Discovery of novel thiazolyl-pyrazolines as dual EGFR and VEGFR-2 inhibitors endowed with in vitro antitumor activity towards non-small lung cancer

Abstract New series of thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) have been synthesised and assessed for their potential EGFR and VEGFR-2 inhibitory activities. Compounds 10b and 10d exerted potent and selective inhibitory activity towards the two receptor tyrosine kinases; EGFR (IC50 = 40.7 ± 1.0 and 32.5 ± 2.2 nM, respectively) and VEGFR-2 (IC50 = 78.4 ± 1.5 and 43.0 ± 2.4 nM, respectively). The best anti-proliferative activity for the examined thiazolyl-pyrazolines was observed against the non-small lung cancer cells (NSCLC). Compounds 10b and 10d displayed pronounced efficacy against A549 (IC50 = 4.2 and 2.9 µM, respectively) and H441 cell lines (IC50 = 4.8 and 3.8 µM, respectively). Moreover, our results indicated that 10b and 10d were much more effective towards EGFR-mutated NSCLC cell lines (NCI-H1650 and NCI-H1975 cells) than gefitinib. Finally, compounds 10b and 10d induce G2/M cell cycle arrest and apoptosis and inhibit migration in A549 cancerous cells.

Accordingly, interruption of the EGFR signalling pathway using small-molecule tyrosine kinase inhibitors (TKIs) is considered as a well-established therapeutic approach in cancer treatment since the approval of gefitinib by the United States Food and Drug Administration (FDA) in 2003 9,10 .
Gefitinib and erlotinib ( Figure 1) are first-generation EGFR inhibitors that have been approved for the treatment of advanced NSCLC with activating mutations in the EGFR tyrosine kinase domain mainly: L858R (EGFR L858R ) [11][12][13][14] . However, some patients develop secondary drug resistance mutations which results in a relapse after nearly one year of treatment 15 . The "gatekeeper" mutation is the predominant one which is a single point substitution of Thr790 with Met in exon 20 (EGFR T790M ) 16 . Hence, afatinib and dacomitinib ( Figure 1) have been developed as irreversible second generation EGFR inhibitors 17,18 because they have electrophilic Michael-acceptor systems, such as acrylamide moiety which forms a covalent bond with Cys797 19 . This resulted in blocking of T790M resistance mutation by increasing target residence time 20 .
Despite the promising in vitro results in patients with erlotinibresistant cancers 21 , they showed poor efficacy at clinically achievable concentration 22 . This is due to the narrow therapeutic window, as the dose required for the inhibition of mutated EGFR T790M resulted in severe side effects as rash and diarrhoea from inhibiting EGFR wt23 . Recently, this problem was solved by the discovery of third generation EGFR inhibitors as osimertinib (TagrissoV R , AZD9291), rociletinib (CO-1686), naquotinib (ASP8273) and nazartinib (EGF816) 24,25 (Figure 1), however, a resistance has been developed too [26][27][28] . So, exploring more efficient EGFR inhibitors is an urgent and critical demand to surmount the continual evolution of resistance to the current inhibitors.
Besides EGFR, there are other RTKs that have been shown to be important targets in cancer 29 . For example, the RTK vascular endothelial growth factor receptor-2 (VEGFR-2) stands out as a key target in cancer treatment due to its important role in angiogenesis 30 . Based on the functional crosstalk of EGFR and VEGFR-2 through shared common downstream signalling pathways 31 , it is inferred that the simultaneous inhibition of both EGFR and VEGFR is an effective approach for overcoming the reported resistance in NSCLC 32 . In fact, it has been shown that VEGFR-2 inhibition enhances the cytotoxic effect of EGFR inhibitors, whereas, VEGFR-2 activation results in accelerated tumour growth independent of EGFR signalling, thereby facilitating the emergence of resistance to EGFR inhibitors 33 . Vandetanib (Caprelsa V R ) is an example of FDA approved quinazoline-based drug with dual EGFR and VEGFR-2 inhibitory activity 34 (Figure 1).
In the current medical era, thiazole is identified as an important heterocyclic motif that emerged as a promising privileged scaffold in the anticancer drug discovery [35][36][37] , Several FDA approved anticancer drug incorporate the heterocycle thiazole, such as dabrafenib and dasatinib. Pyrazoline moiety, on the other hand, is a highly active heterocyclic nucleus possessing interesting biological activities 38 like; anticancer [39][40][41] , anti-inflammatory 42 , antidepressant 43 , anticonvulsant 44 and antimicrobial activities 45 .
Recently, some research groups have devoted their efforts to hybridise both the thiazole and pyrazoline into a single frame work, producing novel series based on the thiazolyl-pyrazoline system which thereafter identified as a substantial scaffold in anticancer drug discovery 46 . Interestingly, there is a large number of reported compounds with potent cytotoxic effect based on thiazolyl-pyrazoline scaffold having EGFR (compounds I, II and III, Figure 2) [47][48][49] and VEGFR-2 (compound IV, Figure 2) 50 suppressing potentials. It is worth mentioning that none of these studies have developed dual or multiple kinase inhibitors, in addition they have not evaluated the biological activity against EGFR-mutated cancer cell lines.
Motivated by the above mentioned findings, here we sought to develop new different sets of novel thiazolyl-pyrazoline-based small molecules (7a-7d, 10a-10d and 13a-13f) as potential dual EGFR/VEGFR-2 inhibitors with improved anticancer activities based on the molecular hybridisation between thiazole and pyrazoline motifs ( Figure 2).
It is worth to mention that the reported pharmacophoric features required for EGFR inhibition include a central flat hetero-aromatic ring scaffold that should occupy the adenine binding pocket, a terminal hydrophobic head that occupies the hydrophobic sub-pocket, as well as a hydrophobic tail to occupy a second hydrophobic region 51,52 . Additionally, certain pharmacophoric features have been identified for the VEGFR-2 inhibitors including a flat hetero-aromatic moiety to be fitted in the ATP binding region, a terminal hydrophobic motif to achieve several hydrophobic interactions in the allosteric hydrophobic pocket, and the presence of H-bond acceptor (HBA) functionalities [53][54][55] . Interestingly, the target thiazolyl-pyrazoline-based small molecules (7a-7d, 10a-10d and 13a-13f) achieved these features required for inhibition of both EGFR and VEGFR-2. Furthermore, the substitution pattern on the pendant phenyl moieties was selected so as to ensure different electronic and lipophilic environments which should manipulate the activity of the target thiazolyl-pyrazolines.
Once created, all derivatives were assessed for their potential inhibitory activity towards EGFR and VEGFR-2 kinases. Also, the inhibitory activities against a panel of 11 kinases (TEK, SYK, EPHB2, ABL1, LCK, CLK1, ROCK1, PKC, AKT1, CDK1, and CDK5) were further investigated to determine the selectivity profile. Then, all compounds were screened for their cytotoxic effect against nine cell lines derived from five tumour subpanels including leukaemia (K562, and KG-1a), breast (MCF-7, BT-549, and HCC70), lung (A549 and H441), colon (HCT116) and liver (HepG2) cancer cell lines. In addition, the anti-proliferative potential of the most potent EGFRWT inhibitors was analysed towards two EGFR mutated cancer cell lines; NCI-H1650 and NCI-H1975. Thereafter, the most efficient cells growth and kinase inhibitors 10b and 10d were selected to explore their possible cellular mechanism of action via cell cycle, apoptosis Annexin-V-FITC and migration assays in NSCLC A549 cells. Finally, in silico studies were carried out to explore the binding interactions of thiazolyl-pyrazolines 10b and 10d within the vicinity of ATP-binding sites on EGFR and VEGFR-2 kinase domains.

Chemistry
The synthetic pathways adopted for preparation of the intermediates 5a-b and the target thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d and 13a-13f) were depicted in Schemes 1 and 2. In Scheme 1, chalcones 3a, b were prepared via reaction of 2,4dichloroacetophenone 1 with the appropriate aromatic aldehydes 2a, b, adopting the base catalysed Claisen-Schmidt condensation reaction as reported 56 (Scheme 1). As active intermediates, chalcones 3a, b were used to prepare pyrazolines 5a, b by the reaction with thiosemicarbazide 4 in the presence of sodium hydroxide as a catalyst (Scheme 1). Pyrazoline formation occurs via two steps; the first one is the condensation reaction between the chalcones 3a, b and thiosemicarbazide 4, whereas, the second step is the intramolecular cyclisation through Michael addition of the NH group to the double bond 57 (Scheme 1). Structures of compounds 5a, b were confirmed by microanalyses and spectral data. IR spectra of 5a, b showed NH 2 bands at 3418, 3444 and 3247, 3322 cm À1 , respectively, in addition to two bands at 1236, 1254 and 828, 833 cm À1 referring to C¼S. 1   the presence of two diastereotopic protons at C-4 (H A and H M ) and one single proton at C-5 (H X ) 58  Furthermore, reaction of carbothioamide derivatives 5a, b with 1-aryl-2-bromoethanones 6a, b in refluxing ethanol yielded the corresponding 4-(aryl)-2-(3-(2,4-dichlorophenyl)-5-(4-aryl)-4,5-dihydro-1H-pyrazol-1-yl)thiazoles 7a-7d, respectively, via Hantzsch thiazole synthesis 54,55 (Scheme 1). Initially, nucleophilic substitution of Br in phenacyl bromide by the S-atom of thioamide generates the isothiourea, which subsequently undergoes cyclocondensation and water elimination to furnish the thiazole ring 57 (Scheme 1). The latter synthesised compounds 7a-7d were confirmed based on their elemental analysis and spectral data. IR spectra showed the disappearance of the characteristic band of NH 2 in the region of 3418, 3444 and 3247, 3322 cm À1 . Their 1 H NMR spectra showed an increase in the aromatic integration due to thiazole proton and protons of the extra phenyl ring at d 6.91-7.82 ppm. 13 C NMR spectra of 7b, c showed peak around d 105 ppm corresponding to C-5 of thiazole ring along with an increase in signals assigned for aromatic carbons at d 111.6-164.8 ppm.
IR spectra of 10a-10d showed a band at 1637-1669 cm À1 due to C¼O group, while the spectra of 10c, d revealed a band at 3257 and 3262 cm À1 , respectively corresponding to NH group. 1 H NMR spectra of 10a-10d showed a signal at d 2.36-2.54 ppm attributed to the protons of the methyl group at the 4-position of the thiazole ring. Moreover, 1 H NMR spectrum of 10a showed a singlet signal at d 2.40 ppm attributed to CH 3 protons of the acetyl group. Furthermore, 1 H NMR spectrum of 10b showed two signals at d 1.35 and 4.29 ppm, respectively attributed to the CH 3 and CH 2 protons of the ethyl group. Additionally, 1 H NMR spectra of 10c, d displayed a D 2 O exchangeable singlet signal of NH proton at d 9.72, 8.14 ppm, respectively, in addition to an increase in the aromatic integration at d 6.78-7.81 ppm assignable to the extra phenyl ring. 13 C NMR of compound 10a showed signals assigned to CH 3 carbon of the thiazole ring, CH 3 and C¼O carbons of the acetyl group at d 19.1, 30.1 and 189.7 ppm, respectively. Additionally, 13 C NMR spectrum of 10b displayed signals attributed to ethyl carbons at d 14.7 and 60.7 ppm along with signal due to ester carbonyl carbon at d 165.3 ppm. Furthermore, 13 C NMR spectrum of 10d revealed signals at d 18.2 and 163.8 ppm due to CH 3 carbon of the thiazole ring and C¼O, respectively.
IR spectra of compounds 13a-13f showed a band at 1582-1589 cm À1 referring to (N ¼ N) in addition to the absence of the band corresponding to NH 2 group at 3418, 3444 and 3247, 3322 cm À1 . 1 H NMR spectra of 13b, e, f revealed singlet signals at d 2.53-2.61 ppm equivalent to CH 3 protons of the thiazole ring in addition to an increase in the integration of the aromatic protons at d 6.78-7.90 ppm. Also, 1 H NMR spectra of 13b, e showed additional singlet signals at d 2.42 and 2.41 ppm attributed to the three protons of 4-CH 3 , respectively. 13 C NMR of 13a-13e showed a signal for CH 3 carbon of the thiazole ring at d 16.1-16.6 ppm along with an increase in the number of signals of aromatic carbons at d 109.4-165.3 ppm. Also, 13 C NMR of 13b, e revealed the presence of a signal at d 21.4 ppm indicating an additional CH 3 carbon at the phenyldiazenyl ring.

EGFR and VEGFR-2 kinase inhibitory activities of thiazolylpyrazoline derivatives
In the current study, the potential inhibitory activity of target thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d and 13a-13f) towards EGFR and VEGFR-2 were examined, with the aim of exploring the plausible mechanism. The IC 50 values of the tested compounds were evaluated compared to reference EGFR inhibitor (gefitinib) and VEGFR-2 inhibitor (vandetanib), Table 1.
The following structure-activity relationships (SARs) can be concluded from the kinases inhibition data displayed in Table 1. Regarding the EGFR inhibitory activity of the first series 7a-7d, it was found that substitution of the pendant phenyl ring at C-4 of the thiazole moiety (compounds 7b and 7d; IC 50 ¼ 114.2 ± 0.4 and 132.9 ± 1.2 nM, respectively) elicited an enhancement of effectiveness towards EGFR in comparison to the unsubstituted analogues (compounds 7a and 7c; IC 50 ¼ 145.1 ± 2.0 and 511.2 ± 3.2 nM, respectively). In contrast, the 3,4-dimethoxy substitution for the pyrazoline C-5 phenyl ring (compounds 7c and 7d; IC 50 ¼ 511.2 ± 3.2 and 132.9 ± 1.2 nM, respectively) resulted in a worsening of inhibitory activity against EGFR in comparison to the 4-methoxy monosubstituted counterparts (compounds 7a and 7b; IC 50 ¼ 145.1 ± 2.0 and 114.2 ± 0.4 nM, respectively).
On the other hand, the SAR outcomes for VEGFR-2 inhibition pointed out that utilisation of the ester group at C-5 of the thiazole moiety within the second series 10a-10d (compound 10 b; IC 50 ¼ 78.4 ± 1.5 nM) led to an enhanced activity more than the acetyl (compound 10a; IC 50 ¼ 450.3 ± 8.3 nM) and amide (compound 10c; IC 50 ¼ 102.2 ± 4.3 nM) groups. Similar to the SAR for EGFR inhibition, grafting a second methoxy group for the amide-bearing counterpart 10c (IC 50 ¼ 102.2 ± 4.3 nM) produced compound 10d that identified as the most potent VEGFR-2 inhibitor in this work (IC 50 ¼ 43.0 ± 2.4 nM). Further analysis of the VEGFR-2 inhibition data for the third series 13, highlighted that both the para-substitution of the appended phenyl ring at C-5 of the thiazole moiety, and the 3,4-dimethoxy substitution for the pyrazoline C-5 phenyl ring resulted in an improvement of the VEGFR-2 inhibitory activity.

Western blotting of EGFR and VEGFR-2 in A549 cells
To confirm the ability of thiazolyl-pyrazolines derivatives to dual inhibit the activity of EGFR and VEGFR-2 kinases, we examined the effect of the most potent compounds 10b and 10d on the expression levels of p-EGFR, EGFR, p-VEGFR-2 and VEGFR-2 proteins in A549 cells using Western blot analysis. As shown in Figure 3, the protein expression levels of p-EGFR and p-VEGFR-2 were significantly downregulated by compounds 10b and 10d.

Anti-proliferative activity of thiazolyl-pyrazoline derivatives
To evaluate the anti-proliferative activity of the synthesised thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d, and 13a-13f), a preliminary in vitro one dose (10 mM) anticancer assay was performed. Nine cell lines derived from five tumour subpanels were examined; leukaemia (K562 and KG-1a), breast (MCF-7, BT-549 and HCC70), lung (A549 and H441), colon (HCT116) and liver (HepG2) cancer cell lines. The compounds were added at a single concentration (10 mM) and the cultures were incubated for 24 h, then the per cent growth inhibition (%GI) was calculated. As shown in Tables 3 and 4, compounds 10b and 10d revealed promising cell growth inhibition activity against almost all the screened cell lines (%GI ranging from 31.0% to 95.3%), especially against the non-small cell lung cancer A549 and H441 cell lines (%GI ranging from 79.1% to 95.3%). Other compounds showed moderate to weak anti-proliferative effect against the tested cell lines; however, the non-small lung cancer cells (A549 and H441) were more sensitive to the target thiazolyl-pyrazoline derivatives.
Overexpression of EGFR and VEGFR-2 is well-reported in the literature to be significantly correlated with the induction of the NSCLC cells proliferation 31 . Thereafter, the quantitative IC 50 values have been determined for thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d and 13a-13f) towards the most susceptible cell lines at the preliminary assay (non-small lung cancer A549 and H441 cell lines), at testing concentrations of 1, 5, 10, 25, 50 or 100 mM for 24 h. The antitumor drugs gefitinib and vandetanib were used as reference drugs in this MTT cytotoxicity assay ( Table 5).
The results illustrated in Table 5 showed that compounds 10b and 10d have the best anti-proliferative activity, herein reported, against A549 (IC 50 ¼ 4.2 and 2.9 mM, respectively) and H441 (IC 50 ¼ 4.8 and 3.8 mM, respectively) cell lines, whereas compounds 7c and 13a have non-significant anti-proliferative activity (IC 50 more than 100 lM). Other compounds showed moderate to low antiproliferative effect against A549 (IC 50 range: 12.0-59.1 mM) and H441 (IC 50 range: 15.2-94.4 mM). Interestingly, the anti-proliferative effect of compounds 10b and 10d was higher than the reference drug gefitinib and was comparable to vandetanib. In addition, compounds 7a, 7b, 7d, 10c, 13e and 13f exerted moderate anticancer activity against A549 cell lines with IC 50 s values spanning between 12.0 and 23.5 mM, whereas compounds 7b, 7d, 13b and 13d-13f showed moderate growth inhibitory activity towards H441 cells with IC 50 s spanning in the range 15.2-29.1 mM, Table 5.

Effect of thiazolyl-pyrazoline derivatives on EGFR-mutated NSCLC cell lines
The prolonged use of the TKIs targeting EGFR or VEGFR-2 kinases led to drug resistance due to mutations development and toxicity. In this study, we analysed the anti-proliferative potential of the most potent EGFRWT inhibitors 10b and 10d towards two EGFR mutated cancer cell lines; NCI-H1650 possesses exon19 deletion (delE746-A750) mutation of EGFR and NCI-H1975 carries L858R/ T790M double mutation of EGFR. Interestingly, compounds 10b and 10d exhibited significant growth inhibitory activity against NCI-H1650 (IC 50 ¼ 5.7 and 3.5 mM, respectively) and NCI-H1975 (IC 50 ¼ 6.2 and 4.4 mM, respectively) Table 7 and Figure 4.
Moreover, the obtained results indicated that both 10b and 10d were much more sensitive towards NCI-H1650 and NCI-H1975 cells than gefitinib (IC 50 > 20 mM towards the two examined cell lines). Accordingly, 10b and 10d may serve as a lead compounds for the treatment of gefitinib-resistant EGFR mutant NSCLC.

Thiazolyl-pyrazoline derivatives inhibited cancer cell migration
As the thiazolyl-pyrazoline derivatives 10b and 10d showed potent inhibitory effect against VEGFR-2 Kinase that plays an important role in cancer cell migration, we investigated the effect of 10b and 10d on the A549 cancer cell migration using wound healing assay. As shown in Figure 5, compounds 10b and 10d were effective in reducing the cancer cell migration compared to the control, highlighting that the VEGFR-2 kinase inhibitory effect of 10b and 10d is the engine for the inhibition of the cancer cell migration.

Thiazolyl-pyrazoline derivatives induced G 2 /M cell cycle arrest
In general, the anticancer agents inhibit the proliferation of cancerous cells by arresting cell division at various checkpoints. These checkpoints present at G 1 /S phase, S-phase and G 2 /M phases. Therefore, we tested the effect of the most potent compounds (10b and 10d) on cell cycle progression in A549 cell line. Results in Figure 6 obviously indicate that compounds 10b and 10d arrested the cell cycle at a G 2 /M phase (29.2% and 33.92%, respectively) when compared to the untreated controls (6.06% and 5.93%, respectively). Parallel to these findings, the cell population in G 1 and S phases decreased after treatment with 10b and 10d ( Figure 6(A,B)).
On the other hand, gefitinib (20 mM) significantly arrested the cell cycle at G1 phase. This difference between the synthesised compounds and gefitinib in the arrested cell cycle phase could be attributed to the moderate inhibitory effect of 10b and 10d on the CDK1 kinase (IC 50 ¼ 310 and 245 nM, respectively) that regulates the G2/M phase transition. This result strongly suggests that the synthesised thiazolyl-pyrazoline derivatives inhibit the proliferation of A549 cells by arresting cell cycle at G 2 /M phase.

Thiazolyl-pyrazoline derivatives induced apoptosis
Furthermore, the pro-apoptotic effect of compounds 10b and 10d was also investigated on A549 cells using the Annexin V-FITC/PI double staining. The proportion of annexin V-positive apoptotic cells increased gradually from 5.4% at control to 16.5% at 5 lM 10b (Figure 7(A,B)) and from 6.3% at control to 28.5% at 5 lM 10d. These data indicate that the synthesised thiazolyl-pyrazoline derivatives treatment provokes the induction of apoptosis in A549 cells.

Molecular docking
In view of the promising kinase inhibitory activity of the thiazolylpyrazoline derivatives, herein reported, a molecular docking study was carried out for the most potent derivatives 10b and 10d to provide insights for their potential binding interactions within the examined kinases binding sites. The molecular operating environment (MOE) 2019.02 was implemented in the conduction, analysis and visualisation of the entire docking studies. The 3D structural co-ordinates of EGFR and VEGFR-2 were downloaded from the protein databank PDB IDs 1M17 and 4ASD, respectively. Both the selected PDB IDs have excellent resolution of 2.6 Å and 2.03 for 1M17 and 4ASD, respectively. In addition, 1M17 contains EGFR in complex with potent inhibitor erlotinib, while 4ASD contains VEGFR-2 in complex with the potent inhibitor sorafenib, making them optimum choice for the docking studies. Pose retrieval step of the co-crystallised ligands resulted in values of 0.85 and 0.63 Å between the docked and the co-crystallised poses for erlotinib and sorafenib, respectively, indicating a valid docking protocol (Figure 8). Table 5. Inhibitory activities of thiazolyl-pyrazoline derivatives (7a-7d, 10a-10d and 13a-13f)   Moreover, the previous step resulted in an energy scores (S) of À11.7 and À14.9 kcal/mol, for erlotinib and sorafenib, respectively. The retrieved docking scores of the co-crystalized ligands were used as comparative means to benchmark the docking score values of compounds 10b and 10d.

Docking of compounds 10b and 10d into EGFR active site
Visual inspection of erlotinib binding with EGFR active site, revealed the formation of two hydrogen bonds with the key residues Leu768 and Met769, in addition, two carbon hydrogen bonds were noticed with Glu738 and Gln767. Through a water molecule, erlotinib was able also to interact with Cys751 and Thr766 (Figure 9).
Docking simulations for thiazolyl-pyrazoline derivatives 10b and 10d showed that they fit well into the EGFR active site with good docking scores (À12.9 and À14.1 kcal/mol, respectively) comparable to that of erlotinib (À11.7 kcal/mol). The general binding patterns of compounds 10b and 10d are consistent with crystallographic binding mode of erlotinib within the EGFR-TK active site (PDB: 1M17), Figure 10.
For instance, the carbonyl group in the amide functionality of compound 10d was engaged in two hydrogen bonds with the key residues Leu768 and Met769. Similarly, the carbonyl group in the ester functionality of compound 10b was engaged in hydrogen bond interaction with Met769, in addition to three hydrogen bonds with Gln767, Cys751 and Thr766 through a water molecule (Figures 10 and 11).
Compounds 10b and 10d formed a carbon hydrogen bond with Cys773 and Val693, respectively. The ortho-chloride atom of compound 10b formed a halogen interaction with Gly695, while the ortho-chloride atom of compound 10d formed a halogen interaction with Lys692. On the other hand, only compound 10d was engaged in arene bond interactions with Leu694, Lys704 and Leu802, which explains the superior activity and docking score of compound 10d over compound 10b. Also, it highlights the important role of the additional phenyl ring attached to the amide linker in compound 10d in EGFR inhibition (Figures 10 and 11).

Docking of compounds 10b and 10d into VEGFR-2 active site
The efficient VEGFR-2 inhibitory activity of compounds 10b and 10d were illustrated by their good docking scores (À13.7 and À14.4 kcal/mol) comparable to that of sorafenib (À14.9 kcal/mol). As depicted in Figure 12, sorafenib exerts its VEGFR-2 inhibition activity through formation of hydrogen bonds with the essential residues; Glu885, Cys1045, Asp1046 and Cys919, in addition to other multiple interactions.  Interestingly, both compounds 10b and 10d were proven to maintain multiple essential interactions upon docking into VEGFR-2 active site (Figures 13 and 14). For instance, compound 10b was engaged in hydrogen bond interactions with Leu889 and Asp1046 via the nitrogen and sulphur atoms of the thiazole ring, respectively. The nitrogen of pyrazoline ring formed two hydrogen bonds with Cys1045, in addition, the carbonyl group in the ester functional contributed to a hydrogen bond with Phe1047. The orthochloride and the para-methoxy substituents contributed to bonding interactions with Ile888 and Glu885, respectively. In a similar manner, all the previous interactions were achieved by compound 10d in addition to an extra hydrogen bond interaction with  Lys868, in addition to an arene interaction with Leu1035 ( Figures  13 and 14).

Conclusion
In conclusion, this study reports the facile synthesis of potent anticancer series of thiazolyl-pyrazoline derivatives acting as dual EGFR/VEGFR-2 inhibitors with significant proapoptotic properties. All the synthesised thiazolyl-pyrazolines have been screened for their inhibitory activities against EGFR and VEGFR-2 kinases and for their cytotoxic effect against nine cell lines derived from five tumour subpanels. Compounds 10b and 10d displayed potent and selective inhibitory activity towards EGFR-TK (IC 50 ¼ 40.7 ± 1.0 and 32.5 ± 2.2 nM, respectively) and VEGFR-2 (IC 50 ¼ 78.4 ± 1.5 and 43.0 ± 2.4 nM, respectively). Furthermore, compounds 10b and 10d showed a promising anticancer activity against non-small lung cell lines as they were the most potent derivatives with IC 50 values equal 4.2 and 2.9 mM against A549 cells, and equal 4.8 and 3.8 mM against H441 cells, respectively. Moreover, our results indicated that 10b and 10d were much effective towards EGFR-mutated NSCLC cell lines (NCI-H1650 and NCI-H1975 cells) than gefitinib. Finally, compounds 10b and 10d induce G 2 /M cell cycle arrest and apoptosis and inhibit migration in A549 cancerous cells. Furthermore, the molecular docking study explored the binding mode and possible different interactions between the target compounds and the active sites of EGFR and VEGFR-2 enzymes. Accordingly, thiazolyl-pyrazoline scaffold can be considered as promising scaffold for further development of more potent dual EGFR and VEGFR-2 inhibitors.

General
Melting points were measured with a Stuart melting point apparatus (Stuart Scientific, Redhill, UK) and were uncorrected. Infra-red (IR) spectra were recorded on FT-IR spectrometer using KBr discs (Perkin Elmer, Waltham, MA). Mass spectra (MS) were performed on a Varian electron impact EI mass spectrometer (EI-MS) at 70 eV at Regional Centre for Mycology and Biotechnology, Al-Azhar University utilising Thermo Scientific ISQ LT mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). NMR Spectra were recorded on a Bruker NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) at Center for Drug Discovery Research and Development, Faculty of Pharmacy, Ain Shams University. 1 H spectra were run at 400 MHz and 13 C spectra were run at 100 MHz in deuterated dimethyl sulfoxide (DMSO-d 6 ) or chloroform CDCl 3 . Chemical shifts are expressed in d values (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Centre for Mycology and Biotechnology, Al-Azhar University utilising Thermo Scientific Flash 2000 elemental analyser (Thermo Fisher Scientific Inc., Waltham, MA). Analytical thin-layer chromatography (TLC) was employed routinely to follow the course of reactions and to check the purity of products using aluminium sheets pre coated with silica gel (Kieselgel, F254, pore size 60 Å, Merck, Darmstadt, Germany) and observed under a UV lamp (short-wavelength, 254 nm). All reagents and solvents were purified and dried by standard techniques.    H), 7.11 (d, J ¼ 16. 8  To a mixture of 3a, b (1 mmol) and thiosemicarbazide 4 (1 mmol) in absolute ethanol (50 ml), sodium hydroxide (0.29 g, 7 mmol) was added. The reaction mixture was heated under reflux with stirring for 6 h. After cooling, the formed product was filtered, washed with ethanol, dried and crystallised from ethanol to give the corresponding pyrazoline derivatives 5a, b, respectively.

General procedure for preparation of compounds 7a-7d
A mixture of carbothioamides 5a, b (1 mmol) and the appropriate 1-aryl-2-bromoethanone 6a, b (1.1 mmol) in absolute ethanol (20 ml) was heated under reflux for 4 h. After cooling, the formed precipitate was filtered and crystallised from ethanol to afford the corresponding compounds 7a-7d.   plated at a density of 1 Â 10 4 cells per well in 96-well plates overnight and then treated with the compounds. After 24 h treatment, 20 lL of MTT solution (2 mg/mL in phosphate-buffered saline [PBS]) was added to each well and the cells were cultured for another 4 h at 37 C. The medium was aspirated and 150 lL DMSO was added to solubilise MTT formazan crystals. The plates were then shaken, and the optical density was determined at 570 nm using an ELISA plate reader (Model 550, Bio-Rad, Hercules, CA). At least three independent experiments were performed. The IC 50 values were calculated using GraphPad Prism 5 (Version 5.01, GraphPad Software, San Diego, CA).

Cell cycle and apoptosis analyses
DNA content was measured using 7-aminoactinmycin D (7AAD) (Biotium, Inc., Hayward, CA) staining and is commonly used for cell cycle phase analysis as previously described 65 . Apoptosis was evaluated using the annexin V/propidium iodide (PI) staining kit (BioLegend, San Diego, CA) according to the manufacturer's instructions and as previously described.

Molecular docking
In this work, all the docking studies were conducted using Molecular Operating Environment (MOE 2019.02) software 67,68 . The crystal structures of EGFR in complex with erlotinib and VEGFR-2 in complex with sorafenib were downloaded from the protein databank PDB IDs 1M17 and 4ASD, respectively. All the receptors and ligands were prepared using the default parameters of MOE software. The binding sites in the two targets were determined by selecting the pocket surrounding the binding domain of the co-crystallised ligands. Prior commencing the docking of compounds 10b and 10d, a pose-retrieval docking experiments for the X-ray coordinates of the co-crystallised ligands in their corresponding binding sites was carried out. The RMSD values between the co-crystallised pose and the docking pose from the previous step were 0.85 and 0.63 Å for erlotinib and sorafenib, respectively. After the positive indication on the docking validity, compounds 10b and 10d were docked into the two receptors using the same conditions. Finally, the results of the docking were visualised using the 2 D and 3 D interaction diagrams generated by MOE.

Scratch wound healing assay
A549 cells were seeded and incubated in 6-well cell culture plates till reaching confluence. Then, the confluent monolayers were scratched to form a "wound" using a sterile needle. The cells were treated with either vehicle as a control, 10b or 10d for 36 h. The images were recorded at 0 and 36 h to monitor the migration of cells into the wounded area using a light photomicroscope.