Pyrazolo-benzothiazole hybrids: Synthesis, anticancer properties and evaluation of antiangiogenic activity using in vitro VEGFR-2 kinase and in vivo transgenic zebrafish model

Article abstract of DOI:10.1016/j.ejmech.2019.111609

A series of new pyrazolo-benzothiazole hybrids (7–26) were synthesised and screened for their cytotoxic activity towards several cancer cell lines [colon (HT-29), prostate (PC-3), lung (A549), glioblastoma (U87MG)] and normal human embryonic kidney cell line (Hek-293T). Compounds 8, 9, 13, 14, 18, 19, 23, and 24 displayed significant activity, with compound 14 being particularly potent towards all the tested cancer cell lines with IC₅₀ values in the range 3.17–6.77 μM, even better than reference drug axitinib (4.88–21.7 μM). Compound 14 also showed the strongest growth inhibition in 3D multicellular spheroids of PC-3 and U87MG cells. The mechanism of cellular toxicity in PC-3 cells was found to be cell cycle arrest and apoptosis induction through depolarisation of mitochondrial membrane potential, increased ROS production and subsequent DNA damage. Further, compound 14 displayed significant in vitro (VEGFR-2 inhibition) and in vivo [transgenic zebrafish Tg(flila:EGFP) model] antiangiogenic properties. Overall, these results provide strong evidence that compound 14 could be considered for a lead candidate in anticancer and antiangiogenic drug discovery.

Full text of DOI:10.1016/j.ejmech.2019.111609

Journal Pre-proof
Pyrazolo-benzothiazole hybrids: Synthesis, anticancer properties and evaluation of
antiangiogenic activity using in vitro VEGFR-2 kinase and in vivo transgenic zebrafish
model
Velma Ganga Reddy, T. Srinivasa Reddy, Chetna Jadala, M. Soumya Reddy, Faria
Sultana, Ravikumar Akunuri, Suresh K. Bhargava, Donald Wlodkowic, P. Srihari,
Ahmed Kamal
PII:
S0223-5234(19)30743-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111609
EJMECH 111609
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2019
Revised Date: 6 August 2019
Accepted Date: 8 August 2019
Please cite this article as: V.G. Reddy, T.S. Reddy, C. Jadala, M.S. Reddy, F. Sultana, R. Akunuri, S.K.
Bhargava, D. Wlodkowic, P. Srihari, A. Kamal, Pyrazolo-benzothiazole hybrids: Synthesis, anticancer
properties and evaluation of antiangiogenic activity using in vitro VEGFR-2 kinase and in vivo transgenic
zebrafish model, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111609.
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Graphical abstract
Pyrazolo-benzothiazole hybrids: Synthesis, anticancer properties
and evaluation of antiangiogenic activity using in vitro VEGFR-2
kinase and in vivo transgenic zebrafish model
a,b,d
d
c
f
Velma Ganga Reddy,
T. Srinivasa Reddy,* Chetna Jadala, M. Soumya Reddy, Faria
b
c
d
e
a,b
Sultana, Ravikumar Akunuri, Suresh K. Bhargava, Donald Wlodkowic, P. Srihari,**
a,b,c,g
Ahmed Kamal***
a
Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource
Development Centre (CSIR-HRDC) Campus, Ghaziabad 201 002, Uttar Pradesh, India
b
Department of Organic Synthesis & Process Chemistry, CSIR-Indian Institute of Chemical
Technology (IICT), Hyderabad 500 007, India
c
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
d
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science,
RMIT University, GPO Box 2476, Melbourne 3001, Australia
e
Phenomics Laboratory, School of Science, RMIT University, Plenty Road, PO Box 71,
Bundoora, Victoria 3083, Australia
f
Department of Chemistry, Keshav Memorial Institute of Commerce and Sciences,
Narayanguda, Hyderabad 500 029, India
g
School of Pharmaceutical Education and Research (SPER), Jamia Hamdard,
New Delhi 110 062, India
Corresponding authors: e-mail addresses:
*
srinivasareddy.telukutla@rmit.edu.au (Dr. T. Srinivasa Reddy),
*
*
*srihari@iict.res.in (Dr. P. Srihari),
**ahmedkamal@iict.res.in; ahmedkamal915@gmail.com (Dr. Ahmed Kamal).

Pyrazolo-benzothiazole hybrids: Synthesis, anticancer properties
and evaluation of antiangiogenic activity using in vitro VEGFR-2
kinase and in vivo transgenic zebrafish model
a,b,d
d
c
f
Velma Ganga Reddy,
T. Srinivasa Reddy,* Chetna Jadala, M. Soumya Reddy, Faria
b
c
d
e
a,b
Sultana, Ravikumar Akunuri, Suresh K. Bhargava, Donald Wlodkowic, P. Srihari,**
a,b,c,g
Ahmed Kamal***
a
Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource
Development Centre (CSIR-HRDC) Campus, Ghaziabad 201 002, Uttar Pradesh, India
b
Department of Organic Synthesis & Process Chemistry, CSIR-Indian Institute of Chemical
Technology (IICT), Hyderabad 500 007, India
c
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
d
Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science,
RMIT University, GPO Box 2476, Melbourne 3001, Australia
e
Phenomics Laboratory, School of Science, RMIT University, Plenty Road, PO Box 71,
Bundoora, Victoria 3083, Australia
f
Department of Chemistry, Keshav Memorial Institute of Commerce and Sciences,
Narayanguda, Hyderabad 500 029, India
g
School of Pharmaceutical Education and Research (SPER), Jamia Hamdard,
New Delhi 110 062, India
Corresponding authors: e-mail addresses:
*
srinivasareddy.telukutla@rmit.edu.au (Dr. T. Srinivasa Reddy),
*
*
*srihari@iict.res.in (Dr. P. Srihari),
**ahmedkamal@iict.res.in; ahmedkamal915@gmail.com (Dr. Ahmed Kamal).

Abstract
A series of new pyrazolo-benzothiazole hybrids (7-26) were synthesised and screened
for their cytotoxic activity towards several cancer cell lines [colon (HT-29), prostate (PC-3),
lung (A549), glioblastoma (U87MG)] and normal human embryonic kidney cell line (Hek-
2
93T). Compounds 8, 9, 13, 14, 18, 19, 23, and 24 displayed significant activity, with
compound 14 being particularly potent towards all the tested cancer cell lines with IC50
values in the range 3.17-6.77 µM, even better than reference drug axitinib (4.88-21.7 µM).
Compound 14 also showed the strongest growth inhibition in 3D multicellular spheroids of
PC-3 and U87MG cells. The mechanism of cellular toxicity in PC-3 cells was found to be
cell cycle arrest and apoptosis induction through depolarisation of mitochondrial membrane
potential, increased ROS production and subsequent DNA damage. Further, compound 14
displayed significant in vitro (VEGFR-2 inhibition) and in vivo [transgenic zebrafish
Tg(flila:EGFP) model] antiangiogenic properties. Overall, these results provide strong
evidence that compound 14 could be considered for a lead candidate in anticancer and
antiangiogenic drug discovery.
Keywords: pyrazole; benzothiazole; anticancer; VEGFR-2; angiogenesis; apoptosis;
zebrafish
1
. Introduction
Of the diverse types of treatments available to treat cancer, chemotherapy is the most
common approach [1] which involves the use of chemotherapeutics to kill cancer cells or
prevent them from growing by various mechanisms [2]. However, the synthesis and
identification of novel, effective and safe chemotherapeutic agents for the treatment of cancer
remains a challenge to medicinal chemists and in pharmaceutical research due to limitations
such as poor solubility and bioavailability, toxicity to normal cells, and the development of

drug resistance towards existing chemotherapeutic drugs [3-5]. Hence, it is essential to
develop potent and effective new chemical entities to overcome these limitations [6].
Angiogenesis plays a pivotal role in new blood vessel formation from pre-existing
vessels, leading to the growth of tumours [7]. Many cancer tissues can produce vascular
endothelial growth factor (VEGF) to initiate angiogenesis from adjacent blood vessels [8].
During the angiogenesis process, VEGF secreted from the solid tumors activates VEGFR-2
on endothelial cells to form the blood vessel network that enhances tumour proliferation and
metastasis [9]. Therefore, inhibition of the VEGFR-2/VEGF signalling pathway is considered
one of the most important and valuable approaches in the development of cancer
chemotherapeutics. Currently, many of the FDA (Food and Drug Administration) approved
VEGFR-2 inhibitors, namely sorafenib, sunitinib, axitinib, vatalanib and regorafenib, are
used in clinical cancer therapy as chemotherapeutic drugs [10-11], however, drug resistance
leads to decreased effectiveness and increased toxicity causing unwanted side effects.
Therefore, it is essential to discover novel VEGFR-2 inhibitors for the treatment of cancer
with low toxicity and to overcome drug resistance [12].
In view of this, nitrogen containing heterocyclic frameworks are a common building
block in nature and often exhibit interesting biological activities in humans [13-14]. Among
them, pyrazoles represent a core structure of numerous biologically active compounds which
display a broad range of biological properties, such as anti-fungal, anti-HIV, anti-
inflammatory, anti-microbial and anti-tuberculosis activities [15-20]. In recent years, several
pyrazole derivatives such as crizotinib (I), axitinib (II) and ibrutinib (III), have found use as
chemotherapeutic drugs [21] (Fig. 1). Many pyrazole congeners possess potent anti-cancer
and anti-angiogenesis activities by targeting numerous receptors, including vascular
endothelial growth factor (VEGF), fibroblast growth factor (FGF), tumour growth factor
(TGF), and various kinases that are essential for the progression of cancer (IV, V) [22-23].

On the other hand, heterocyclic rings containing nitrogen and sulfur atoms denote an
exclusive and universal scaffold for the design of chemotherapeutic agents (VI-IX) [24-26].
Benzothiazoles are a class of sulfur containing heterocyclic ring compounds which show
unique properties that are not possessed by other heterocyclic compounds [27]. In this
context, 2-aminobenzothiazoles represent an important heterocyclic family that have been
reported to have a wide range of pharmaceutical applications, including antimicrobial,
antifungal, anti-inflammatory, chronic pain suppression and significant anticancer properties
[
28-31].
<
Figure 1>
Considering the above research findings and efforts by our research group to find
potent pyrazole based cytotoxic molecules (X-XIII) [32], herein we report the synthesis of
new pyrazolo-benzothiazole hybrid molecules (7-26) and their evaluation of biological
activities towards different cancer cell lines.
2
2
. Results and Discussions
.1. Chemistry
The synthetic procedure for the N-(benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-
carboxamide hybrids (7-26) is depicted in Scheme 1. The pyrazole carboxylic acid precursors
5a–d) were prepared from the reaction of (un)substituted acetophenones with phenyl
(
hydrazine followed by cyclocondensation and oxidation. Compounds 5a-d were then coupled
with the appropriate amines (6a-e) in the presence of N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropyl-ethylamine (DIPEA) and 1-
hydroxybenzotriazole (HOBt) in dry DMF under a nitrogen atmosphere to produce the
desired pyrazolo-benzothiazole hybrids (7-26) in yields of 75-90%. The compounds were

1
13
purified by column chromatography and characterised by H, C spectroscopy NMR and
ESI-MS.
<
Scheme 1>
2
.2. Cytotoxicity
Evaluation of the growth-inhibitory properties of the pyrazolo-benzothiazole
compounds was carried out in four human cancer cell lines, HT-29 (colon), PC-3 (prostate),
A549 (lung), and U87MG (brain), using the MTT assay [33] following 72 h of drug exposure
and the results are presented in Table 1 as IC50 values (µM). The results revealed that
compounds 8, 9, 12, 13, 14, 18, 19, 23 and 24 displayed promising growth inhibition
properties towards all the tested cancer cells with IC values ranging from 3.17 to 10.7 µM.
5
0
Interestingly, hybrid 14 showed the most potent activity among the series, with IC values of
5
0
3
.17 µM (PC-3), 3.32 µM (HT-29), 3.87 µM (A549) and 6.77 µM (U87MG), and was more
active than axitinib, a clinically employed drug. The pyrazolo-benzothiazole derivatives were
also evaluated for their cytotoxicity against a normal cell line (Hek-293T) to investigate the
selectivity towards cancer cells. Most of the synthesised derivatives were non-toxic to normal
cells except 8, 13, 19, 23 and 24 which displayed moderate toxicity to Hek-293T cells.
Moreover, the potent compound 14 was 9-15-fold more selective to cancer cells compared to
axitinib, which is only 2-3 times more selective, thereby demonstrating the high selectivity of
this compound towards cancer cells.
<
Table 1>
From the MTT results, the structural activity relationships were investigated.
Compounds bearing electron withdrawing groups such as fluoro and chloro on the phenyl
ring B (8, 9, 13, 14, 18, 19, 23, and 24) displayed prominent growth inhibition properties on

the tested cancer cell lines whereas electron donating substituents (methoxy and methyl) lead
to a decrease or loss of cytotoxic activity (compounds 10, 11, 15, 16, 20, 21, 25 and 26).
Similarly, compounds with electron withdrawing groups on ring phenyl ring A (13, 14, 18
and 19) showed excellent growth inhibition towards various cancer cell lines while the
presence of methoxy groups significantly decreased the cytotoxic activity (23 and 24). In
detail, compound 8 displayed cytotoxic activities towards the human cancer cell lines, namely
HT-29 (colon), PC-3 (prostate), A549 (lung) and U87MG (brain), with IC values in the
5
0
range 3.25-10.2 µM; 9 with 3.69-7.81 µM; 13 with 4.16-12.5 µM; 14 with 3.32-6.77; 18 with
.97-8.86; 19 with 3.23-5.87; 23 with 4.93-8.99 and compound 24 with 4.71-8.71 µM. These
4
activities were more significant than the postive control axitinib (4.87-21.7 µM). However,
the compounds with no substituents, an electron donating methoxy group on ring A or
electron donating substituents (methyl and methoxy) on ring B (10, 11, 25 and 26) did not
shown any notable effects towards the tested cancer cell lines. Additionally, it is worth noting
that compound 7 in which both rings A and B are unsubstituted showed moderate activity
with IC values in the range 8.96-16.8 µM. From these results it was clear that the compound
5
0
1
4 containing electron withdrawing groups on ring A (fluoro) and B (chloro) showed the
highest toxicity towards cancer cells with minimal cytotoxicity towards non-cancerous (Hek-
2
93T) cells.
2
.3. Colony forming assay
Inspired by the potent toxicity towards PC-3 cells, compound 14 was selected to
further analyse the antitumor potential in PC-3 cells. The proliferation ability of a single
cancer cell to grow into a colony can be assessed using the colony formation assay [34]. In
this assay, PC-3 cells were grown in media containing different concentrations (1, 3 and 5
µM) of compound 14 over a period of 7 days to form colonies. The cells were then stained
with crystal violet to visualise colonies. The CFU (colony-forming unit) assay results are

presented in Fig. 2. The colony forming potential of PC-3 was inhibited by treatment with 14
at all tested concentrations, thereby indicating its cytotoxic activity.
<
Figure 2>
2
.4. 3D Multicellular spheroids (MSCs) inhibition assay
D Multicellular spheroids of cancer cells have similar features to in vivo tumor
3
environments with respect to cell permeability, nutrient and oxygen consumption, phenotypic
heterogeneity, micro metastases and genetic expression profiles [35]. In this regard, we next
investigated the effect of compound 14 on 3D multicellular spheroids of PC-3 and U87MG
cell growth inhibition. Spheroids were grown for three days and were incubated with
different concentrations of compound 14. After 72 h treatment, images of the spheroids were
captured to observe the morphological changes induced by the compound. The results
showed that compound 14 effectively penetrated the multiple layers of both PC-3 and
U87MG spheroids, as the outer-layer cells detached from the spheroids, possibly due to
reduced cell-cell adhesion as a result of compound exposure (Fig. 3).
<
Figure 3>
2
.5. Wound healing assay (Migration assay)
Migration and invasion of cancer cells are major incidences that occur in the later
period of cancer progression [36]. Therefore, the inhibition of migration and invasion is
essential for efficient cancer treatment. To examine the inhibitory effect of compound 14 on
migration ability, a wound healing assay was performed on PC-3 cells. In this assay, wounds
were made by scraping highly metastases confluent PC-3 cell monolayers with a sterile
pipette tip and visualising the response using phase contrast microscopy. As shown in Fig. 4,
after 48h, cells treated with 3 µM of compound 14 showed significant inhibition of cell

migration compared to the control cells, whereas migration was almost completely stopped
after treatment with a 5 µM solution.
<
Figure 4>
2
.6. Cell cycle analysis
The effects of compound 14 on the cell cycle checkpoints of PC-3 cells were
determined by flow cytometry analysis. PC-3 cells incubated with different concentrations (1,
and 5 µM) of compound 14 were stained with propidium iodide (PI) for DNA content
analysis. The flow cytometry results, shown in Fig. 5, reveal that following 48 h exposure to
µM solution of 14 resulted in an increased G0/G1 population and simultaneous reduction of
3
1
S and G2/M phase populations. For instance, 65.5% of cells were in the G0/G1 phase after 1
µM treatment with 14, compared to 51.3% of cells in the control. The effect was more
significant with 3 and 5 µM exposure, with 74.8% and 81.4% of cells in the G0/G1 phase,
respectively, indicating the dose dependent G0/G1 phase cell cycle arrest in PC-3 cells.
<
Figure 5>
2
2
.7. Apoptosis
.7.1. Hoechst staining
Apoptosis is a process of programmed cell death which is critical for maintaining
tissue homeostasis. The disruption or inactivation of apoptosis is considered responsible for
cancer cell progression and development of drug resistance. Therefore, apoptosis induction in
cancer cells has emerged as an effective strategy for the treatment of cancer [37]. As cell
cycle arrest in cancer cells could lead to apoptosis induction [38], we investigated the ability
of compound 14 to induce apoptosis in prostate cancer (PC-3) cells. To examine this, cells
exposed to 1, 3 and 5 µM concentrations of compound 14 were stained with Hoechst 33242

and observed by fluorescence microscopy for morphological changes to the nucleus. The
results showed that 1 µM treatment resulted in condensation of the nuclei and after 3 µM
treatment, condensation and fragmentation of the nuclei had taken place. Both changes can
also be seen in the cells treated with the 5 µM dose (Fig. 6). These results indicate that
compound 14 induced apoptosis in a concentration dependent manner.
<
Figure 6>
2
.7.2. Assessment of mitochondrial membrane potential
Mitochondria play a key role in signal transmission during the apoptosis of cancer
cells. It is known that dissipation of the mitochondrial electrochemical potential gradient
occurs in the early stage of apoptosis [39]. Therefore, we examined the changes in the
mitochondrial membrane potential (MMP or DJm) of PC-3 cells treated with compound 14
using JC-1 cationic dye. JC-1 is a mitochondrial selective fluorescent dye that aggregates in
the intact mitochondria (normal DJm) and emits a red fluorescence, or forms monomers with
a green fluorescence in depolarised mitochondria. As is evident from Fig. 7, PC-3 cells
incubated with compound 14 displayed an increased green/red light ratio, disruption of
membrane potential. Moreover, the depolarisation was also related to the concentration of 14.
These results demonstrate that the induction of apoptosis by compound 14 is associated with
a mitochondrial pathway.
<
Figure 7>
2
.7.3. Effect of compound 14 on intracellular Reactive oxygen species (ROS) levels
It has been demonstrated that depolarisation of mitochondrial membrane potential
leads to increased intracellular ROS levels and is considered to play a double sword role in
the toxicity of cancer cells [40]. Therefore, it was considered of interest to investigate

whether compound 14 leads increased cellular ROS levels. PC-3 cells were incubated with
different concentrations of compound 14 for 48 h and stained with DCFDA (2’,7’-
dichlorofluorescin diacetate). The stained cells were analysed for a green fluorescence using
flow cytometry. As shown in Fig. 8, treatment with 1 µM concentration of compound 14
resulted in 1.78 times higher generation of intracellular ROS compared to the control; at 3
and 5 µM concentrations, ROS levels increased to 2.5 and 4.2 times respectively.
<
Figure 8>
2
.7.4. Annexin V- FITC/ Propidium Iodide (AV-PI) staining assay
Externalisation of phosphatidylserine (PS) on the outer layer of the plasma membrane
is known to occur in the early stages of apoptosis and can be determined using Aannexin V
Annexin V-FITC), which has a high affinity for PS [41]. The dual staining with Annexin V
(
and propidium iodide facilitates the differentiation of necrotic and apoptotic cells. As shown
in Fig. 9, the population of total apoptotic cells increased with increasing concentration of
compound 14. At 1 µM treatment very few cells were positive with either Annexin or PI.
However, by increasing the dose to 3 µM, a higher number of cells underwent apoptosis (Q2-
LR; AV+/PI-) and late apoptosis (Q2-UR-AV+/PI+) and the effect was much higher with 5
µM. The percentage of total apoptotic cells (sum of early and late apoptotic cells) in PC-3
cells increased to 12, 16 and 18% after treatment with 1, 3 and 5 µM concentrations of
compound 14, respectively (Fig. 9). From these results, it was concluded that the compound
1
4 induced apoptosis in a dose dependent manner.
<
Figure 9>
2
.8. VEGFR-2 inhibition assay

The most active compounds 8, 9, 13, 14, 19, 20, 25 and 26 were further evaluated for
their inhibitory activities against the VEGFR-2 protein, along with the known positive control
axitinib (VEGFR-2 inhibitor). The results (Table 2) indicated that the tested compounds
prominently inhibited VEGFR-2, with IC50 values of 97 to 980 nM. Among the tested
compounds, 14 and 18 showed significant inhibition of VEGFR-2 with IC values of 97 nM
5
0
and 109 nM, respectively, in comparison to the positive control (39 nM). These results
indicate that compound 14 can be considered the most potent of all the prepare compounds
because of the distinguished cytotoxic activities towards cancer cell lines and the comparable
inhibition of VEGFR-2 with the positive control axitinib.
<
Table 2>
2
.9. Molecular docking
Docking algorithms provide essential structural information about protein-drug
interactions which play a pivotal role in drug development. Molecular docking tools were
used to predict the orientation of the newly designed pyrazolo-benzothiazole hybrids within
the constraints of protein binding pockets. The VEGFR-2 inhibition results of these hybrids
(especially hybrids 14 and 18) encouraged us to perform molecular docking studies and
compare the results with the VEGFR inhibitor axitinib.
The docking studies showed that the most active hybrids (14 and 18) fit well in the
AXI binding pocket of VEGFR-2 (PDB ID - 4AGC), shown in Figure 10A. For comparison,
the different binding pose of axitinib is shown in Figure 10B. As axitinib is a linear molecule,
its carboxamide portion nestles into the hydrophobic pocket while the pyridin-2-yl terminal
protrudes. On the other hand, in hybrids 14 and 18, both the pyrazole ring and the aryl
rd
substituent in the 3 position optimally fit into the hydrophobic core.

Hybrids 14 and 18 show similar interactions with the target protein (Fig. 10C and
1
0D). The carbonyl oxygen present in both hybrids show hydrogen bonding with the peptide
and side chain amide N-H of Asn923 (red dashed lines). In addition, the side chain N-H also
interacts with the nitrogen atom present in the benzothiazole ring. The amide N-H present in
both hybrids also shows an interaction with the carbonyl oxygen of Leu840 and the hydrogen
bond lengths are shown in Figure 10C and 10D. Hybrids 14 and 18 also display strong
hydrophobic interactions with VEGFR-2 (Fig. 10C and 10D). The substituted
benzo[d]thiazol-2-amine motif present in both hybrids show π-interactions with Phe1047 and
hydrophobic interactions with the Gly841, Arg1032 and Asp1056 residues. The substituted
pyrazole ring in these hybrids shows interactions with Lys838, Leu840, Val848, Glu850,
Ala866, Phe918, Cys919, Lys920, Phe921, Gly922, Asn923 and Leu1035 amino acid
residues.
<
Figure 10>
2
.10. In vivo zebrafish angiogenesis assay
Angiogenesis is a key step involved in the formation of new blood vessels that leads
to tumour growth and metastasis [42]. Primarily, we used transgenic Tg(fli1a:EGFP)
zebrafish models to identify the antiangiogenic effects of compound 14. Zebrafish embryos
[
Tg(fli1a:EGFP)] were exposed to various concentrations of 14 (0.1, 0.5 and 1 µM) at 24-
hour post fertilisation (hpf) stage. The VEGFR-2 inhibitor drug axitinib was used as a
positive control and treatment with 1% DMSO served as the vehicle control [43].
From the results (Fig. 11A and B), no significant inhibition of intersegmental vessels
(
ISV) was observed in the embryos treated with the control or vehicle treated control (A-B
and A'-B', respectively). After treatment with 0.1 µM of 14, 16% defective ISVs were
observed (C-C'). This was more prominent in the 0.5 (D-E) and 1 µM treatments (D'-E')

which showed 47.3 and 54.5% defective ISVs, respectively. At the same concentrations,
embryos treated with axitinib were affected much more strongly (63.1-90.4% defective ISVs)
than those treated with compound 14 (F-H and F'-H'). The obtained results indicate that the
inhibition of angiogenesis in transgenic zebrafish models treated with 14 is dose dependent.
<
Figure 11>
3
. Conclusions
In this study, a series of new pyrazolo-benzothiazole hybrids (7-26) were prepared
and their in vitro anticancer activity against four human tumour cell lines were investigated.
Potent growth-inhibitory effects were observed for compounds 8, 9, 13, 14, 18, 19, 23 and 24
on most of the tested cell lines which were also tested against the VEGFR-2 protein.
Amongst these, compound 14 displayed the most potent inhibition of VEGFR-2 (0.097 µM),
showed significant cytotoxicity towards PC-cells (3.17 µM) and possessed high selectivity
towards cancer cells compared to the standard drug axitinib. Docking results indicated that
compound 14 exhibited strong binding interactions with the active sites of the VEGFR-2
protein. Additionally, treatment of prostate (PC-3) cancer cells with compound 14 produced
the highest percentage of G0/G1 population, indicative of cell cycle arrest, and induced
apoptosis through a mitochondrial dependent pathway. Moreover, compound 14 also
displayed inhibitory activity towards the growth of the more physiologically relevant 3D
multicellular spheroids of PC-3 and U87MG spheroids in a dose-dependent manner,
demonstrating the potential of 14 in the discovery and development of cancer
chemotherapeutics. Furthermore, in in vivo models, compound 14 strongly inhibited the
formation of intersegmental vessels in transgenic zebrafish indicating anti-angiogenesis
characteristics. From the above findings, compound 14 could be considered a lead molecule
for the further development of novel VEGFR-2 inhibitors for cancer therapy.

4
. Experimental Section
Starting materials and reagents were purchased from Alfa Aesar and Aldrich and used
without further purification. Reactions were monitored by TLC analysis using Merck 9385
aluminium plates pre-coated with silica gel containing F254 indicator, which were visualised
using UV light. Column chromatography was performed using Merck silica gel of 60-120
mesh with hexane and ethyl acetate as eluents. Melting points were determined on an
electrothermal melting point apparatus and are uncorrected. Nuclear Magnetic Resonance
1
13
spectra ( H and C) were recorded on 400 and 500 MHz Bruker, AVANCE spectrometers,
using TMS as an internal reference. The chemical shifts values are expressed in parts per
million (ppm) and coupling constants (J) in Hertz. Splitting patterns of multiplicities are
designated as: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet.
High-resolution mass spectra were obtained using an ESI-QTOF mass spectrometer (70 eV).
4
.1. General procedure for the synthesis of 1,3-diphenyl-1H-pyrazole-4-carboxylic acids
(5a-d)
A general procedure: A para-substituted acetophenone (1a-d, 10 mmol) and phenyl
hydrazine (2, 12.5 mmol) were added to anhydrous ethanol and heated to form the substituted
acetophenone phenylhydrazone (3a-d). The solid was filtered off, dried, and added to a cold
solution of POCl (3 mL) in DMF (15 mL). The mixture was stirred at 50-60 ºC for 5 h to
3
give the pyrazole carbaldehyde derivatives (4a-d). The reaction mixture was poured into ice-
cold water and saturated solution of NaHCO was added slowly to neutralize the reaction
3
mixture. The precipitated solid (4a-d) was collected by filtration and dried. The aldehyde
(
4a-d, 10 mmol) was dissolved in 15 mL of acetone, sodium chlorite (NaClO 11 mmol) and
2,
sulfamic acid (NH HSO 11 mmol) was added and the reaction was stirred for 5 h at 40-50
2
3,
ºC. Acetone was removed by evaporation; the residue was dissolved in ethylacetate and the
solution washed with water. The organic layer was separated, dried (Na SO ) and the solvent
2
4

removed by evaporation to give the pyrazole carboxylic acid derivative (5a-d) in good yields
85-90%).
.2. General procedure for the synthesis of N-(benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-
(
4
pyrazole-4-carboxamide hybrids (7-26)
To a solution of pyrazole carboxylic acid (5a-d, 1.0 mmol) and EDCI (1.2 mmol) in
anhydrous dimethylformamide was added N,N-diisopropylethylamine (1.2 mmol) and HOBT
(1.2 mmol) at 0 ºC under a nitrogen atmosphere. The reaction mixture was stirred for 10 min,
substituted 2-aminobenzothiazole (6a-e, 1.0 mmol) was added and the reaction was stirred at
room temperature for an additional 12 h. The reaction mixture was poured into ice-cold water
(25 mL), extracted with ethyl acetate (3x15 mL) and the combined extracts were washed with
NaCl solution. The organic layer was separated, dried over anhydrous Na SO and the
2
4
solvent removed by evaporation. The crude product was purified by column chromatography
eluting with ethyl acetate-hexane (30-50%) give the desired pyrazolo-benzothiazole hybrid
(7-26) in 75-90% yield.
4
.2.1. N-(benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (7)
1
White solid, yield 88%, Mp: 180-182 ºC; H NMR (400 MHz, CDCl ): δ 8.93 (s, 1H),
3
8
7
1
.09 (d, J = 8.43 Hz, 1H), 7.95-7.91 (m, 2H), 7.87 (d, J = 7.70Hz, 2H), 7.60-7.53 (m, 3H),
1
3
.49-7.41 (m, 7H). C NMR (100 MHz, CDCl ): δ 160.6, 158.7, 155.4, 143.4, 138.7, 133.2,
3
30.5, 129.7, 128.9, 128.6, 128.3, 124.7, 123.0, 120.4, 119.8, 113.6, 108.4, 106.3; MS (ESI):
+
+
m/z 397 [M+H] . HRMS (ESI) calcd for C H N OS [M+H] 397.1123; found: 397.1127.
2
3
16
4
4
.2.2. N-(6-fluorobenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (8)
1
White solid, yield 86%, Mp: 164-166 ºC; H NMR (500 MHz, CDCl ): δ 8.93 (s, 1H),
3
8
7
.08 (d, J = 7.54 Hz, 1H), 7.95-7.92 (m, 2H), 7.87 (d, J = 7.78 Hz, 2H), 7.59-7.52 (m, 3H),
1
3
.49-7.42 (m, 6H). C NMR (100 MHz, CDCl ): δ 158.5, 155.6, 143.4, 138.6, 133.3, 130.5,
3

1
1
29.7, 129.4, 129.1, 128.8, 128.7, 128.3, 128.2, 124.7, 121.0 (d, JCF = 5.86 Hz), 120.4, 119.8,
+
19.4, 108.3, 106.7; MS (ESI): m/z 415 [M+H] .
4
.2.3. N-(6-chlorobenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (9)
1
White solid, yield 79%, Mp: 170-172 ºC; H NMR (500 MHz, CDCl ): δ 8.92 (s, 1H),
3
8
7
1
.08 (d, J = 8.39 Hz, 1H), 7.95-7.92 (m, 2H), 7.87 (d, J = 7.93 Hz, 2H), 7.59-7.52 (m, 3H),
1
3
.49-7.42 (m, 6H). C NMR (100 MHz, CDCl ): δ 158.5, 155.7, 143.4, 138.7, 133.3, 130.6,
3
29.8, 129.5, 129.1, 128.9, 128.7, 128.4, 128.2, 124.7, 120.5, 119.9, 108.3, 106.7; MS (ESI):
+
m/z 431 [M+H] .
4
.2.4. N-(6-methoxybenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (10)
1
White solid, yield 82%, Mp: 160-162 ºC; H NMR (400 MHz, CDCl ): δ 8.75 (s, 1H),
3
7
1
8
1
1
4
.82 (d, J = 7.82 Hz, 1H), 7.76-7.70 (m, 4H), 7.59 (d, J = 8.07 Hz, 1H), 7.48 (t, J = 7.58,
5.77 Hz, 2H), 7.36 (t, J = 7.21, 13.93 Hz, 2H), 7.29 (t, J = 6.96, 15.28 Hz, 1H), 7.04 (d, J =
1
3
.80 Hz, 2H), 3.87 (s, 3H). C NMR (100 MHz, CDCl ): δ 160.7, 160.6, 158.9, 152.4, 147.5,
3
39.0, 131.7, 131.4, 130.5, 129.5, 127.6, 126.2, 123.9, 123.7, 121.3, 120.2, 119.4, 115.0,
+
+
14.3, 55.3; MS (ESI): m/z 427 [M+H] . HRMS (ESI) calcd for C H N O S [M+H]
2
4
18
4
2
27.1229; found: 427.1222.
4
.2.5. N-(6-methylbenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide (11)
1
White solid, yield 89%, Mp: 206-208 ºC; H NMR (400 MHz, CDCl ): δ 11.04 (bs,
3
1
1
H), 8.44 (s, 1H), 7.72 (d, J = 7.94 Hz, 2H), 7.59 (s, 1H), 7.53-7.40 (m, 7H), 7.34 (t, J = 7.21,
1
3
4.30 Hz, 1H), 7.23 (d, J = 8.19 Hz, 1H), 7.04 (d, J = 8.31 Hz, 1H), 2.39 (s, 3H). C NMR
(
100 MHz, CDCl ): δ 161.0, 158.5, 152.3, 145.4, 138.8, 133.9, 131.8, 131.3, 131.0, 129.4,
3
1
29.2, 128.7, 128.7, 127.7, 127.5, 121.0, 119.6, 119.3, 115.6, 21.3; MS (ESI): m/z 411
+
+
[
M+H] . HRMS (ESI) calcd for C H N OS [M+H] 411.1280; found: 411.1280.
24 18 4

4
.2.6. N-(benzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-carboxamide
(12)
1
White solid, yield 83%, Mp: 172-174 ºC; H NMR (400 MHz, CDCl ): δ 8.92 (s, 1H),
3
8
.09 (dt, J = 8.43 Hz, 1H), 7.95 (dd, J = 5.38, 8.92 Hz, 2H), 7.87-7.85 (m, 2H), 7.60-7.53 (m,
+
3
H), 7.49-7.43 (m, 3H), 7.12 (t, J = 8.80, 17.60 Hz, 2H); MS (ESI): m/z 415 [M+H] .
4
.2.7. N-(6-fluorobenzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (13)
1
White solid, yield 80%, Mp: 179-181 ºC; H NMR (400 MHz, CDCl ): δ 8.92 (s, 1H),
3
8
.09 (dt, J = 8.43 Hz, 1H), 7.94 (dd, J = 5.38, 8.92 Hz, 2H), 7.88-7.84 (m, 2H), 7.59-7.53 (m,
+
3
H), 7.49-7.42 (m, 3H), 7.12 (t, J = 8.80, 17.60 Hz, 2H); MS (ESI): m/z 433 [M+H] . HRMS
+
(
ESI) calcd for C H F N OS [M+H] 433.0935; found: 433.0934.
2
3
14
2
4
4
.2.8.
N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (14)
1
White solid, yield 78%, Mp: 218-220 ºC; H NMR (400 MHz, CDCl ): δ 8.93 (s, 1H),
3
8
3
1
1
.10 (d, J = 8.54 Hz, 1H), 7.95 (dd, J = 5.34, 8.85 Hz, 2H), 7.89-7.85 (m, 2H), 7.59-7.54 (m,
1
3
H), 7.49-7.42 (m, 3H), 7.12 (t, J = 8.69, 17.39 Hz, 2H). C NMR (100 MHz, CDCl ): δ
3
63.5 (d, JCF = 250.15 Hz), 158.6, 154.7, 143.4, 138.6, 133.3, 131.1 (d, JCF = 8.80 Hz),
29.8, 129.6, 128.8, 128.8, 128.5, 126.7 (d, JCF = 2.93 Hz), 124.8, 120.5, 119.9, 119.4, 115.3
+
(
d, JCF = 22.00 Hz), 108.3, 106.6; MS (ESI): m/z 449 [M+H] . HRMS (ESI) calcd for
+
C H ClFN OS [M+H] 449.0639; found: 449.0652.
23
14
4
4
.2.9.
3-(4-fluorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (15)
1
White solid, yield 80%, Mp: 248-250 ºC; H NMR (400 MHz, CDCl ): δ 8.50 (s, 1H),
3
7
.77 (dd, J = 5.25, 8.68 Hz, 2H), 7.60 (d, J = 7.45 Hz, 2H), 7.46 (t, J = 7.45, 15.65 Hz, 2H),

7
.40 (d, J = 8.80 Hz, 1H), 7.37 (t, J = 7.33, 14.67 Hz, 1H), 7.28 (d, J = 2.56 Hz, 1H), 7.21 (t,
13
J = 8.68, 17.36 Hz, 2H), 6.92 (dd, J = 2.44, 8.80 Hz, 1H), 3.84 (s, 3H). C NMR (100 MHz,
CDCl ): δ 163.4 (d, J = 249.41 Hz), 160.7, 157.1, 156.8, 151.5, 141.7, 138.7, 133.0, 130.
3
CF
(d, JCF = 8.80 Hz ), 127.6, 127.5 (d, JCF = 2.93 Hz), 120.8, 119.3, 115.8 (d, JCF = 21.27 Hz),
+
1
15.3, 115.1, 104.3, 55.7; MS (ESI): m/z 445 [M+H] .
4
.2.10.
3-(4-fluorophenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (16)
1
White solid, yield 83%, Mp: 230-232 ºC; H NMR (400 MHz, CDCl ): δ 8.40 (s, 1H),
3
7
2
2
2
1
.78 (dd, J = 5.25, 8.68 Hz, 2H), 7.60 (s, 2H), 7.46 (d, J = 7.82, 2H), 7.40 (t, J = 7.21, 15.28,
H), 7.33 (t, J = 7.21, 14.30 Hz, 1H), 7.28 (d, J = 8.43 Hz, 1H), 7.17 (t, J = 8.68, 17.23 Hz,
1
3
H), 7.06 (d, J = 8.31 Hz, 1H), 2.39 (s, 3H). C NMR (100 MHz, CDCl ): δ 163.3 (d, J
=
3
CF
49.76 Hz), 160.9, 158.6, 151.7, 145.3, 138.7, 134.1, 131.8, 130.8 (d, JCF = 8.17 Hz), 129.4,
27.8, 127.6, 127.5, 121.1, 119.7, 119.3, 115.7 (d, JCF = 21.99 Hz), 115.3, 21.3; MS (ESI):
+
+
m/z 429 [M+H] . HRMS (ESI) calcd for C H FN OS [M+H] 429.1185; found: 429.1186.
2
4
17
4
4
.2.11.
N-(benzo[d]thiazol-2-yl)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (17)
1
White solid, yield 88%, Mp: 174-176 ºC; H NMR (400 MHz, CDCl ): δ 8.93 (s, 1H),
3
8
7
.09 (d, J = 8.43 Hz, 1H), 7.91 (d, J = 8.68 Hz, 2H), 7.87-7.84 (m, 2H), 7.60-7.53 (m, 3H),
+
.49-7.43 (m, 3H), 7.41 (d, J = 8.68 Hz, 2H). MS (ESI): m/z 431 [M+H] .
4
.2.12.
3-(4-chlorophenyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (18)
1
White solid, yield 82%, Mp: 178-180 ºC; H NMR (400 MHz, CDCl ): δ 8.92 (s, 1H),
3
8
7
.09 (d, J = 8.43 Hz, 1H), 7.90 (d, J = 8.68 Hz, 2H), 7.87-7.84 (m, 2H), 7.60-7.53 (m, 3H),
1
3
.48 (t, J = 0.85, 1.72 Hz, 1H), 7.47-7.43 (m, 2H), 7.40 (d, J = 8.68 Hz, 2H). C NMR (100

MHz, CDCl ): δ 158.5, 154.5, 143.4, 138.5, 135.6, 133.4, 130.6, 130.4, 129.8, 129.6, 129.0,
3
+
1
28.8, 128.5, 128.4, 124.8, 120.5, 119.8, 108.3, 106.6; MS (ESI): m/z 449 [M+H] .
4
.2.13.
N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (19)
1
White solid, yield 79%, Mp: 188-190 ºC; H NMR (400 MHz, CDCl ): δ 8.93 (s, 1H),
3
8
7
1
1
.09 (d, J = 8.43 Hz, 1H), 7.91 (d, J = 8.55 Hz, 2H), 7.87-7.84 (m, 2H), 7.60-7.53 (m, 3H),
1
3
.49-7.43 (m, 3H), 7.41 (d, J = 8.68 Hz, 2H). C NMR (100 MHz, CDCl ): δ 158.5, 154.5,
3
43.4, 138.5, 135.6, 133.4, 130.4, 129.8, 129.0, 128.8, 128.7, 128.5, 128.4, 124.8, 120.5,
+
19.9, 108.2, 106.7; MS (ESI): m/z 465 [M+H] .
4
.2.14. 3-(4-chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (20)
1
White solid, yield 76%, Mp: 193-195 ºC; H NMR (400 MHz, CDCl ): 8.45 (s, 1H),
3
7
.72 (d, J = 8.31 Hz, 2H), 7.53 (d, J = 7.70 Hz, 2H), 7.46-7.41 (m, 4H), 7.35 (t, J = 7.21,
13
1
4.55 Hz, 1H), 7.31-7.27 (m, 2H), 6.87 (dd, J = 2.44, 8.80 Hz, 1H), 3.82 (s, 3H). C NMR
(
100 MHz, CDCl ): δ 160.5, 156.8, 151.2, 141.8, 138.8, 135.5, 133.1, 131.0, 130.2, 129.8,
3
+
1
29.5, 129.0, 127.7, 120.9, 119.3, 115.5, 115.1, 104.3, 55.8; MS (ESI): m/z 461 [M+H] .
4
.2.15.
3-(4-chlorophenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (21)
1
White solid, yield 82%, Mp: 220-222 ºC; H NMR (400 MHz, CDCl ): δ 8.42 (s, 1H),
3
7
7
.73 (d, J = 8.43 Hz, 2H), 7.60 (s, 1H), 7.47-7.37 (m, 6H), 7.34 (t, J = 7.21, 13.69 Hz, 1H),
13
.25 (d, J = 5.62 Hz, 1H), 7.05 (d, J = 8.12 Hz, 1H), 2.39 (s, 3H). C NMR (100 MHz,
CDCl ): δ 160.0, 159.1, 151.7, 145.0, 138.7, 135.2, 134.1, 131.6, 130.7, 130.2, 130.0, 129.4,
3
+
1
28.6, 127.8, 127.6, 121.1, 119.4, 119.2, 115.3, 21.3; MS (ESI): m/z 445 [M+H] . HRMS
+
(
ESI) calcd for C H ClN OS [M+H] 445.1134; found: 445.1136.
2
4
17
4

4
.2.16.
N-(benzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (22)
1
White solid, yield 80%, Mp: 198-200 ºC; H NMR (400 MHz, CDCl ): δ 8.75 (s, 1H),
3
7
1
8
1
1
4
.82 (d, J = 7.58 Hz, 1H), 7.76-7.71 (m, 4H), 7.59 (d, J = 8.07 Hz, 1H), 7.48 (t, J = 7.58,
5.77 Hz, 2H), 7.37 (t, J = 7.21, 15.03 Hz, 2H), 7.29 (t, J = 6.96, 15.28 Hz, 1H), 7.03 (d, J =
1
3
.80 Hz, 2H), 3.87 (s, 3H). C NMR (100 MHz, CDCl ): δ 160.7, 160.6, 158.9, 152.4, 147.5,
3
39.0, 131.7, 131.4, 130.5, 129.5, 127.6, 126.2, 123.9, 123.7, 121.3, 120.2, 119.4, 115.0,
+
+
14.3, 55.3; MS (ESI): m/z 427 [M+H] . HRMS (ESI) calcd for C H N O S [M+H]
2
4
18
4
2
27.1229; found: 427.1222.
.2.17. N-(6-fluorobenzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
4
carboxamide (23)
1
White solid, yield 78%, Mp: 169-171 ºC; H NMR (400 MHz, CDCl ): δ 8.91 (s, 1H),
3
8
.08 (d, J = 8.43 Hz, 1H), 7.91 (d, J = 8.92 Hz, 2H), 7.86 (d, J = 7.58 Hz, 2H), 7.59-7.52 (m,
1
3
3
H), 7.50-7.41 (m, 3H), 6.96 (d, J = 8.80 Hz, 2H), 3.83 (s, 3H). C NMR (100 MHz,
CDCl ): δ 160.6, 158.7, 155.4, 143.4, 138.7, 133.2, 130.5, 129.7, 129.5, 128.9, 128.6, 128.2,
3
1
24.7, 123.0, 120.4, 119.8, 119.4, 114.3, 113.6, 108.4, 106.3, 55.2; MS (ESI): m/z 445
+
[
M+H] .
4
.2.18. N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-
carboxamide (24)
1
White solid, yield 81%, Mp: 193-195 ºC; H NMR (400 MHz, CDCl ): δ 8.90 (s, 1H),
3
8
.08 (d, J = 8.39 Hz, 1H), 7.91 (d, J = 8.85 Hz, 2H), 7.86 (d, J = 7.78 Hz, 2H), 7.59-7.52 (m,
1
3
3
H), 7.50-7.41 (m, 3H), 6.95 (d, J = 8.85 Hz, 2H), 3.82 (s, 3H). C NMR (100 MHz,
CDCl ): δ 160.6, 158.6, 155.4, 143.4, 138.7, 133.2, 130.5, 129.7, 128.9, 128.6, 128.3, 124.7,
3

+
1
23.0, 120.4, 119.8, 119.3, 114.4, 113.6, 108.3, 106.3, 55.2; MS (ESI): m/z 461 [M+H] .
+
HRMS (ESI) calcd for C H ClN O S [M+H] 461.0839; found: 461.0842.
2
4
17
4
2
4
.2.19. 3-(4-methoxyphenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (26)
1
White solid, yield 88%, Mp: 237-239 ºC; H NMR (400 MHz, CDCl ): δ 10.88 (bs,
3
1
H), 8.45 (s, 1H), 7.65 (d, J = 8.55 Hz, 2H), 7.59 (s, 1H), 7.53 (d, J = 7.82 Hz, 2H), 7.42 (t, J
=
7.58, 14.52 Hz, 2H), 7.33 (t, J = 7.21, 14.18 Hz, 1H), 7.28 (d, J = 8.55 Hz, 1H), 7.05 (d, J =
1
3
8
.31 Hz, 1H), 6.99 (d, J = 8.55 Hz, 2H), 3.83 (s, 3H), 2.40 (s, 3H). C NMR (100 MHz,
CDCl ): δ 161.1, 160.3, 158.4, 152.1, 145.4, 138.9, 133.9, 131.8, 131.0, 130.1, 129.3, 127.7,
3
+
1
27.4, 123.7, 121.0, 119.6, 119.3, 115.3, 114.1, 55.2, 21.3; MS (ESI): m/z 441 [M+H] .
+
HRMS (ESI) calcd for C H N O S [M+H] 441.1385; found: 441.1386.
2
5
20
4
2
4
4
.3. Biology
.3.1. Cell Culture
Colon (HT-29), prostate (PC-3), lung (A549), glioblastoma (U87MG) and embryonic
kidney (Hek-293T) cells were purchased from ATCC. PC-3 and A549 cells were grown in
RPMI medium whereas HT-29, U87MG and Hek-293T were maintained in DMEM
supplemented with 10% foetal bovine serum (FBS) and 1% pencillin-streptomycin (PS). All
the cell lines were grown in an incubator with 75% humidity and 5% CO at 37 ºC. 0.25%
2
trypsin-ethylenediaminetetraacetic acid (EDTA, Life Technologies) was used for harvesting
the cells. For all the assays, stock solutions of the compounds were prepared in DMSO (10
mM)
4
.3.2. MTT assay
In this assay, colon (HT-29), prostate (PC-3), lung (A549), glioblastoma (U87MG)
and embryonic kidney (Hek-293T) cells were seeded in 96 well plates depending on their

doubling time and were grown overnight. The cells were exposed to different concentrations
of pyrazolo-benzothiazole hybrids (100, 10, 1, 0.1 and 0.01 µM) for 72 h. Then, the medium
containing compounds was removed and replaced with 100 µL of MTT solution (5 mg/mL)
and the cells were further incubated for 4 h in dark at 37 ºC. The unreacted MTT solution was
removed and 100 µL DMSO was added to each well to solubilise the produced formazan
crystals. The absorbance of the purple formazan solution was recorded using a plate reader
(
SpectraMax) at 570 nm and the IC values for each compound were calculated. All the
50
experiments were repeated three times and the standard deviations are reported in Table 1.
4
.3.3. Colony Forming Assay
PC-3 cells in exponential growth phase were seeded into 6-well plates at 4000
cells/well. After 24 hours incubation, the culture medium was replaced with medium
containing increasing concentrations (1, 3, 5 and 10 µM) of compound 14 and 1% DMSO
(control). The cells were incubated for 7 days and the drug-containing medium was
replenished after 3 days. Each treatment was performed in triplicate. After incubation, the
cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min and stained
with crystal violet for a further 15 min. Scans of 6-well plates were generated on an Epson
Perfection V700 Photo scanner using Epson scan software.
4
.3.4. Spheroid Formation Assay
PC-3 cells were seeded (25000/well) in ultra-low attachment 24 well plates (Corning)
in complete growth medium and allowed to grow for three days in an incubator to produce
spheroids. The spheroids were treated with different concentrations of compound 14 and
monitored for morphological changes after 48 h by phase contrast microscopy.
4
.3.5. Wound healing assay

Confluent PC-3 monolayers in 30 mm petri dishes were wounded with 200 µL pipette
tips, giving rise to 1 mm wide lanes per well. The cell debris was removed by washing with
PBS and cells were supplied with 2 mL of complete medium (controls) or complete medium
containing different concentrations (1, 3 and 5 µM) of compound 14. The wounds were
observed by phase contrast microscopy immediately and after 48 h incubation.
4
.3.6. Cell Cycle analysis
PC-3 cells in 6 well plates were seeded at a density of 1x10 /well and were grown
6
overnight in an incubator. The cells were then incubated with 1, 3 and 5 µM concentrations of
compound 14 and after 48 h, collected using 0.25% trypsin-EDTA. The obtained cell pellets
were washed and resuspended in PBS. The cells were fixed by pipetting the resuspended cell
suspension into 9 mL of 70% ethanol. After 30 min fixation at 4 ºC, the ethanol was removed
by centrifugation and the cells washed with PBS. After centrifugation, the cells were
incubated with propidium iodide staining solution for 15 min in the dark at room temperature.
1
0000 cells from each sample were analysed for DNA content (propidium iodide
fluorescence) using a BD Accuri C6 flow cytometer.
4
.3.7. Hoechst staining
Changes in the nuclear morphology of PC-3 cells were determined using Hoechst
3
1
3242. In this assay, PC-3 cells were grown on cover slips in a 6 well plate at a density of
6
X10 cells/well and were incubated with different concentrations of compound 14 for 48 h.
The cells were washed with PBS and 4% paraformaldehyde solution was added. The cells
were incubated with 2 µg/mL Hoechst 33242 for 20 minutes then washed three times with
PBS to remove excess dye. The morphological changes in the nuclei were observed using a
ZOE™ Fluorescent Cell Imager (BIO-RAD).
4
.3.8. Assessment of mitochondrial membrane potential

5
PC-3 Cells were grown in 24-well plates (5x10 cells/mL) and incubated with
different concentrations (1, 3 and 5 µM) of compound 14. After 48 h incubation, the medium
containing the compound was replaced with 500 µL of fresh medium containing 5 µg/mL JC-
1
and further incubated for 20 min. The cells were washed three times with PBS to remove
excess dye and photographed in red and green channels using a ZOE™ Fluorescent Cell
Imager (BIO-RAD).
4
.3.9. Intracellular reactive oxygen species
The intracellular ROS levels in PC-3 cells were determined by DCFDA staining. In
this assay, PC-3 cells were incubated with increasing concentrations of compound 14 (1, 3
and 5 µM) for 48 h. After incubation, the cells were harvested and stained with a 10 µM
solution of carboxy-DCFDA in PBS for 20 min at 37 ºC. The intensity of the green
fluorescence was analysed using a BD-Accuri C6 flow cytometer [44].
4
.3.10. Annexin-V FITC/ Propidium Iodide double staining
6
PC-3 cells (1x10 /well) were grown in 6 well plate and treated with increasing
concentrations of compound 14 for 48 h. After incubation, the cells were trypsinised and
washed with PBS. The obtained cell pellet was resuspended in 1x annexin binding buffer. 5
µL of annexin V and 1 µL of PI was added to the resuspended cells and incubated for 15 min
at room temp. 10000 cells from each sample were used for analysis using a BD Accuri C6
flow cytometer.
4
.3.11. VEGFR-2 inhibition assay
The VEGFR-2 tyrosine kinase activity of the compounds was performed according to
BPS Bioscience Corporation, San Diego, CA, USA (www.bpsbioscience.com) protocol,
where VEGFR-2 (KDR) (BPS#40301) served as the enzyme source and Poly (Glu, Tyr)
sodium salt, (4:1, Glu:Tyr) (Sigma#P7244) served as the standardised substrate and Kinase-

Glo Plus Luminescence kinase assay kit (Promega#V3772) [45]. In this assay, 5 µL of the
compound (1 nM, 10 nM, 100 nM, 1 µM, 10 µM) was added to a 45 µL of reaction mixture
(
40 mM Tris, pH 7.4, 10 mM MgCl , 0.1 mg/mL BSA, 1 mM DTT, 10 µM ATP, Kinase
2
substrate and VEGFR) and incubated at 30 ºC for 45 min. After the enzymatic reaction, 50
µL of Kinase-Glo Plus Luminescence kinase assay solution (Promega) was added to each
reaction well and the plate was further incubated for 15 min at room temperature in dark [46].
The luminescent signal was measured using a microplate reader (SpectraMax). The intensity
of the ATP luminescence is inversely proportional to the amount of kinase activity. The
assays were performed in triplicate for each concentration and the IC50 values were
determined from non-linear regression analysis of the Sigmoidal dose-response curve
generated in Graph pad prism.
4
.3.12. Molecular docking
The molecular docking studies were performed at the AXI binding site of VEGFR-2
(PDB ID: 4AGC) [47]. The coordinates of the crystal structure were obtained from RCSB-
Protein Data Bank and suitable corrections were made using Protein Preparation Wizard from
the Schrödinger package. Regarding the ligands, molecules were constructed using
ChemBio3D Ultra 12.0 and their geometries were optimised using molecular mechanics.
Finally, docking studies were performed on the most active molecules (14 and 18) using
AutoDock 4.2 docking software [48] and the results were visualised through PyMOL [49].
4
.3.13. In vivo zebrafish angiogenesis assay
Zebrafish embryos from the Tg(fil-1:EGFP) transgenic line expressing enhanced
green fluorescent protein (EGFP) were used in this study to evaluate blood vessel formation.
Freshly fertilised eggs were collected immediately upon natural spawning and placed in
embryo medium E3 at 28.5 °C [50]. Fertilised embryos were sorted and developmentally

staged before undertaking any experiments as described previously [51]. At 1 dpf (day post
fertilisation), the embryos were placed in 5 mL of E3 medium with different concentrations
(0.1, 0.5 and 1 µM) of 14 and axitinib in 6 well plate (10 embryos/well) and incubated for 72
h at 28.5 °C. After the incubation period, the hatched embryos were anaesthetised with 0.01%
tricaine (Sigma-Aldrich), immobilised in 1% low melting agarose gel and the intersegmental
blood vessels (ISVs) of the embryos were observed using a fluorescence microscope (Nikon
SMZ18) [52].
Acknowledgements
V.G.R. acknowledges the UGC, New Delhi for the award of a research fellowship and
th
the authors are thankful to CSIR, New Delhi for the financial support under the 12 Five
Year Plan projects “Affordable Cancer Therapeutics (ACT)” (CSC0301) & ORIGIN (CSC-
1
08). CSIR-IICT Communication No. IICT/Pubs./2019/245. This work was performed in part
at the RMIT Micro Nano Research Facility (MNRF) in the Victorian Node of the Australian
National Fabrication Facility (ANFF). The authors are also thankful to Dr. Steven H. Privér
(RMIT University, Australia) for proof reading the manuscript.
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