, 118.30, 116.77,
110.99, 54.30 (benzylic CH2), 34.75 (CH2 – amide); MS (ESI): m/z 541 [M + H]+; HRMS calcd
for C28H22ClN6O2S [M + H]+541.1208, found 541.1211.
4.1.5.24. 6-(4-Chlorophenyl)-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[2,1-
b]thiazole-3-carboxamide (4x)
1
Light brown solid; 78% yield; mp: 219-221 °C; H NMR (300 MHz, CDCl3+DMSO-d6): δ 9.29
(s, 1H, NH), 8.54 (s, 1H, Ar-H of thiazole), 8.09 (s, 1H, Ar-H of imidazole), 7.98 (s, 1H, –
C=CH of triazole), 7.82 (d, J = 8.0 Hz, 2H, Ar-H), 7.36 (d, J = 6.8 Hz, 4H,Ar-H), 7.09 (t, J = 8.3
Hz, 2H, Ar-H), 5.54 (s, 2H, benzylic CH2), 4.59 (d, J = 4.6 Hz, 2H, CH2 - amide); 13C NMR (75
MHz, DMSO-d6): δ 163.57 (–CO), 160.31, 157.54, 148.13, 145.15, 144.55, 132.74, 131.73,
131.48, 130.04, 129.93, 128.34, 126.10, 125.79, 122.78, 117.45, 115.48, 115.19, 110.82, 52.25
(benzylic CH2), 34.41 (CH2 – amide); MS (ESI): m/z 467 [M + H]+; HRMS calcd for
C22H17ClFN6OS [M + H]+467.0852, found 467.0859.

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4.2. Biology
4.2.1. Anticancer activity
The cytotoxic activity of the compounds was determined using MTT assay [42-44]. Cells
were seeded in 100 µl DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 °C in a CO2 incubator. After incubation cells
were treated with test compounds for 48 h. After 48 h of incubation, 10 µl MTT (3-(4,5-
dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to each well and
the plates were further incubated for 4 h. Then the supernatant from each well was carefully
removed, formazon crystals were dissolved in 100 µl of DMSO and absorbance at 570 nm
wavelength was recorded.
4.2.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
through the cell-cycle phases. A549 cells were incubated for 48 h with compounds 4g and 4h at
concentrations of 0.5 and 1 µM. Untreated and treated cells were harvested, washed with
phosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol, and stained with propidium
iodide (Sigma–Aldrich). Cell-cycle analysis was performed by flow cytometry (Becton
Dickinson FACS Caliber instrument) [45].
4.2.3. Tubulin polymerization assay
A fluorescence based in vitro tubulin polymerization assay was performed according to
the manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total
volume of 10 µl contained PEM buffer, GTP (1 µM) in the presence or absence of test
compounds. Tubulin polymerization was followed by a time dependent increase in fluorescence
due to the incorporation of a fluorescence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured by using a
Varioscan multimode plate reader (Thermo scientific Inc.). Nocodazole was used as reference
compound in this study. To determine the IC50 values of the compounds against tubulin
polymerization, the compounds were pre-incubated with tubulin at varying concentrations.
Assays were performed under similar conditions as employed for polymerization assays as
described above [34, 35].
4.2.4. Immunohistochemistry

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MCF-7 cells were seeded on glass cover slips and incubated for 48 h in the presence or
absence of the test compounds 4g and 4h (0.5 µM). After treatment, the cover slips were fixed
with a paraformaldehyde solution (4% in 1×PBS) for 20 min at room temperature. Cell
permeabilizationwas achieved by administration of a Triton X-100 solution (0.2% in 1×PBS) for
5 min. The cover slips were left in 100% MeOH overnight at 4 °C. Subsequently, the cover slips
were blocked with a 1% bovine serum albumin (BSA) solution for 60 min and then incubated
with α-tubulin antibody (1:1000) at room temperature for 2 h. The slides were washed three
times for 5 min each with PBST. Next, the cover slips were incubated with FITC-conjugated
anti-mouse secondary antibody (Cell signaling technology) for 1 h and then washed three times
with PBST solution. Finally, the cells were observed under a fluorescence microscope
(Olympus), and the pictures were analyzed for the integrity of the microtubule network [35].
4.2.5. Morphological analysis for apoptosis with Hoechst staining
A549 cells were seeded at a density of 10,000 cells over 18- mm cover slips and
incubated for 24 h. The medium was replaced, and cells were treated with compounds, 4g and 4h
at 0.5 µM concentration for 48 h. Cells treated with vehicle (0.001% DMSO) were included as
controls for all experiments. After treatment, Hoechst 33258 (Sigma Aldrich) was added to
medium at a concentration of 0.5 mg/Ml. After incubation for 30 min, cells from each dish were
captured from randomly selected fields under fluorescent microscope (Olympus) to qualitatively
determine the proportion of viable and apoptotic cells based on their relative fluorescence and
nuclear fragmentation.
4.2.6. Mitochondrial membrane potential
A549 cells were cultured in six-well plates after treatment with compounds 4g and 4h at
0.5 and 1 µM concentrations for 48 h. After 48 h of treatment, cells were collected by
trypsinization and washed with PBS followed by resuspending in JC-1 (5,5,6,6-tetrachloro-
1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide-5µg/mL) and incubated at 37 0C for 15 min.
Cells were rinsed three times with medium and suspended in pre warmed medium. The cells
were then subjected to flow cytometric analysis on a flow cytometer (Becton Dickinson) in the
FL1, FL2 channel to detect mitochondrial potential [46].
4.2.7. Annexin staining assay for apoptosis
A549 cells were seeded in six-well plates and allowed to grow overnight. The medium
was then replaced with complete medium containing compounds 4g and 4h at 0.5 and 1 µM

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concentrations. After 48 h of drug treatment, cells from the supernatant and adherent monolayer
cells were harvested by trypsinization, washed with PBS at 5000 rpm. Then the cells were
stained with Annexin VFITC and propidium iodide using the Annexin-V-FITC apoptosis
detection kit (Sigma aldrich). Flow cytometry was performed for this study as described earlier
[47].
4.2.8. Western blot analysis
After treatment with test compounds 4g and 4h at 0.5 µM concentration for 48 h. Protein
was isolated with RIPA (radioimmunoprecipitation assay) buffer. Protein (50 µg per lane) was
applied in 10 % SDS-PAGE (Sodium dodecyl sulfate Polyacrylamide gel electrophoresis). After
electrophoresis, the protein was transferred to a polyvinylidine difluoride (PVDF) membrane
(Thermo Scientific Inc.) and blocked with BSA (bovine serum albumin). The membrane was
washed with TBST for 5 min, then primary antibody was added. After 24 h, the membrane was
incubated with the corresponding horseradish peroxidase labeled secondary antibody and
incubated for another 1 h. Membranes were washed with TBST three times for 15 min and the
protein blots were visualized with chemiluminescence reagent (Thermo Scientific Inc.). Images
were captured by using the chemiluminescence (vilber lourmat) [48].
4.3. Molecular Modeling
AutoDock4 was employed to dock lead pyrazole linked benzimidazoles in colchicine
binding site of tubulin [49,50]. Initial Cartesian coordinates for the protein-ligand complex
structure were derived from crystal structure of tubulin (PDB ID: 3E22). The protein targets
were prepared for molecular docking simulation by removing water molecules, bound ligands.
Hydrogen atoms and Kollman charges were added to each protein atom. Auto-Dock Tools-1.5.6
(ADT) was used to prepare and analyze the docking simulations for the AutoDock4. Coordinates
of each compound were generated using Chemdraw11.0 followed by MM2 energy minimization.
The interaction of protein and ligands in binding pocket for Autodock4 was defined by a Grid
box. The grid box was created with 60 points equally in each direction of x, y, and z. AutoGrid4
was used to produce grid maps for AutoDock4 calculations where the search space size utilized
grid points of 0.375 Å. The Lamarckian genetic algorithm was opted to search for the best
conformers. Each docking experiment was performed 100 times, yielding 100 docked
conformations. Parameters used for the docking were as follows: population size of 150; random
starting position and conformation; maximal mutation of 2 Å in translation and 50 degrees in

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rotations; elitism of 1; mutation rate of 0.02 and crossover rate of 0.8; and local search rate of
0.06. Simulations were performed with a maximum of 1.5 million energy evaluations and a
maximum of 50000 generations. Final docked conformations were clustered using a tolerance of
1.0 Å root mean square deviation. The best model was picked based on the best stabilization
energy. Final figures for molecular modeling were visualized by using PyMol [51].
Acknowledgements
S. P. S., F. S., A. V. S., A. B. S and K. S. B thanks CSIR-UGC, New Delhi for the award
of senior research fellowship. We thank CSIR for financial support under the 12th Five Year
plan project “Affordable Cancer Therapeutics” (CSC0301). This project was also supported by
King Saud University, Deanship of Scientific Research, Research Chair.
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