key: cord-0025692-7upx28an
authors: Abdallah, Abdallah E.; Mabrouk, Reda R.; Al Ward, Maged Mohammed Saleh; Eissa, Sally I.; Elkaeed, Eslam B.; Mehany, Ahmed B. M.; Abo-Saif, Mariam A.; El-Feky, Ola A.; Alesawy, Mohamed S.; El-Zahabi, Mohamed Ayman
title: Synthesis, biological evaluation, and molecular docking of new series of antitumor and apoptosis inducers designed as VEGFR-2 inhibitors
date: 2022-01-10
journal: nan
DOI: 10.1080/14756366.2021.2017911
sha: db4a3dbc575c677569bc9bd12f210793ed8c88aa
doc_id: 25692
cord_uid: 7upx28an

Based on quinazoline, quinoxaline, and nitrobenzene scaffolds and on pharmacophoric features of VEGFR-2 inhibitors, 17 novel compounds were designed and synthesised. VEGFR-2 IC(50) values ranged from 60.00 to 123.85 nM for the new derivatives compared to 54.00 nM for sorafenib. Compounds 15(a), 15(b), and 15(d) showed IC(50) from 17.39 to 47.10 µM against human cancer cell lines; hepatocellular carcinoma (HepG2), prostate cancer (PC3), and breast cancer (MCF-7). Meanwhile, the first in terms of VEGFR-2 inhibition was compound 15(d) which came second with regard to antitumor assay with IC(50) = 24.10, 40.90, and 33.40 µM against aforementioned cell lines, respectively. Furthermore, Compound 15(d) increased apoptosis rate of HepG2 from 1.20 to 12.46% as it significantly increased levels of Caspase-3, BAX, and P53 from 49.6274, 40.62, and 42.84 to 561.427, 395.04, and 415.027 pg/mL, respectively. Moreover, 15(d) showed IC(50) of 253 and 381 nM against HER2 and FGFR, respectively.

Cancer is a life-threatening disease and is reported as a leading cause of death worldwide, accounting for approximately 10 million deaths in 2020 1 . It was reported that non-selective chemotherapeutic agents cause toxicity to normal cells 2,3 . These two points clearly reveal the need to develop new potent anticancer agents with relatively better selectivity.

Cancer cells are characterised by biochemical abnormalities rather than normal cells. We suggest that development of anticancer agents should primarily be directed to address such defects. One of these specific features of cancer cells is the abnormality of tyrosine kinases. Tyrosine kinases are important enzymes that play a pivotal role in signal transduction, cell survival, proliferation, and migration 4 .

Tyrosine kinases are differentiated into two types: receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs) 5 . Twenty different types of RTKs have been recognised 3 . They primarily consist of insulin-like receptor and growth factor receptor such as VEGFR, PDGFR, EGFR, FGFR, and NGFR 6 . Among the targets of RTKs, vascular endothelial growth factor receptors (VEGFRs) have attracted attention of researchers as excellent targets to develop new anticancer agents 7, 8 .

VEGF family includes five factors, namely VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (PLGF) 9 . Many types of tumour have shown overexpression of VEGFs and their receptors 10 . Accordingly, high rate of angiogenesis is obtained to provide nutrition and oxygen required for tumour growth 9 . Regarding VEGF receptors, they are categorised into three types: VEGFR-1, VEGFR-2, and VEGFR-3 11 . In physiological and pathological angiogenesis, VEGFR-1 and VEGFR-2 play an essential role. While VEGFR-3 is associated with embryogenesis and lymphangiogenesis 12, 13 .

An essential receptor for VEGF-dependent angiogenesis is VEGFR-2 which binds primarily all VEGF-A isoforms 9 . There is some evidence that metastasis of many solid tumours is attributed to overexpression of VEGFR-2 14, 15 . It was reported that tumour growth can be efficiently inhibited by blocking of angiogenesis 16 . Hence, VEGFR-2 inhibition is an efficient approach to obtain effective anticancer agents 17 . There are some FDA-approved VEGFR-2 inhibitors such as Sorafenib 1 18 , Regorafenib 2 19 , Sunitinib 3 20 , Pazopanib 4 21 , and Vatalanib 5 22 (Figure 1 ).

VEGFR-2 inhibitors are divided into two major types 23 . Type I inhibitors are characterised by their ability to accommodate the region, of the active conformation of the receptor that primarily occupied by the adenine moiety of ATP 24 . While type II kinase inhibitors are known to occupy a hydrophobic allosteric site appeared just close to the ATP-binding domain in the inactive conformation (DFG-out) of the enzyme 25 . Type-II inhibitors such as Sorafenib outweigh type-I inhibitors by many advantages such as relatively higher selectivity to the enzyme as well as slower off-rates 26 .

Type II VEGFR-2 inhibitors have common four pharmacophoric features on which the design of the new derivatives of the current work was based for the present work. Figure 2 illustrates the four regions that were reported to be occupied by these features 27, 28 . We can see that the ATP-binding domain (Hinge region) is accommodated by a flat heteroaromatic moiety. The interaction between the heteroaromatic moiety and Glu917 and/or Cys919 residues in this region is important 6, 29, 30 . Secondly, the area between ATPbinding domain and DFG domain requires a linker with suitable length to fit 31 . Thirdly, Glu885 and Asp1046 residues in the DFG domain are essential residues to which the hydrogen bond acceptors and donors interact 29 . Finally, there are hydrophobic interactions between the allosteric region of the inactive confirmation of the receptor and the terminal hydrophobic group 29, 32 . Figure 3 shows a model for the designed compounds and demonstrates the consistency of their structures with the pharmacophoric features that reported to type II VEGFR-2 inhibitors. With respect to the flat heteroaromatic moiety, three nuclei were taken into account in our design: quinazoline, quinoxaline, and nitrobenzene. AZD-2932 6 (IC 50 ¼ 8 nM) 33 is a quinazoline-based VEGFR-2 inhibitor (Figure 1 ). It clearly reveals the significance of quinazoline as flat heteroaromatic moiety in VEGFR-2 inhibitors. As a bioisoster to quinazoline, quinoxaline was also designed to study the effect of changing benzodiazine nitrogen positions on the activity. 2-Nitrobenzene was also designed in this work because that benzene is an isostere to pyridine of Sorafenib 1 and Regorafenib 2 ( Figure 1 ). It will show to what extent heterocycle is important for activity. Additionally, there is a nitro group at position 2 which may interact with the essential residues Glu917 and/or Cys919 in Hinge region.

Urea as well as amide moiety was found to be effective hydrogen bond acceptors and donors in some VEGFR-2 inhibitor drugs as Regorafenib and Sunitinib, respectively (Figure 1 ). So that urea and amide moieties were designed in this work as hydrogen bond acceptors and donors. We aimed to study which moiety will be of more significant effect.

Herein, the phenoxy group was designed as an aryl linker in all new derivatives. This group was selected because it was an effective linker in some VEGFR-2 inhibitor drugs as Sorafenib, Regorafenib, and AZD-2932 6 ( Figure 1) .

Finally, the terminal hydrophobic moiety was planned to be phenyl, 3-chlorophenyl, 4-chlorophenyl as well as pyridyl. Furthermore, butyl as aliphatic group was introduced to test whether the aliphatic hydrophobic moiety could play a good substitute for the aromatic hydrophobic groups or not.

All melting points were obtained by open capillary method on a Gallen lamp Melting point apparatus and were uncorrected. Infrared spectra were recorded on Pye Unicam SP 1000 IR spectrophotometer (KBr discs) and were expressed in wave number (cm -1 ). 1 H NMR and 13 C NMR spectra were recorded on a BRUKER 400 MHZ-NMR spectrophotometer. TMS was used as internal standard in deuterated DMSO, and chemical shifts were measured in d ppm. Elemental analyses were performed on a PerkinElmer 2400 series II CHN elemental analyser. Progresses of the reaction was monitored by TLC using TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates and were visualised using UV lamp.

2.1.1. General method for synthesis of compounds (14 a&b ) and (15 a-d ) Equimolar amounts of 2,4-dichloroquinazoline (0.12 g, 0.60 mmol) and the appropriate intermediate (8 a&b and 10 a-d ) were mixed with K 2 CO 3 (0.10 g, 0.72 mmol) in isopropanol. The reaction mixture was refluxed for about 1 h. Upon cooling, the obtained precipitate was collected by filtration and dried. Then, it was washed by water, dried, and recrystallized from isopropanol. 13 d (ppm): 0.90 (t, J ¼ 7.2 Hz, 3H, CH 3 ), 1.29 (m, 2H, CH 2 CH 3 ), 1.42 (m, 2H, CH 2 CH 2 CH 3 ), 3.08 (dt, J ¼ 6.5 Hz, 2H, CH 2 NH), 6.11 (t, J ¼ 5. 7 13 13 13 50 of all new derivatives against VEGFR-2 kinase. The antibody specific to the enzyme was planted on a 96 well microplate to which different micromolar concentrations of the positive control and the new compounds was added. After incubation at 25 C for about 2.5 h, the biotin antibody (100 mL) was added. The plates were further incubated for 1 h at 25 C and then washed. A solution of streptavidin (100 mL) was added to the microplates which were incubated for another 45 min at 25 C and then washed. Similarly, a solution of tetramethybenzidine (TMB) substrate (100 mL) was added with incubation at 25 C for half an hour. Finally, stop solution (50 mL) was added and the reading was immediately determined at 450 nm. To get the standard curve, the concentrations used were presented on the X-axis and the corresponding absorbances were on the Y-axis.

The antitumor assay was performed against the human cancer cell lines: HepG2, PC3, and MCF-7. The chemicals used in this test were supplied by Sigma-Aldrich with high analytical grade. National Cancer Institute (Cairo, Egypt) was the supplier of the cell lines tested which originally obtained from American Type Culture Collection (ATCC). MTT protocol was applied to quantitatively measure the antiproliferative activity of the new derivatives 34 . In this test, the colour change is attributed to that the yellow tetrazolium bromide (MTT) is converted to formazan derivative which has purple colour. This biochemical reaction occurs only in viable cells by mitochondrial succinate dehydrogenase. The tested cells were initially cultured in RPMI-1640 medium with foetal bovine serum (10%). To the incubated cell, streptomycin (100 mg/mL) and penicillin (100 units/mL) were added in a 5% CO 2 incubator at 37 C. Then seeding of the cells was performed in a 96-well plate under 5% CO 2 and at density of 1.0 Â 10 4 cells/well at 37 C for 48 h. Various non-toxic concentrations of the tested candidates were added and the cells were then incubated for further 24 h to the cells treated with the new compounds, MTT solution (20 mL at 5 mg/mL) was added, followed by 4 h incubation. One hundred microlitres of Dimethyl sulfoxide (DMSO) were added into each well to dissolve the purple formazan formed which is colorimetrically absorbed at 570 nm using a plate reader (EXL 800, USA). Finally, the percentages of cell viability was obtained as (A570 of treated samples/A570 of untreated sample) Â 100.

The protocol reported by Wang et al. 35 was followed to determine the effect of 15 d on cell cycle of HepG2. The cancer cells (2 Â 10 5 cells) were seeded and incubated in each of six-well plates for 24 h. The cells were further incubated at 37 C and 5% CO 2 after addition of foetal bovine serum (FBS, 10%). The medium was replaced by DMSO solution (1% v/v) in which 9.6 mg of compound 15 d was dissolved. Well plates were then incubated for 48 h and then rinsed with cold phosphate-buffered saline (PBS), fixed with ethanol (70%) and washed again with PBS. DNA fluorochrome propidium iodide (PI) was used to stain the cells which were then kept for 15 min at 37 C. FACS Calibre flow cytometer was used to analyse the samples.

Apoptosis ratio of HepG2 treated with 9.6 mg of compound 15 d in comparison with untreated HepG2 cells was calculated according to the reported method using Annexin V fluorescein isothiocyanate (V-FITC)/PI kit 36 .

HepG2 cells were cultured and treated with compound 15 d for 24 h. after that cells were collected and washed with PBS, then were collected and lysed by adding it to the extraction buffer containing protease inhibitors (1 ml per 1 Â 10 6 cells.) then the lysate was diluted immediately before the assay. Caspase-3, BAX, Bcl-2, and p53 level was measured by using Quantikine-Human active kits (R&D Systems, Inc. Minneapolis, USA) according to the manufacturer protocols.

2.2.6. Effect of 15d on multi types of kinases Inhibitory activity of compound 15 d against HER2 and FGFR was performed using Homogeneous time resolved fluorescence (HTRF) assay method. Different kinases and ATP were obtained from Sigma. The specified kinase and its substrate was incubated with compound for 5 min in buffer solution to start the enzymatic reaction; ATP was then added to the reaction mixture and was maintained for 30 min at room temperature. The reaction was stopped by adding detection reagents containing EDTA for 1 h and then the IC 50 values were determined by GraphPad Prism 5.0.

Molecular docking studies for the new molecules were performed by using Discovery Studio 2.5 software (Accelrys software Inc., USA) against VEGFR-2. The 3D structure of the VEGFR-2 kinase combined with sorafenib was downloaded from Protein Data Bank (PDB) website (PDB ID: 4ASD 37 ).

To prepare the obtained structure, water molecules were deleted. Then correction of the valence of atoms was achieved by running the valence monitor method. To minimise the energy, force fields CHARMM and MMFF94 were applied 38-40 . The active site was then defined and prepared for docking. The new derivatives were sketched using ChemBioDraw Ultra 14.0 software 41 and saved as MDL-SD files. The co-crystallized ligand was similarly drawn. The drawn structures were opened and prepared for docking by 3 D protonation, and energy was minimised through running force fields CHARMM and MMFF94. Protocol selected for docking process was CDOCKER. Meanwhile, CHARMM-based molecular dynamics (MD) was used to dock the co-crystallized ligand 42-44 . Ten docked poses were obtained for each molecule. Finally, the conformers of the most minimal free energy were selected to study the binding pattern of each molecule.

Firstly, preparation of the intermediates 8 a&b and 10 a-d was achieved as illustrated in Scheme 1. Heating of 4-aminophenol with phenylisocyanate and butylisocyanate in absolute ethanol afforded 8 a&b , respectively, 45 while the intermediates 10 a-d [46] [47] [48] were obtained by reaction of 4-aminophenol with benzoic acid derivatives namely, benzoic acid, 4-chlorobenzoic acid and 3-chlorobenzoic acid and with nicotinic acid, respectively. Synthesis of these intermediates was afforded by treating an appropriate acid with ethyl chloroformate and triethyl amine in DCM at about -5.0 C for 1 h. Then 4-aminophenol was added to the reaction mixture which stirred at r.t. for 1 h.

Secondly, synthesis of dichloroquinazoline (13) and dichloroquinoxaline (18) Moreover, amidic carbonyl revealed a peak at about 166 ppm in 13 C NMR charts. While urea carbonyl group appeared at about 153 ppm, regarding quinazoline nucleus, it showed one peak at about 168 ppm and two peaks above 150 ppm which are due to 3 C ¼ N. While quinoxaline nucleus demonstrated one peak above 150 ppm, it showed no peaks above 160 ppm.

The synthesised compounds were evaluated biologically according to the following strategy; initially, all compounds were screened for in vitro inhibition of VEGFR-2 kinase. Then, antiproliferative assay of the best nine compounds was performed against three cancer cell lines namely: hepatocellular carcinoma (HepG2), prostate cancer (PC3), and breast cancer (MCF-7) and against one normal cell line as well. Finally, HepG2 cells treated with compound 15 d were evaluated for apoptosis and cell cycle kinetics compared to untreated HepG2 cells.

The novel derivatives were investigated in vitro for the inhibitory activities against VEGFR-2 as a possible mechanism of its antitumor activity. In the meantime, sorafenib was used as a positive control. Half-maximal inhibitory concentration (IC 50 ) values as presented in Table 1 were calculated from the obtained concentration-inhibition response curve. As can be seen from 

The potential antitumor activity of the selected nine compounds 14 a , 15 a , 15 b , 15 c , 15 d , 19 b , 20 c , 20 d , and 23 a was further investigated against HepG2, PC3, and MCF-7. As is presented in Table 2 as IC 50 good selectivity. So that the effects of 15 d on cell cycle and apoptosis rate of HepG2 were evaluated.

HepG2 cell line was selected to evaluate the effect of compound 15 d on its different cell cycle phases as shown in Figure 4(A,B) where we can see that compound 15 d (at its IC 50 ) has the ability to stop the growth of HepG2 in both G2/M and pre G1 phases leading to significant increase in cell accumulation percentage. Table 3 and Figure 5(A,B) show that compound 15 d (at IC 50 ) caused a 10 folds increase in apoptosis of HepG2 from 1.20 to 12.46%. We can see the significant increase in the percentages of early apoptosis from 0.87 to 5.33% and late apoptosis from 0.33 to 7.13%, while necrosis was slightly increased from 1.17 to 2.35%.

Furthermore, it is also noted that in the control cells, induction of necrosis was almost at the same rate of apoptosis. But when the cells were treated with compound 15 d , apoptosis became about five times higher than necrosis. Table 4 and Figure 6 illustrate that compound 15 d (at IC 50 ) caused remarkable increase in apoptotic markers (i.e. Caspase-3, BAX, and P53) in HepG2 cells. We can see the significant increase in the expression levels of these markers from 49.6274, 40.62, and 42.84 to 561.427, 395.04, and 415.027 pg/mL, respectively. Meanwhile, considerable decrease was seen in the level of Bcl-2 from 5.761 to 1.385 pg/mL. These obtained data gave an explanation for the ability of compound 15 d to induce apoptosis in cancer cells.

Further exploration for the activity of compound 15 d was performed against two other types of kinases: human epidermal growth factor receptor 2 (HER2) and fibroblast growth factor receptor (FGFR). Table 5 shows the obtained IC 50 of compound 15 d compared to erlotinib as a reference drug. It was found that IC 50 of the 15 d is much higher for HER2 and FGFR than erlotinib. However, the new derivative was able to show considerable inhibition for HER2 and FGFR with IC 50 ¼ 253 and 381 nM, respectively. 

Using the C-Docker protocol in the Discovery Studio 2.5 software for studying docking mood of the new molecules and their orientation in VGEFR-2 kinase (PDB ID: (4ASD)) using sorafenib as a reference ligand. The docking was validated by incorporating the C-Docker algorithm to redock the co-crystallized ligand. RMSD obtained was 0.59 which indicated the validation of the applied docking protocol (Figure 7) . In this section of the work, we consider the binding free energy (DG) and the binding modes of the new derivatives. Critical interactions with various key residues, including Cys919 in the hinge region and Glu885 and Asp1046 in the DFG-binding domain were reported for potent VEGFR-2 inhibitors 32, 51 . The docking results demonstrated that the studied molecules displayed correct binding patterns into the active site of VEGFR-2 kinase. At the same time, there was a significant correlation between the biological results and DG of the new derivatives. Table 6 shows the values of DG for the new derivatives as well as for the active control (sorafenib).

The proposed sorafenib binding mode as a reference ligand revealed an affinity value of -74.23 C-Docker energy score. Nmethylpicolinamide moiety was found to form two hydrogen bonds with Cys919 and an aromatic stacking interaction (pi-pi interaction) with Phe918 in the Hinge region. Also, the phenoxy group was observed in the linker region forming cation-pi interaction with Lys868. Urea moiety, meanwhile, displayed two hydrogen bonds with Glu885 and Asp1046 in the DFG binding domain. Finally, the terminal substituted phenyl moiety accommodated the allosteric region formed by Lue889, Glu885, His1026, Leu1019, and Ile888. The results obtained were consistent with the reported 32,51 (Figure 8(A,B) .

With regard to the new molecules, compound 14 a revealed a high binding affinity (DG ¼ -66.86). This high affinity for VEGFR-2 is likely due to the formation of three hydrogen bonds as illustrated in Figure 9(A,B) . As can be seen from Figure 9 , the quinazoline moiety correctly occupied the Hinge region. Additionally, the phenoxy group was fitted to the linker region and formed cation-pi interaction with Lys868. It is also evident from Figure 9 that Ile892, and Val899. The spatial orientation of compound 14 a in comparison with sorafenib is shown in Figure 9 (C). Compound 15 c exhibited a good fitting to VEGFR-2 with binding energy of 66.52 C-Docker energy score. The quinazoline moiety accommodated the Hinge region as shown in Figure 10(A,B) . The Figure 7 . Overlay of the co-crystallized ligand (magenta) and the redocking one (turquoise) of sorafenib into VEGFR-2 kinase active site. Table 6 . Binding free energies (calculated DG in C-Docker energy score) of the studied compounds and sorafenib as a positive control into VEGFR-2.

phenoxy group was fitted to the linker region and exhibited cation-pi interaction with Lys868. In DFG region, the amide showed two hydrogen bonding interactions with Asp1046 and Glu885. Furthermore, the terminal 3-chlrophenyl group occupied the allosteric binding region and formed hydrophobic interactions with Leu889, Ile892, and Lue1019. Figure 10 (C) shows a superimposition of sorafenib and 15 c in order to compare their spatial orientations. Investigation of the best pose of compound 15 d (-64.11 C-Docker energy score) demonstrated the formation of hydrogen bonds as well as electrostatic and hydrophobic interactions. Figure 11(A,B) shows pi-cation interaction between the phenoxy group and Lys868 of the linker region. Meanwhile, N-H of the amide group demonstrated a hydrogen bond with Glu885, and the oxygen of carbonyl group formed anther hydrogen bond with Asp1046 at the DFG binding domain. Moreover, the chloride atom at position 2 of quinazoline formed hydrogen bond with Cys919 of the ATP-binding domain. Finally, pyridyl moiety accommodated the allosteric binding region showing hydrophobic interactions. Figure 11 (C) shows a superimposition of 15 d and sorafenib.

The docking results revealed the ability of the new derivatives to accommodate VEGFR-2 pocket and form interactions in a mode similar to that of sorafenib. These results are not only consistent with biological data obtained from the VEGFR-2 assay but also give an explanation to them. For example, quinazoline based derivatives (14 a,b and 15 a-d ) displayed the best docking energy (from -53.63 to -66.52 C-Docker energy score) as presented in Table 6 . Regarding VEGFR-2 inhibition, the same derivatives were the most potent (IC 50 ranged from 60 to 94.22 nM) as listed in Table 1 . The most potent candidates for VEGFR-2 inhibition were 15 d and 15 c with IC 50 ¼ 60.00 and 65.24 nM, respectively. Similarly, these two compounds were the most promising in terms of docking score showed the highest binding free energies 64.11 and 66.52 C-Docker energy score, respectively. At the same time, quinoxaline-based derivatives (19 a,b and 20 a-d ) came the second with regard to both VEGFR-2 inhibition and docking score as illustrated in Tables 1 and 6 . It can also be seen that nitrobenzenebased derivatives came last in both biological activity and docking results.

According to the results given, the following relationships can be established for the new derivatives 1. Quinazoline nucleus as a planar heteroaromatic moiety is much better than both quinoxaline and benzene ring. It can be noticed from Table 1 2. The nitrogen atom in the heteroaromatic moiety plays a key role in the activity. For example, the data of VEGFR-2 inhibitory assay revealed IC 50 ¼ 67.05 nM for 14 a which is quinazoline-based candidate. Meanwhile, the corresponding nitrobenzene-based derivative (22 a ) displayed IC 50 ¼ 123.85 nM. In a similar manner, compound 15 a (quinazoline derivative) showed significantly better antiproliferative results than 23 a (a nitrobenzene derivative). displayed far more potent antiproliferative activity than 15 a which contain plain phenyl ring. 7. Pyridine nucleus is better than benzene ring. The nitrogen atom increased the basicity of the pyridine ring than benzene ring which makes a chance to form pi-cation interaction more than benzene ring. Figures 12(A,B) and 13(A,B) in Supplementary data. Moreover, compound 15 d was found to be the most promising derivative as VEGFR-2 inhibitor with IC 50 ¼ 60.00 nM. It also demonstrated antiproliferative activity better than that of 15 a (Table 1) .

On the basis of quinazoline, quinoxaline, and benzene nuclei, we designed and synthesised 17 new compounds in order to meet the reported pharmacophoric features of type II VEGFR-2 inhibitors. Biological results showed that compounds 15 d and 15 b are of particular interest as anticancer agents targeting VEGFR-2 kinase. In addition to their considerable inhibition of VEGFR-2, they have shown promising antitumor effects especially against hepatocellular cancer cell line (HepG2) with high degree of selectively. The data obtained from VEGFR-2 inhibitory assay revealed IC 50 ¼ 60.00 and 86.36 nM for 15 d and 15 b , respectively compared to IC 50 ¼ 54.00 nM for the positive control sorafenib. At the same time, the IC 50 values obtained for the two candidates against HepG2 were 24.10 and 17.39 mM, respectively. Further examination of compound 15 d revealed its importance as an apoptosis inducer at HepG2 cells where it raised the apoptosis rate from 1.20 to 12.46%. The mechanism of apoptosis induction was proved to compound 15 d through increasing the expression levels of caspase-3, BAX, and P53. Additionally, Bcl-2 level was decreased. Accordingly, this work suggests that compounds 15 b and 15 d should be considered for further evaluation and/or modification as promising anticancer candidates.

Chloroquinazolin-4-yl)oxy)phenyl)benzamide (15 a )

1558, 1485, 1446 (C ¼ C aromatic), 1531 (amide II band), 1203 (C-O)

13 C NMR (DMSOd 6 , 100 MHz) d (ppm): 114

1562, 1489, 1446 (C ¼ C aromatic), 1531 (amide II band), 1207 (C-O); 1 H NMR (DMSO-d 6 , 400 MHz) d (ppm): 7.35 (d, J ¼ 8.4 Hz

50%); m.p. ¼ 229-230 C; IR (KBr, cm -1 ): 3290, 3128 (NH), 3055 (C-H aromatic), 1654 (CO amide), 1616 (C ¼ N), 1566, 1489, 1450 (C ¼ C aromatic), 1531 (amide II band), 1211 (C-O); 1 H NMR (DMSO-d 6

Ar-H), 8.11 (ddd, J ¼ 8.5, 6.9, 1.5 Hz, 1H, Ar-H quinazoline), 8.42 (dd, J ¼ 8.3, 1.5 Hz, 1H, Ar-H quinazoline

Chloroquinazolin-4-yl)oxy)phenyl)nicotinamide

1580, 1562, 1489, 1450 (C ¼ C aromatic), 1531 (amide II band), 1211 (C-O); 1 H NMR (DMSO-d 6 , 400 MHz) d (ppm): 7.40 (d, J ¼ 8.9 Hz, 2H, Ar-H), 7.60 (dd

60 mmol) and the appropriate intermediate (8 a&b and 10 a-d ) along with K 2 CO 3 (0.10 g, 0.72 mmol) as a base were mixed in isopropanol. The reaction mixture was refluxed for approximately 5 h then allowed to cool. The obtained precipitate was collected by filtration, washed by water, dried, and recrystallized from isopropanol. 2.1.2.1. 1-(4-((3-Chloroquinoxalin-2-yl)oxy)phenyl)-3-phenylurea (19 a )

Ar-H), 7.75 (m, 3H, Ar-H quinoxaline), 8.03 (dd, J ¼ 8.3, 1.6 Hz, 1H, Ar-H quinoxaline)

-chloroquinoxalin-2-yl)oxy)phenyl)urea

White solid: (yield: 0.16 g, 70.90%)

CH 2 CH 2 CH 3 ), 3.10 (td, J ¼ 6.6, Hz, 2H), 6.16 (t, J ¼ 5.6 Hz, 1H, D 2 O exchangeable

Chloroquinoxalin-2-yl)oxy)phenyl)benzamide (20 a )

1608 (C ¼ N), 1562, 1508, 1446 (C ¼ C aromatic), 1531 (amide II band)

40 (s, 1H, NH). 13 C NMR (DMSO-d 6

Found: C, 66

mM) > 14 a (IC 50 ¼ 41.49 mM) > 15 c (IC 50 ¼ 50.41 mM) > 19 b (IC 50 ¼ 73.38 mM) > 20 c (IC 50 ¼ 144.29 mM) > 23 a

58 mM) > 15 a (IC 50 ¼ 30.28 mM) > 15 d (IC 50 ¼ 40.90 mM) > 14 a (IC 50 ¼ 50.67 mM) > 15 c (IC 50 ¼ 55.95 mM) > 19 b (IC 50 ¼ 84.41 mM) > 20 c (IC 50 ¼ 157.00 mM) > 23 a

88 mM) > 15 d (IC 50 ¼ 33.40 mM) > 15 a (IC 50 ¼ 47.10 mM) > 14 a (IC 50 ¼ 58.26 mM) > 15 c (IC 50 ¼ 62.68 mM) > 19 b (IC 50 ¼ 105.48 mM) > 20 c (IC 50 ¼ 134.85 mM) > 23 a

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Sidel'nikova S. Efficient one-stage procedure of Beckmann ketones rearrangement in the presence of hydroxylamine

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The authors extend their appreciation to the Research Center at AlMaarefa University for funding this work under TUMA project agreement number (TUMA-2021-4).

No potential conflict of interest was reported by the author(s).

It can be noticed that compound 15 b was ranked first in terms of activity against all the tested cancer cell lines. It showed potency equal to 0.74, 0.76, and 0.73 of sorafenib potency against HepG2, PC3, and MCF-7, respectively. Meanwhile compound 15 d came second against HepG2 and MCF-7 and third against PC3. The second compound against PC3 was 15 a which came third against both HepG2 and MCF-7. The other derivatives displayed the same descending order of activity against all cancer cell lines as follows: 14 a > 15 c > 19 b > 20 c > 23 a > 20 d .Of note, the cytotoxicity against normal human lung fibroblasts cell line (WI-38) were examined for the nine selected compounds. It is evident from Table 2 that the tested compounds have a significantly higher IC 50 versus WI-38 than the IC 50 versus cancer cell lines. It was found that compounds 15 b and 15 d showed the best selectivity. Compound 15 b showed selectivity indices of 13.57, 9.50, and 11.87 to HepG2, PC3, and MCF-7 cell lines, respectively, while it was found that selectivity indices of compound 15 d were 9.22, 5.53, and 6.92 to the cell lines, respectively.On the basis of the obtained data, Compound 15 d was the most promising derivative in VEGFR-2 inhibitory testing and showed promising antitumor activity against HepG2 cells with