## 4 Discussion  
The structures of the synthesized Compounds **1** and **2** were confirmed using IR, NMR (\({}^{1}\)H and \({}^{13}\)C), mass spectral data and physical properties, and compared with those reported values [5]. As reported [5], 2,6-dichloro Compound **1** exhibited excellent cytotoxicity (IC\({}_{50}\) = 1.51 \(\pm\) 0.09 uM (Table 1, entry 1) against human breast adenocarcinoma (MCF7) cell lines, which is two-fold more than the control anticancer drug doxorubicin (IC\({}_{50}\) = 3.10 \(\pm\) 0.29 uM) (Table 1, entry doxorubicin) and 2-chloro,6-fluoro substituted Compound **2** exhibited similar cytotoxicity (IC\({}_{50}\) = 3.56 \(\pm\) 0.31 uM) comparing to doxorubicin. In addition, both compounds were exhibited good inhibitory activity against CDK2, IC\({}_{50}\) = 0.246 \(\pm\) 0.05 uM and 0.301 \(\pm\) 0.02 uM, respectively, which is half fold comparing to the known kinase inhibitor imatinib (IC\({}_{50}\) = 0.131 \(\pm\) 0.24 uM). As we know, isatin moiety containing compounds shows multiple protein kinase enzymes inhibitory activity and numbers of drugs are already in the market (Figure 1). On the other hand, both of our synthesized compounds (**1** and **2**) having isatin moiety in the structures. In addition, considering their excellent cytotoxicity as well as CDK2 inhibitory activity, we, therefore, assume that, Compounds **1** and **2** might show multiple protein kinase enzymes inhibitory activity, this led us to do further multiple protein kinase enzymes (EGFR, VEGFR-2 and FLT-3) inhibitory assay. The IC\({}_{50}\) values interpolated from dose-response data with five different concentrations were 0, 0.01, 0.1, 1 and 10 uM for all the protein kinase enzymes, EGFR, VEGFR and FLT-3, respectively. Highest IC\({}_{50}\) value was observed for Compound **1**, which showed IC\({}_{50}\) = 1.51 \(\pm\) 0.09 uM and it was twofold than the IC\({}_{50}\) values of doxorubicin (3.10 \(\pm\) 0.29 uM). Compound **1** also showed strong enzyme inhibitory activities against two protein kinases enzymes EGFR and VEGFR with IC\({}_{50}\) values of 0.269 (Figure S1) and 0.232 uM, respectively, whereas CDK2 reported [5] value showed 0.246 uM. The control drug imatinib showed 0.131 uM, erlotinib showed 0.056 uM and sorafenibibly showed 0.091 uM, against CDK2, EGFR, VEGFR-2 enzymes, respectively. On the other hand, Compound **2** also showed promising cytotoxicity and protein kinase inhibitory activities against all the three proteins kinase evaluated. IC\({}_{50}\) value of Compound **2** was 3.56 \(\pm\) 0.31 uM, which is similar to doxorubicin IC\({}_{50}\) value 3.10 \(\pm\) 0.29 uM [5], meanwhile its enzyme inhibitory activities against CDK2, EGFR, VEGFR-2 enzymes were 0.301, 0.369 and 0.266 uM, respectively. In case of FLT-3, Compound **1** showed 1.535 uM against FLT-3, interestingly Compound **2** showed better activity than Compound **1**, which was 0.546 uM against first FLT-3 inhibitor sunitinib (sunitinib was 0.262 uM), which prolonged haematoxicity and hand-foot syndrome caused by FLT-3 mutated acute myeloid leukaemia (AML) [70]. In our previous study [5] we have reported that, Compounds **1** and **2** inhibited CDK2 kinase in an ATP dependent manner and acted as type II inhibitor by lacking DFG motif interaction in the activation loop. Interaction with DFG motif residues is crucial for an inhibitor to define as active or inactive state kinase inhibition. Similarly, in this experiment, the interaction of Compounds **1** and **2** with EGFR, VEGFR-2 and FLT-3 protein kinases was evaluated by molecular docking analysis. Since Compound **1** with EGFR kinase domain showed several important interactions and involved hydrogen bonding interactions with ATP binding site and DFG motif residue, thus, it can be said that, Compound **1** might act on active kinase by interacting with DFG motif residues. It also formed several \(\pi\)-\(\pi\) stacked and \(\pi\)-\(\pi\) T shaped interactions with Met766, Met790, Phe723, Leu747, Leu777, Leu788, Ile759, Leu858 and Leu861.