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 182 (2019) 111609

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Research paper

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 a, b, d, T. Srinivasa Reddy d, *, Chetna Jadala c, M. Soumya Reddy f, Faria Sultana b, Ravikumar Akunuri c, Suresh K. Bhargava d, Donald Wlodkowic e, P. Srihari a, b, **, Ahmed Kamal a, b, c, g, *** 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

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 May 2019 Received in revised form 6 August 2019 Accepted 8 August 2019 Available online 8 August 2019

A series of new pyrazolo-benzothiazole hybrids (7e26) 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 3.17e6.77 mM, even better than reference drug axitinib (4.88e21.7 mM). 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. © 2019 Published by Elsevier Masson SAS.

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

* Corresponding author. ** Corresponding author. Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource Development Centre (CSIR-HRDC) Campus, Ghaziabad, 201 002, Uttar Pradesh, India. *** Corresponding author. Department of Organic Synthesis & Process Chemistry, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad, 500 007, India. E-mail addresses: [email protected] (T.S. Reddy), srihari@ iict.res.in (P. Srihari), [email protected] (A. Kamal). https://doi.org/10.1016/j.ejmech.2019.111609 0223-5234/© 2019 Published by Elsevier Masson SAS.

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 [3e5]. 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

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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 [15e20]. 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 antiangiogenesis 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) [24e26]. 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 [28e31]. 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 pyrazolobenzothiazole hybrid molecules (7e26) and their evaluation of biological activities towards different cancer cell lines. 2. Results and discussions 2.1. Chemistry The synthetic procedure for the N-(benzo[d]thiazol-2-yl)-1,3diphenyl-1H-pyrazole-4-carboxamide hybrids (7e26) is depicted in Scheme 1. The pyrazole carboxylic acid precursors (5aed) 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)-N0 -ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropyl-ethylamine

Fig. 1. Examples of pyrazole and benzothiazole based anticancer drugs and the design strategy for the target hybrids (7e26).

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Scheme 1. General synthesis of N-(benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives (7e26). Reagents and conditions: (i) Ethanol, 50e60  C, 3 h; (ii) DMF, POCl3, 50e60  C, 5 h; (iii) Acetone, NaClO2/NH2HSO3, 40e50  C, 5 h; (iv) DMF, EDC, DIPEA, HOBt, 0  C-rt, 12 h.

(DIPEA) and 1-hydroxybenzotriazole (HOBt) in dry DMF under a nitrogen atmosphere to produce the desired pyrazolobenzothiazole hybrids (7e26) in yields of 75e90%. The compounds were purified by column chromatography and characterised by 1H, 13C spectroscopy NMR and ESI-MS. 2.2. Cytotoxicity Evaluation of the growth-inhibitory properties of the pyrazolobenzothiazole 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 (mM). 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 IC50 values ranging from 3.17 to 10.7 mM. Interestingly, hybrid 14 showed the most potent activity among the series, with IC50 values of 3.17 mM (PC-3), 3.32 mM (HT-29), 3.87 mM (A549) and 6.77 mM (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 2e3 times more selective, thereby demonstrating the high selectivity of this compound towards cancer cells. 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 IC50 values in the range 3.25e10.2 mM; 9 with 3.69e7.81 mM; 13 with 4.16e12.5 mM; 14 with 3.32e6.77; 18 with 4.97e8.86; 19 with 3.23e5.87; 23 with 4.93e8.99 and compound 24 with 4.71e8.71 mM. These activities were more significant than the positive control axitinib (4.87e21.7 mM). 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 IC50 values in the range 8.96e16.8 mM. From these results it was clear that the compound 14 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-293T) 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 PC3 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 mM) 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.

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Table 1 Cytotoxicity (IC50)a of pyrazolo-benzothiazole hybrids (7e26). Compound

R1

R2

HT29b (colon)

PC3c (prostate)

A549d (lung)

U87MGe (brain)

Hek293Tf

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Cisplatin Axitinib

H H H H H F F F F F Cl Cl Cl Cl Cl OMe OMe OMe OMe OMe -

H F Cl OMe Me H F Cl OMe Me H F Cl OMe Me H F Cl OMe Me

16.8 ± 1.36 9.8 ± 1.27 7.81 ± 0.83 >50 >50 9.06 ± 1.26 4.16 ± 0.37 3.32 ± 0.25 33.2 ± 2.94 22.9 ± 1.48 13.7 ± 2.75 4.97 ± 0.18 4.55 ± 0.26 18.6 ± 3.64 23.67 ± 1.89 10.5 ± 0.88 5.56 ± 0.27 6.57 ± 0.38 >50 21.5 ± 2.57 10.3 ± 0.81 13.12 ± 1.84

10.4 ± 2.57 3.25 ± 0.25 3.69 ± 0.25 17.5 ± 2.69 15.7 ± 2.66 7.13 ± 0.82 5.26 ± 0.51 3.17 ± 0.19 12.8 ± 1.25 20.5 ± 3.98 7.75 ± 1.56 5.79 ± 0.26 3.23 ± 0.12 29.3 ± 3.31 14.5 ± 2.4 6.67 ± 0.26 4.93 ± 0.85 6.51 ± 0.47 13.5 ± 1.93 15.9 ± 2.74 3.62 ± 0.35 16.43 ± 1.4

13.5 ± 1.48 10.2 ± 2.71 7.67 ± 1.13 >50 26.5 ± 2.71 9.01 ± 0.57 6.65 ± 0.26 3.87 ± 0.12 >50 24.6 ± 3.65 10.3 ± 1.28 8.86 ± 0.63 4.36 ± 0.27 31.4 ± 3.53 18.6 ± 1.78 13.7 ± 1.56 7.56 ± 2.53 4.71 ± 1.23 18.87 ± 3.65 27.65 ± 2.55 4.54 ± 0.27 4.88 ± 0.83

8.96 ± 0.87 5.57 ± 0.63 4.98 ± 0.89 40.6 ± 3.91 23.2 ± 3.27 10.7 ± 1.37 12.5 ± 0.97 6.77 ± 0.38 >50 >50 11.5 ± 0.92 7.77 ± 0.27 5.87 ± 0.39 21.2 ± 2.64 33.2 ± 4.36 11.5 ± 1.13 8.99 ± 0.57 8.71 ± 0.83 5.89 ± 0.11 >50 8.21 ± 0.36 21.7 ± 2.37

>50 17.6 ± 2.57 21.3 ± 1.88 >50 >50 33.4 ± 2.69 16.7 ± 2.35 45.7 ± 3.81 >50 >50 39.6 ± 4.67 >50 15.7 ± 1.43 29.6 ± 3.41 35.6 ± 2.58 18.9 ± 1.36 15.4 ± 2.54 12.6 ± 1.93 >50 >50 6.36 ± 0.57 46.82 ± 3.17

a IC50 values are the concentrations (mM) that cause 50% inhibition of cancer cell growth. Data represent the average of three independent experiments performed in quadruplet. b Colon cell line. c Prostate cancer cell line. d Lung cancer cell line. e Glioblastoma cell line. f Embryonic kidney cell line.

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). 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 PC3 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 48 h, cells treated with 3 mM 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 mM solution. Fig. 2. Colony formation inhibition effect of compound 14 on PC-3 cells. The assay was performed in triplicate and a representative result is shown.

2.4. 3D multicellular spheroids (MSCs) inhibition assay 3D Multicellular spheroids of cancer cells have similar features to in vivo tumor 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

2.6. Cell cycle analysis The effects of compound 14 on the cell cycle checkpoints of PC3 cells were determined by flow cytometry analysis. PC-3 cells incubated with different concentrations (1, 3 and 5 mM) 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 1 mM solution of 14 resulted in an increased G0/G1 population and simultaneous reduction of S and G2/M phase populations. For instance, 65.5% of cells were in the G0/

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Fig. 3. Effect of compound 14 on the growth of 3D multicellular spheroids of PC-3 and U87MG cells. Scale bar represents 35 mm.

Fig. 4. Effect of compound 14 on PC-3 cell migration. The cells were cultured in the absence and presence of compound 14 (1, 3 and 5 mM). The wounds were created in the confluent monolayers of PC-3 with a sterile micro pipette tip.

G1 phase after 1 mM treatment with 14, compared to 51.3% of cells in the control. The effect was more significant with 3 and 5 mM 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.

2.7. Apoptosis 2.7.1. 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 mM 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 mM treatment resulted in condensation of the nuclei and after 3 mM treatment, condensation and fragmentation of the nuclei had taken place. Both changes can also be seen in the cells treated with the 5 mM dose (Fig. 6). These results indicate that

compound 14 induced apoptosis in a concentration dependent manner. 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. 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

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Fig. 5. Cell cycle distribution of PC-3 cells after treatment with compound 14 for 48 h. Results are expressed as the mean ± the standard deviation of three independent experiments. Significance was determined using the Student's T-test where n ¼ 3 and * p < 0.01, **p < 0.001, ***p < 0.0001.

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. PC3 cells were incubated with different concentrations of compound

14 for 48 h and stained with DCFDA (20 ,70 -dichlorofluorescin diacetate). The stained cells were analysed for a green fluorescence using flow cytometry. As shown in Fig. 8, treatment with 1 mM concentration of compound 14 resulted in 1.78 times higher

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Fig. 6. Apoptosis induced by compound 14 in PC-3 cells, observed by fluorescence microscopy using Hoechst 33242 staining after 48 h incubation with the compound. The cells were assessed for morphological changes, such as chromatin condensation and nuclear fragmentation, which are hallmarks of cell apoptosis.

generation of intracellular ROS compared to the control; at 3 and 5 mM concentrations, ROS levels increased to 2.5 and 4.2 times respectively.

distinguished cytotoxic activities towards cancer cell lines and the comparable inhibition of VEGFR-2 with the positive control axitinib.

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 VFITC), 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 mM treatment very few cells were positive with either Annexin or PI. However, by increasing the dose to 3 mM, a higher number of cells underwent apoptosis (Q2-LR; AVþ/PI-) and late apoptosis (Q2-UR-AVþ/PIþ) and the effect was much higher with 5 mM. 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 mM concentrations of compound 14, respectively (Fig. 9). From these results, it was concluded that the compound 14 induced apoptosis in a dose dependent manner.

2.9. Molecular docking

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 97e980 nM. Among the tested compounds, 14 and 18 showed significant inhibition of VEGFR-2 with IC50 values of 97 nM 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

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 Fig. 10A. For comparison, the different binding pose of axitinib is shown in Fig. 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 substituent in the 3rd position optimally fit into the hydrophobic core. Hybrids 14 and 18 show similar interactions with the target protein (Fig. 10C and D). The carbonyl oxygen present in both hybrids show hydrogen bonding with the peptide and side chain amide NeH of Asn923 (red dashed lines). In addition, the side chain NeH also interacts with the nitrogen atom present in the benzothiazole ring. The amide NeH present in both hybrids also shows an interaction with the carbonyl oxygen of Leu840 and the hydrogen bond lengths are shown in Fig. 10C and D. Hybrids 14 and 18 also display strong hydrophobic interactions with VEGFR-2 (Fig. 10C and D). The substituted benzo[d]thiazol-2-amine motif present in both hybrids show p-interactions with Phe1047 and hydrophobic interactions with the Gly841, Arg1032 and Asp1056 residues. The

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Fig. 7. Compound 14 disrupted mitochondrial membrane integrity. PC-3 cells were treated with 1, 3 and 5 mM concentrations of compound 14 for 48 h, stained with JC-1 and imaged by fluorescence microscopy. Scale bar represents 25 mm.

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. 2.10. In vivo zebrafish angiogenesis assay

Fig. 8. Compound 14 induced production of intracellular ROS in PC-3 cancer cells. Cells were treated with increasing concentrations of compound 14 for 48 h and stained with 10 mM DCFDA. The intensity of the green fluorescence due to the production of ROS was analysed by flow cytometry. Data represent the mean ± the standard deviation from three independent experiments. Significance was determined using the Student's T-test, where n ¼ 3 and * p < 0.01, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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 mM) at 24-h post fertilisation (hpf) stage. The VEGFR2 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 A0 -B0 , respectively). After treatment with 0.1 mM of 14, 16% defective ISVs were observed (CeC0 ). This was more prominent in the 0.5 (D-E) and 1 mM treatments (D0 -E0 ) which showed 47.3 and 54.5% defective ISVs, respectively. At the same concentrations, embryos treated with axitinib were affected much more strongly (63.1e90.4%

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Fig. 9. Analysis of apoptotic cells induced by compound 14 by flow cytometry. PC-3 cells exposed to increasing concentrations of compound 14 (1, 3 and 5 mM) were stained with Annexin V-FITC and PI. (LL: live; LR: early apoptotic; UR: late apoptotic; UL: necrotic).

Table 2 Inhibitory activity of the active compounds against VEGFR-2 (IC50, nM). Compound

VEGFR-2 (nM)

8 9 13 14 18 19 23 24 Axitinib

980 647 132 97 109 256 187 212 39

defective ISVs) than those treated with compound 14 (FeH and F0 H0 ). The obtained results indicate that the inhibition of angiogenesis in transgenic zebrafish models treated with 14 is dose dependent. 3. Conclusions In this study, a series of new pyrazolo-benzothiazole hybrids (7e26) were prepared and their in vitro anticancer activity against four human tumour cell lines were investigated. Potent growthinhibitory 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 mM), showed significant cytotoxicity towards PC-cells (3.17 mM) 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 PC3 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

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Fig. 10. A) Surface binding pose of hybrid 14. B) Binding pose comparison of hybrid 14 and 18 with axitinib. C) and D) Docking pose for hybrid 14 and 18, respectively, (hydrogen bonding and hydrophobic interactions) with the AXI binding pocket of 4AGC. Hybrid 14, 18 and axitinib are shown as stick models and coloured by the atom type. Carbon: cyan (14), yellow (18), pink (axitinib); Oxygen: red; Hydrogen: white; Nitrogen: blue; Chlorine: green; Fluorine: ice blue; Sulphur: yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

monitored by TLC analysis using Merck 9385 aluminium plates precoated with silica gel containing F254 indicator, which were visualised using UV light. Column chromatography was performed using Merck silica gel of 60e120 mesh with hexane and ethyl acetate as eluents. Melting points were determined on an electrothermal melting point apparatus and are uncorrected. Nuclear Magnetic Resonance spectra (1H and 13C) 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-1Hpyrazole-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 POCl3 (3 mL) in DMF (15 mL). The mixture was stirred at 50e60  C for 5 h to give the pyrazole

carbaldehyde derivatives (4a-d). The reaction mixture was poured into ice-cold water and saturated solution of NaHCO3 was added slowly to neutralize the reaction 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 (NaClO2, 11 mmol) and sulfamic acid (NH2HSO3, 11 mmol) was added and the reaction was stirred for 5 h at 40e50  C. Acetone was removed by evaporation; the residue was dissolved in ethylacetate and the solution washed with water. The organic layer was separated, dried (Na2SO4) and the solvent removed by evaporation to give the pyrazole carboxylic acid derivative (5a-d) in good yields (85e90%). 4.2. General procedure for the synthesis of N-(benzo[d]thiazol-2yl)-1,3-diphenyl-1H-pyrazole-4-carboxamide hybrids (7e26) To a solution of pyrazole carboxylic acid (5a-d, 1.0 mmol) and EDCI (1.2 mmol) in anhydrous dimethylformamide was added N,Ndiisopropylethylamine (1.2 mmol) and HOBT (1.2 mmol) at 0  C 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 (3  15 mL) and the

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column chromatography eluting with ethyl acetate-hexane (30e50%) give the desired pyrazolo-benzothiazole hybrid (7e26) in 75e90% yield. 4.2.1. N-(benzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole-4carboxamide (7) White solid, yield 88%, Mp: 180e182  C; 1H NMR (400 MHz, CDCl3): d 8.93 (s, 1H), 8.09 (d, J ¼ 8.43 Hz, 1H), 7.95e7.91 (m, 2H), 7.87 (d, J ¼ 7.70 Hz, 2H), 7.60e7.53 (m, 3H), 7.49e7.41 (m, 7H). 13C NMR (100 MHz, CDCl3): d 160.6, 158.7, 155.4, 143.4, 138.7, 133.2, 130.5, 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 [MþH]þ. HRMS (ESI) calcd for C23H16N4OS [MþH]þ 397.1123; found: 397.1127. 4.2.2. N-(6-fluorobenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole4-carboxamide (8) White solid, yield 86%, Mp: 164e166  C; 1H NMR (500 MHz, CDCl3): d 8.93 (s, 1H), 8.08 (d, J ¼ 7.54 Hz, 1H), 7.95e7.92 (m, 2H), 7.87 (d, J ¼ 7.78 Hz, 2H), 7.59e7.52 (m, 3H), 7.49e7.42 (m, 6H). 13C NMR (100 MHz, CDCl3): d 158.5, 155.6, 143.4, 138.6, 133.3, 130.5, 129.7, 129.4, 129.1, 128.8, 128.7, 128.3, 128.2, 124.7, 121.0 (d, JCF ¼ 5.86 Hz), 120.4, 119.8, 119.4, 108.3, 106.7; MS (ESI): m/z 415 [MþH]þ. 4.2.3. N-(6-chlorobenzo[d]thiazol-2-yl)-1,3-diphenyl-1H-pyrazole4-carboxamide (9) White solid, yield 79%, Mp: 170e172  C; 1H NMR (500 MHz, CDCl3): d 8.92 (s, 1H), 8.08 (d, J ¼ 8.39 Hz, 1H), 7.95e7.92 (m, 2H), 7.87 (d, J ¼ 7.93 Hz, 2H), 7.59e7.52 (m, 3H), 7.49e7.42 (m, 6H). 13C NMR (100 MHz, CDCl3): d 158.5, 155.7, 143.4, 138.7, 133.3, 130.6, 129.8, 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 [MþH]þ. 4.2.4. N-(6-methoxybenzo[d]thiazol-2-yl)-1,3-diphenyl-1Hpyrazole-4-carboxamide (10) White solid, yield 82%, Mp: 160e162  C; 1H NMR (400 MHz, CDCl3): d 8.75 (s, 1H), 7.82 (d, J ¼ 7.82 Hz, 1H), 7.76e7.70 (m, 4H), 7.59 (d, J ¼ 8.07 Hz, 1H), 7.48 (t, J ¼ 7.58, 15.77 Hz, 2H), 7.36 (t, J ¼ 7.21, 13.93 Hz, 2H), 7.29 (t, J ¼ 6.96, 15.28 Hz, 1H), 7.04 (d, J ¼ 8.80 Hz, 2H), 3.87 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.7, 160.6, 158.9, 152.4, 147.5, 139.0, 131.7, 131.4, 130.5, 129.5, 127.6, 126.2, 123.9, 123.7, 121.3, 120.2, 119.4, 115.0, 114.3, 55.3; MS (ESI): m/ z 427 [MþH]þ. HRMS (ESI) calcd for C24H18N4O2S [MþH]þ 427.1229; found: 427.1222.

Fig. 11. Angiogenesis assay in transgenic Tg(fli1a:EGFP) zebrafish embryos. Embryos were treated with different concentrations of compound 14 and axitinib (positive control) at 24 hpf. A) The images of zebrafish larvae at 4 dpf were captured using a fluorescence microscope in the green channel. (A-A0 ) control embryos, (BeB0 ) vehicle treated control embryos, (CeC0 to E-E0 ) compound 14 treated embryos at 0.1, 0.5 and 1 mM, respectively, (FeF0 to HeH0 ) axitinib treated embryos at 0.1, 0.5 and 1 mM concentrations, respectively. Inhibition of Intersegmental vessels (ISV) sprouting is marked with an asterisk. The magnified portion of images A-H are shown in A0 -H0 , respectively. Scale bar for A-H; 250 mm, A0 -H'; 450 mm. B) the percentage of defective intersegmental vessel (ISV) were calculated manually. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

combined extracts were washed with NaCl solution. The organic layer was separated, dried over anhydrous Na2SO4 and the solvent removed by evaporation. The crude product was purified by

4.2.5. N-(6-methylbenzo[d]thiazol-2-yl)-1,3-diphenyl-1Hpyrazole-4-carboxamide (11) White solid, yield 89%, Mp: 206e208  C; 1H NMR (400 MHz, CDCl3): d 11.04 (bs, 1H), 8.44 (s, 1H), 7.72 (d, J ¼ 7.94 Hz, 2H), 7.59 (s, 1H), 7.53e7.40 (m, 7H), 7.34 (t, J ¼ 7.21, 14.30 Hz, 1H), 7.23 (d, J ¼ 8.19 Hz, 1H), 7.04 (d, J ¼ 8.31 Hz, 1H), 2.39 (s, 3H). 13C NMR (100 MHz, CDCl3): d 161.0, 158.5, 152.3, 145.4, 138.8, 133.9, 131.8, 131.3, 131.0, 129.4, 129.2, 128.7, 128.7, 127.7, 127.5, 121.0, 119.6, 119.3, 115.6, 21.3; MS (ESI): m/z 411 [MþH]þ. HRMS (ESI) calcd for C24H18N4OS [MþH]þ 411.1280; found: 411.1280. 4.2.6. N-(benzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1-phenyl-1Hpyrazole-4-carboxamide (12) White solid, yield 83%, Mp: 172e174  C; 1H NMR (400 MHz, CDCl3): d 8.92 (s, 1H), 8.09 (dt, J ¼ 8.43 Hz, 1H), 7.95 (dd, J ¼ 5.38, 8.92 Hz, 2H), 7.87e7.85 (m, 2H), 7.60e7.53 (m, 3H), 7.49e7.43 (m, 3H), 7.12 (t, J ¼ 8.80, 17.60 Hz, 2H); MS (ESI): m/z 415 [MþH]þ.

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4.2.7. N-(6-fluorobenzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1phenyl-1H-pyrazole-4-carboxamide (13) White solid, yield 80%, Mp: 179e181  C; 1H NMR (400 MHz, CDCl3): d 8.92 (s, 1H), 8.09 (dt, J ¼ 8.43 Hz, 1H), 7.94 (dd, J ¼ 5.38, 8.92 Hz, 2H), 7.88e7.84 (m, 2H), 7.59e7.53 (m, 3H), 7.49e7.42 (m, 3H), 7.12 (t, J ¼ 8.80, 17.60 Hz, 2H); MS (ESI): m/z 433 [MþH]þ. HRMS (ESI) calcd for C23H14F2N4OS [MþH]þ 433.0935; found: 433.0934.

4.2.13. N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-chlorophenyl)-1phenyl-1H-pyrazole-4-carboxamide (19) White solid, yield 79%, Mp: 188e190  C; 1H NMR (400 MHz, CDCl3): d 8.93 (s, 1H), 8.09 (d, J ¼ 8.43 Hz, 1H), 7.91 (d, J ¼ 8.55 Hz, 2H), 7.87e7.84 (m, 2H), 7.60e7.53 (m, 3H), 7.49e7.43 (m, 3H), 7.41 (d, J ¼ 8.68 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 158.5, 154.5, 143.4, 138.5, 135.6, 133.4, 130.4, 129.8, 129.0, 128.8, 128.7, 128.5, 128.4, 124.8, 120.5, 119.9, 108.2, 106.7; MS (ESI): m/z 465 [MþH]þ.

4.2.8. N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-1phenyl-1H-pyrazole-4-carboxamide (14) White solid, yield 78%, Mp: 218e220  C; 1H NMR (400 MHz, CDCl3): d 8.93 (s, 1H), 8.10 (d, J ¼ 8.54 Hz, 1H), 7.95 (dd, J ¼ 5.34, 8.85 Hz, 2H), 7.89e7.85 (m, 2H), 7.59e7.54 (m, 3H), 7.49e7.42 (m, 3H), 7.12 (t, J ¼ 8.69, 17.39 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 163.5 (d, JCF ¼ 250.15 Hz), 158.6, 154.7, 143.4, 138.6, 133.3, 131.1 (d, JCF ¼ 8.80 Hz), 129.8, 129.6, 128.8, 128.8, 128.5, 126.7 (d, JCF ¼ 2.93 Hz), 124.8, 120.5, 119.9, 119.4, 115.3 (d, JCF ¼ 22.00 Hz), 108.3, 106.6; MS (ESI): m/z 449 [MþH]þ. HRMS (ESI) calcd for C23H14ClFN4OS [MþH]þ 449.0639; found: 449.0652.

4.2.14. 3-(4-chlorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (20) White solid, yield 76%, Mp: 193e195  C; 1H NMR (400 MHz, CDCl3): 8.45 (s, 1H), 7.72 (d, J ¼ 8.31 Hz, 2H), 7.53 (d, J ¼ 7.70 Hz, 2H), 7.46e7.41 (m, 4H), 7.35 (t, J ¼ 7.21, 14.55 Hz, 1H), 7.31e7.27 (m, 2H), 6.87 (dd, J ¼ 2.44, 8.80 Hz, 1H), 3.82 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.5, 156.8, 151.2, 141.8, 138.8, 135.5, 133.1, 131.0, 130.2, 129.8, 129.5, 129.0, 127.7, 120.9, 119.3, 115.5, 115.1, 104.3, 55.8; MS (ESI): m/z 461 [MþH]þ.

4.2.9. 3-(4-fluorophenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (15) White solid, yield 80%, Mp: 248e250  C; 1H NMR (400 MHz, CDCl3): d 8.50 (s, 1H), 7.77 (dd, J ¼ 5.25, 8.68 Hz, 2H), 7.60 (d, J ¼ 7.45 Hz, 2H), 7.46 (t, J ¼ 7.45, 15.65 Hz, 2H), 7.40 (d, J ¼ 8.80 Hz, 1H), 7.37 (t, J ¼ 7.33, 14.67 Hz, 1H), 7.28 (d, J ¼ 2.56 Hz, 1H), 7.21 (t, J ¼ 8.68, 17.36 Hz, 2H), 6.92 (dd, J ¼ 2.44, 8.80 Hz, 1H), 3.84 (s, 3H). 13 C NMR (100 MHz, CDCl3): d 163.4 (d, JCF ¼ 249.41 Hz), 160.7, 157.1, 156.8, 151.5, 141.7, 138.7, 133.0, 130. (d, JCF ¼ 8.80 Hz), 127.6, 127.5 (d, JCF ¼ 2.93 Hz), 120.8, 119.3, 115.8 (d, JCF ¼ 21.27 Hz), 115.3, 115.1, 104.3, 55.7; MS (ESI): m/z 445 [MþH]þ. 4.2.10. 3-(4-fluorophenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (16) White solid, yield 83%, Mp: 230e232  C; 1H NMR (400 MHz, CDCl3): d 8.40 (s, 1H), 7.78 (dd, J ¼ 5.25, 8.68 Hz, 2H), 7.60 (s, 2H), 7.46 (d, J ¼ 7.82, 2H), 7.40 (t, J ¼ 7.21, 15.28, 2H), 7.33 (t, J ¼ 7.21, 14.30 Hz, 1H), 7.28 (d, J ¼ 8.43 Hz, 1H), 7.17 (t, J ¼ 8.68, 17.23 Hz, 2H), 7.06 (d, J ¼ 8.31 Hz, 1H), 2.39 (s, 3H). 13C NMR (100 MHz, CDCl3): d 163.3 (d, JCF ¼ 249.76 Hz), 160.9, 158.6, 151.7, 145.3, 138.7, 134.1, 131.8, 130.8 (d, JCF ¼ 8.17 Hz), 129.4, 127.8, 127.6, 127.5, 121.1, 119.7, 119.3, 115.7 (d, JCF ¼ 21.99 Hz), 115.3, 21.3; MS (ESI): m/z 429 [MþH]þ. HRMS (ESI) calcd for C24H17FN4OS [MþH]þ 429.1185; found: 429.1186. 4.2.11. N-(benzo[d]thiazol-2-yl)-3-(4-chlorophenyl)-1-phenyl-1Hpyrazole-4-carboxamide (17) White solid, yield 88%, Mp: 174e176  C; 1H NMR (400 MHz, CDCl3): d 8.93 (s, 1H), 8.09 (d, J ¼ 8.43 Hz, 1H), 7.91 (d, J ¼ 8.68 Hz, 2H), 7.87e7.84 (m, 2H), 7.60e7.53 (m, 3H), 7.49e7.43 (m, 3H), 7.41 (d, J ¼ 8.68 Hz, 2H). MS (ESI): m/z 431 [MþH]þ. 4.2.12. 3-(4-chlorophenyl)-N-(6-fluorobenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (18) White solid, yield 82%, Mp: 178e180  C; 1H NMR (400 MHz, CDCl3): d 8.92 (s, 1H), 8.09 (d, J ¼ 8.43 Hz, 1H), 7.90 (d, J ¼ 8.68 Hz, 2H), 7.87e7.84 (m, 2H), 7.60e7.53 (m, 3H), 7.48 (t, J ¼ 0.85, 1.72 Hz, 1H), 7.47e7.43 (m, 2H), 7.40 (d, J ¼ 8.68 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 158.5, 154.5, 143.4, 138.5, 135.6, 133.4, 130.6, 130.4, 129.8, 129.6, 129.0, 128.8, 128.5, 128.4, 124.8, 120.5, 119.8, 108.3, 106.6; MS (ESI): m/z 449 [MþH]þ.

4.2.15. 3-(4-chlorophenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (21) White solid, yield 82%, Mp: 220e222  C; 1H NMR (400 MHz, CDCl3): d 8.42 (s, 1H), 7.73 (d, J ¼ 8.43 Hz, 2H), 7.60 (s, 1H), 7.47e7.37 (m, 6H), 7.34 (t, J ¼ 7.21, 13.69 Hz, 1H), 7.25 (d, J ¼ 5.62 Hz, 1H), 7.05 (d, J ¼ 8.12 Hz, 1H), 2.39 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.0, 159.1, 151.7, 145.0, 138.7, 135.2, 134.1, 131.6, 130.7, 130.2, 130.0, 129.4, 128.6, 127.8, 127.6, 121.1, 119.4, 119.2, 115.3, 21.3; MS (ESI): m/z 445 [MþH]þ. HRMS (ESI) calcd for C24H17ClN4OS [MþH]þ 445.1134; found: 445.1136. 4.2.16. N-(benzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1-phenyl1H-pyrazole-4-carboxamide (22) White solid, yield 80%, Mp: 198e200  C; 1H NMR (400 MHz, CDCl3): d 8.75 (s, 1H), 7.82 (d, J ¼ 7.58 Hz, 1H), 7.76e7.71 (m, 4H), 7.59 (d, J ¼ 8.07 Hz, 1H), 7.48 (t, J ¼ 7.58, 15.77 Hz, 2H), 7.37 (t, J ¼ 7.21, 15.03 Hz, 2H), 7.29 (t, J ¼ 6.96, 15.28 Hz, 1H), 7.03 (d, J ¼ 8.80 Hz, 2H), 3.87 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.7, 160.6, 158.9, 152.4, 147.5, 139.0, 131.7, 131.4, 130.5, 129.5, 127.6, 126.2, 123.9, 123.7, 121.3, 120.2, 119.4, 115.0, 114.3, 55.3; MS (ESI): m/ z 427 [MþH]þ. HRMS (ESI) calcd for C24H18N4O2S [MþH]þ 427.1229; found: 427.1222. 4.2.17. N-(6-fluorobenzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1phenyl-1H-pyrazole-4-carboxamide (23) White solid, yield 78%, Mp: 169e171  C; 1H NMR (400 MHz, CDCl3): d 8.91 (s, 1H), 8.08 (d, J ¼ 8.43 Hz, 1H), 7.91 (d, J ¼ 8.92 Hz, 2H), 7.86 (d, J ¼ 7.58 Hz, 2H), 7.59e7.52 (m, 3H), 7.50e7.41 (m, 3H), 6.96 (d, J ¼ 8.80 Hz, 2H), 3.83 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.6, 158.7, 155.4, 143.4, 138.7, 133.2, 130.5, 129.7, 129.5, 128.9, 128.6, 128.2, 124.7, 123.0, 120.4, 119.8, 119.4, 114.3, 113.6, 108.4, 106.3, 55.2; MS (ESI): m/z 445 [MþH]þ. 4.2.18. N-(6-chlorobenzo[d]thiazol-2-yl)-3-(4-methoxyphenyl)-1phenyl-1H-pyrazole-4-carboxamide (24) White solid, yield 81%, Mp: 193e195  C; 1H NMR (400 MHz, CDCl3): d 8.90 (s, 1H), 8.08 (d, J ¼ 8.39 Hz, 1H), 7.91 (d, J ¼ 8.85 Hz, 2H), 7.86 (d, J ¼ 7.78 Hz, 2H), 7.59e7.52 (m, 3H), 7.50e7.41 (m, 3H), 6.95 (d, J ¼ 8.85 Hz, 2H), 3.82 (s, 3H). 13C NMR (100 MHz, CDCl3): d 160.6, 158.6, 155.4, 143.4, 138.7, 133.2, 130.5, 129.7, 128.9, 128.6, 128.3, 124.7, 123.0, 120.4, 119.8, 119.3, 114.4, 113.6, 108.3, 106.3, 55.2; MS (ESI): m/z 461 [MþH]þ. HRMS (ESI) calcd for C24H17ClN4O2S [MþH]þ 461.0839; found: 461.0842.

V.G. Reddy et al. / European Journal of Medicinal Chemistry 182 (2019) 111609

4.2.19. 3-(4-methoxyphenyl)-N-(6-methylbenzo[d]thiazol-2-yl)-1phenyl-1H-pyrazole-4-carboxamide (26) White solid, yield 88%, Mp: 237e239  C; 1H NMR (400 MHz, CDCl3): d 10.88 (bs, 1H), 8.45 (s, 1H), 7.65 (d, J ¼ 8.55 Hz, 2H), 7.59 (s, 1H), 7.53 (d, J ¼ 7.82 Hz, 2H), 7.42 (t, J ¼ 7.58, 14.52 Hz, 2H), 7.33 (t, J ¼ 7.21, 14.18 Hz, 1H), 7.28 (d, J ¼ 8.55 Hz, 1H), 7.05 (d, J ¼ 8.31 Hz, 1H), 6.99 (d, J ¼ 8.55 Hz, 2H), 3.83 (s, 3H), 2.40 (s, 3H). 13C NMR (100 MHz, CDCl3): d 161.1, 160.3, 158.4, 152.1, 145.4, 138.9, 133.9, 131.8, 131.0, 130.1, 129.3, 127.7, 127.4, 123.7, 121.0, 119.6, 119.3, 115.3, 114.1, 55.2, 21.3; MS (ESI): m/z 441 [MþH]þ. HRMS (ESI) calcd for C25H20N4O2S [MþH]þ 441.1385; found: 441.1386. 4.3. Biology 4.3.1. 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% pencillinstreptomycin (PS). All the cell lines were grown in an incubator with 75% humidity and 5% CO2 at 37  C. 0.25% trypsinethylenediaminetetraacetic 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 mM) for 72 h. Then, the medium containing compounds was removed and replaced with 100 mL of MTT solution (5 mg/mL) and the cells were further incubated for 4 h in dark at 37  C. The unreacted MTT solution was removed and 100 mL 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 IC50 values for each compound were calculated. All the 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 h incubation, the culture medium was replaced with medium containing increasing concentrations (1, 3, 5 and 10 mM) 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

13

wounded with 200 mL 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 mM) 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 1  106/ well and were grown overnight in an incubator. The cells were then incubated with 1, 3 and 5 mM 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  C, 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. 10000 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 33242. In this assay, PC-3 cells were grown on cover slips in a 6 well plate at a density of 1  106 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 mg/mL Hoechst 33242 for 20 min 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 PC-3 Cells were grown in 24-well plates (5  105 cells/mL) and incubated with different concentrations (1, 3 and 5 mM) of compound 14. After 48 h incubation, the medium containing the compound was replaced with 500 mL of fresh medium containing 5 mg/ 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 (BIORAD). 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 mM) for 48 h. After incubation, the cells were harvested and stained with a 10 mM solution of carboxy-DCFDA in PBS for 20 min at 37  C. 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 PC-3 cells (1  106/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 mL of annexin V and 1 mL 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)

14

V.G. Reddy et al. / European Journal of Medicinal Chemistry 182 (2019) 111609

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 mL of the compound (1 nM, 10 nM, 100 nM, 1 mM, 10 mM) was added to a 45 mL of reaction mixture (40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 10 mM ATP, Kinase substrate and VEGFR) and incubated at 30  C for 45 min. After the enzymatic reaction, 50 mL 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 nonlinear 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 €dinger package. Regarding the ligands, molecules from the Schro 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 mM) 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 the authors are thankful to CSIR, New Delhi for the financial support under the 12th Five Year Plan projects “Affordable Cancer Therapeutics (ACT)” (CSC0301) & ORIGIN (CSC108). 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 r Facility (ANFF). The authors are also thankful to Dr. Steven H. Prive (RMIT University, Australia) for proof reading the manuscript.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111609.

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