Synthesis, Anticancer Screening of Some Novel Trimethoxy Quinazolines and VEGFR2, EGFR Tyrosine Kinase Inhibitors Assay; Molecular Docking Studies

A new series of 8-methoxy-2-trimethoxyphenyl-3-substituted quinazoline-4(3)-one compounds were designed, synthesized, and screened for antitumor activity against three cell lines, namely, Hela, A549, and MDA compared to docetaxel as reference drug. The molecular docking was performed using Autodock Vina program and 20 ns molecular dynamics (MD) simulation was performed using GROMACS 2018.1 software. Compound 6 was the most potent antitumor of the new synthesized compounds and was evaluated as a VEGFR2 and EGFR inhibitor with (IC50, 98.1 and 106 nM respectively) compared to docetaxel (IC50, 89.3 and 56.1 nM respectively). Compounds 2, 6, 10, and 8 showed strong cytotoxic activities against the Hela cell line with IC50 of, 2.13, 2.8, 3.98, and 4.94 µM, respectively, relative to docetaxel (IC50, 9.65 µM). Compound 11 showed strong cytotoxic activity against A549 cell line (IC50, 4.03 µM) relative to docetaxel (IC50, 10.8 µM). Whereas compounds 6 and 9 showed strong cytotoxic activity against MDA cell line (IC50, 0.79, 3.42 µM, respectively) as compared to docetaxel (IC50, 3.98 µM).

Quinazoline has become an important pharmacophore for anticancer the recent FDA approval of some quinazoline derivatives as anticancer Raltitrexed (TomudexTM) and nolatrexed (thymitaqTM) as shown in Figu gefitinib (IressaTM), lapatinib (TykerbTM also known as GW-572016), Va timaTM), afatinib (GilotrifTM), and erlotinib (TarcevaTM). In our present study, we designed a number of quinazoline deriv trimethoxy phenyl moiety in position 2 and insertion of methoxy at pos different electronic, steric, and lipophilic characters that might help imp tumor activity of the newly designed scaffolds. In addition, we kept the position 3 in an attempt to get optimized quinazoline derivatives with p cancer activity ( Figure 2). In our present study, we designed a number of quinazoline derivatives bearing trimethoxy phenyl moiety in position 2 and insertion of methoxy at position 8 to give different electronic, steric, and lipophilic characters that might help improve the antitumor activity of the newly designed scaffolds. In addition, we kept the substitution at position 3 in an attempt to get optimized quinazoline derivatives with promising anticancer activity ( Figure 2).  Reported quinazoline with GI50 µg/mL [15] and designed quinazoline derivatives as antitumor compounds.

Chemistry
The synthesis of the target compounds is depicted in scheme 1. The starting material 8-methoy-2-trimethoxyphenyl-4H-benzo[d](1,3)oxazin-4-one (3) was prepared according to a reported procedure [28] by allowing N-(trimethoxybenzoyl)-3-methoxyanthranilic acid to boil with acetic anhydride, then compound 3 allowed was fused with hydrazine to get 3-amino-8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (4). IR spectra of compound 4 showed absorption bands at 3221.35, 3387.83, and 1655.19 cm −1 due to stretching vibration of the NH2 group and CO, respectively, and 1 H NMR led to the appearance of a singlet of two protons for the NH2 group at 5.842. Compound 3 reacted with formamide to give 8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (5), the latter compound showed a stretching band for CO at 1655.67 cm −1 , and 1 H NMR showed a singlet peak at 8.292 ppm for the NH group. Compound 3 reacted with ammonia to create benzamide derivative 6, IR of compound 6 showed stretching vibration bands for NH2 and NH at 3193. 13 (10), the latter compound was confirmed by the presence of the CH3 group at 17.08 and 2.637 ppm in 13 C NMR and 1 H NMR, respectively. Compound 3 reacted with 3, 4, 5-trimethoxy aniline to Reported quinazoline with GI 50 µg/mL [15] and designed quinazoline derivatives as antitumor compounds.

Chemistry
The synthesis of the target compounds is depicted in scheme 1. The starting material 8-methoy-2-trimethoxyphenyl-4H-benzo[d](1,3)oxazin-4-one (3) was prepared according to a reported procedure [28] by allowing N-(trimethoxybenzoyl)-3-methoxyanthranilic acid to boil with acetic anhydride, then compound 3 allowed was fused with hydrazine to get 3-amino-8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (4). IR spectra of compound 4 showed absorption bands at 3221.35, 3387.83, and 1655.19 cm −1 due to stretching vibration of the NH 2 group and CO, respectively, and 1 H NMR led to the appearance of a singlet of two protons for the NH 2 group at 5.842. Compound 3 reacted with formamide to give 8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (5), the latter compound showed a stretching band for CO at 1655.67 cm −1 , and 1 H NMR showed a singlet peak at 8.292 ppm for the NH group. Compound 3 reacted with ammonia to create benzamide derivative 6, IR of compound 6 showed stretching vibration bands for NH 2 and NH at 3193.13, 3367.91, and 3389.62 and stretching bands at 1672.66 and 1663.23 for two CO groups. Moreover the 1 H NMR data showed a singlet peak for the NH 2 group at 6.069 ppm and a singlet peak at 9.782 ppm for NH moiety. Compound 3 reacted with hydrazine hydrate in the presence of ethanol to get hydrazide derivative 7. Compound 7 showed characteristic CO bands of diamide at 1670.63 and 1655.19 and NH, NH 2 absorption at 3376.45, 3382.21, 3175.43, and 3295.34 cm −1 , in addition to an 1 H NMR singlet peak at 4.479 ppm for NH 2 and two singlets for NH of diamide at 10.134 and 10.167 ppm. Compound 7 reacted with benzene sulphonyl chloride, which afforded compound 8 with absorption bands of NH groups at 3366.45, 3372.52, and 3385.43 cm −1 and CO band at 1670.65 and 1655.37 cm −1 on the IR spectrum, as well as singlets at 8.5497 and 12.615 ppm for NH protons in 1 H NMR. Additionally, compound 4 reacted with benzene sulphonyl chloride to give compound 9 with absorption bands at 1655.82 and 3385.56 cm −1 due to Co and NH, respectively, beside the 1 H NMR singlet for NH at 11.885 ppm. Moreover, compound 3 refluxed with methyl amine to give 3-methyl-8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (10), the latter compound was confirmed by the presence of the CH 3 group at 17.08 and 2.637 ppm in 13 C NMR and 1 H NMR, respectively. Compound 3 reacted with 3, 4, 5-trimethoxy aniline to create 3-(3, 4, 5-trimethoxyphenyl)-8-methoxy-2-trimethoxyphenylquanazoline-4(3H)-one (11), which was confirmed by the presence of singlet peaks at 56.18, 59.56, and 60.23 ppm and 3.624, 3.768, and 3.964 ppm due to one methoxy group and two trimethoxy groups in 13 C NMR and 1 H NMR spectrum, respectively (Scheme 1).

VEGFR2 and EGFR Inhibitory Activity
The synthesized compounds were evaluated for anticancer activity by adapting a reported procedure [29][30][31][32][33] using two enzymes, VEGFR2 and EGFR, as well as three cell lines. The inhibitory activity for VEGFR2 and EGFR was assessed for the most active one (compound 6) and the results are reported as IC 50 values. Compound No. 6 proved to have almost 91% inhibitory activity for enzyme VEGFR2 compared to the inhibitory activity of the reference drug, with IC 50 values 98.1 to compound 6 and 89.3 for docetaxel for the positive control. Meanwhile, the inhibitory activity for enzyme EGFR was almost 53%, with IC 50 value 106 compared to IC 50 value 56.1 for docetaxel as reference drug (Table 1). The synthesized compounds were also subjected to anticancer assay using three cell line panels. The first cell line was Hela cells using docetaxel as positive control and the results were recorded as IC 50 values. Compounds 2, 6, 10, and 8 proved to be the most active Hela cells inhibitors, with IC 50 values indicating that they are more potent than docetaxel, with scores of IC 50 , 2.13, 2.8, 3.98, 4.94 µM, respectively, and 3-methoxy-N-(trimethoxyphenyl) anthranilic acid was the most potent of them as a Hela inhibitor, with almost fivefold inhibitory activity. Compounds 9, 11, and 5 proved to have good inhibitory activity, with IC 50 values of 11.70, 12.00, and 12.90 µM, while compounds 4 and 3 had moderate activity as Hela cell inhibitors, with IC 50 values 29.80 and 66.50 as compared with that of docetaxel IC 50 , 9.65 µM ( Table 2 and Figure 3). The second one was A549, using docetaxel as positive control, and the results were determined as IC 50 values. 8-methoxy-2trimethoxymethyl-3-trimethoxyphenyl quanazoline-4(3H)-one (11) proved to be the most potent one of them, with almost two-and-a-half-fold more than the reference drug, with an IC 50 value of 4.03 µM. Compounds 10, 8, 4, and 9 showed good A549 inhibitory activity, with IC 50 values 12.30, 13.00, 18.00, and 19.90 µM, respectively. Compounds 5, 2, 3, and 7 showed moderate A549 inhibitory activity, with IC 50 values of 26.30, 29.70, 43.60, and 46.30 µM, respectively. In addition, compound 6 proved to be the least active one, with IC 50 , 81.00 µM in comparison to docetaxel (IC 50 , 10.8 µM) ( Table 2 and Figure 3). The third cell line was MDA cells using a positive control and the results are reported as IC 50 values. Compounds 6 and 9 proved to be the ones that were more potent than the positive control, with IC 50 values of 0.79 and 3.42 µM, respectively, and compound 6 (3-methoxy-2-(trimethoxybenzamido) benzamide) was the most active of all as MDA inhibitor, with a fourfold higher inhibitory activity compared to the reference drug. Re-spectively, compounds 4 and 5 had good inhibitory activity with MDA cells with IC 50 , 4.12 and 4.39 µM. Compounds 11, 7, 2, and 8 showed moderate inhibitory activity, with IC 50 values 7.06, 9.00, 9.24, and 11.70 µM, respectively. In addition, compound 3 showed the least activity, with an IC 50 value of 17.40 µM as compared with docetaxel IC 50 , 3.98 µM. (Table 2 and Figure 3). The third cell line was MDA cells using a positive control and the results are reported as IC50 values. Compounds 6 and 9 proved to be the ones that were more potent than the positive control, with IC50 values of 0.79 and 3.42 µ M, respectively, and compound 6 (3-methoxy-2-(trimethoxybenzamido) benzamide) was the most active of all as MDA inhibitor, with a fourfold higher inhibitory activity compared to the reference drug. Respectively, compounds 4 and 5 had good inhibitory activity with MDA cells with IC50, 4.12 and 4.39 µ M. Compounds 11, 7, 2, and 8 showed moderate inhibitory activity, with IC50 values 7.06, 9.00, 9.24, and 11.70 µ M, respectively. In addition, compound 3 showed the least activity, with an IC50 value of 17.40 µ M as compared with docetaxel IC50, 3.98 µ M. (Table 2 and Figure 3).

Molecular Docking Analysis
In order to get an insight into the mechanism of interaction between M6 and EGFR and VEGFR2 tyrosine kinases, a molecular docking analysis was performed using docetaxel and co-crystalized ligands as references. The docking of M6 into the ATP pocket of EGFR L858R/T790M indicates that M6 adapted a binding mode similar to docetaxel as well as the co-crystal ligand; compound 1 had a docking energy score of −7.4 kcal/mol ( Figure 4A). Compound 1 is a potent non-covalent inhibitor of EGFR del19 T790M C797S , with an IC50 = 790 nM [34].

Molecular Docking Analysis
In order to get an insight into the mechanism of interaction between M6 and EGFR and VEGFR2 tyrosine kinases, a molecular docking analysis was performed using docetaxel and co-crystalized ligands as references. The docking of M6 into the ATP pocket of EGFR L858R/T790M indicates that M6 adapted a binding mode similar to docetaxel as well as the co-crystal ligand; compound 1 had a docking energy score of −7.4 kcal/mol ( Figure 4A). Compound 1 is a potent non-covalent inhibitor of EGFR del19 T790M C797S , with an IC 50 = 790 nM [34].  Figure 3).

Molecular Docking Analysis
In order to get an insight into the mechanism of interaction between M6 and EGFR and VEGFR2 tyrosine kinases, a molecular docking analysis was performed using docetaxel and co-crystalized ligands as references. The docking of M6 into the ATP pocket of EGFR L858R/T790M indicates that M6 adapted a binding mode similar to docetaxel as well as the co-crystal ligand; compound 1 had a docking energy score of −7.4 kcal/mol ( Figure 4A). Compound 1 is a potent non-covalent inhibitor of EGFR del19 T790M C797S , with an IC50 = 790 nM [34].  M6 was able to form a hydrogen bond between the oxygen atom of methoxy-group and T854 within the hinge region and hydrophobic interaction with side chains of F723, L718, and L844 ( Figure 4B). Notably, a mutation of L718 within the P-loop has been reported as a mutation conferring resistance to osimertinib [35]. It was also in close proximity to catalytic lysine residue, K745. In contrast, the docetaxel was involved in two hydrogen bonds, with the nitrogen of N842 and oxygen of D800, as well as several hydrophobic interactions with L844, G796, and L718 ( Figure 4C). To confirm the docking results, M6-EGFR complex was subjected to a 20 ns MD simulation to further analyze the molecular mechanism of the ligand-protein interaction. The backbone RMSD of the M6-EGFR complex was calculated to predict the M6-EGFR complex stability over time. As shown in Figure 4A, the complex reached equilibration after~1.5 ns with a normal fluctuation pattern for the rest of the time (~18.5 ns) between 0.2 and 0.3 Å, indicating a stable dynamic behavior ( Figure 5A). The hydrogen bonds are major contributors to the stability of the binding between the protein and the ligand. M6 was found to form up to three hydrogen bonds with the active site of EGFR over the simulation time ( Figure 5B). pound 1 (white) to EGFR tyrosine kinase. Ribbon representation for the binding interaction of (B) M6 (green) and (C) docetaxel (pink) with EGFR tyrosine kinase. Hydrogen bonds are shown in green dashed lines.
M6 was able to form a hydrogen bond between the oxygen atom of methoxy-group and T854 within the hinge region and hydrophobic interaction with side chains of F723, L718, and L844 ( Figure 4B). Notably, a mutation of L718 within the P-loop has been reported as a mutation conferring resistance to osimertinib [35]. It was also in close proximity to catalytic lysine residue, K745. In contrast, the docetaxel was involved in two hydrogen bonds, with the nitrogen of N842 and oxygen of D800, as well as several hydrophobic interactions with L844, G796, and L718 ( Figure 4C). To confirm the docking results, M6-EGFR complex was subjected to a 20 ns MD simulation to further analyze the molecular mechanism of the ligand-protein interaction. The backbone RMSD of the M6-EGFR complex was calculated to predict the M6-EGFR complex stability over time. As shown in Figure 4A, the complex reached equilibration after ~1.5 ns with a normal fluctuation pattern for the rest of the time (~18.5 ns) between 0.2 and 0.3 Å , indicating a stable dynamic behavior ( Figure 5A). The hydrogen bonds are major contributors to the stability of the binding between the protein and the ligand. M6 was found to form up to three hydrogen bonds with the active site of EGFR over the simulation time ( Figure 5B). Similarly, the docking of M6 against the wild type VEGFR2 was performed using sorafenib as a reference. Sorafenib is a nonselective potent ATP-competitive inhibitor of VEGFR2 in which IC50 = 2.3 nM [36]. The results indicate that M6 binds to the ATP binding pocket with a docking energy score of −7.3 kcal/mol. This compound binds to the conserved regions of the ATP site, similarly to docetaxel as well as the co-crystalized ligand, sorafenib ( Figure 6A). However, unlike M6, docetaxel was able to bind the regulatory domain pocket (RDP), as was the sorafenib, as both molecules have larger substituents [37].
In term of interactions, M6 was involved in two hydrogen bonds between the nitrogen and oxygen atoms within the benzamide moiety and E885 of the αC helix; as well as with the D1046 residue in the DFG region of the VEGFR2, respectively; and in hydrophobic bonds with the side chain of L1019 ( Figure 6B). On the other hand, docetaxel made four hydrogen bonds with V916, L1025, D1046, AND L1049, as well as hydrophobic interactions with K868, L882, G885 L889, I892, and H1026 (Figure 6 C). The stability of the M6-VEGFR2 complex was confirmed by MD simulation. The backbone RMSD of the complex showed a normal dynamic behavior with a fluctuation range between 0.1 and Similarly, the docking of M6 against the wild type VEGFR2 was performed using sorafenib as a reference. Sorafenib is a nonselective potent ATP-competitive inhibitor of VEGFR2 in which IC 50 = 2.3 nM [36]. The results indicate that M6 binds to the ATP binding pocket with a docking energy score of −7.3 kcal/mol. This compound binds to the conserved regions of the ATP site, similarly to docetaxel as well as the co-crystalized ligand, sorafenib ( Figure 6A). However, unlike M6, docetaxel was able to bind the regulatory domain pocket (RDP), as was the sorafenib, as both molecules have larger substituents [37].
In term of interactions, M6 was involved in two hydrogen bonds between the nitrogen and oxygen atoms within the benzamide moiety and E885 of the αC helix; as well as with the D1046 residue in the DFG region of the VEGFR2, respectively; and in hydrophobic bonds with the side chain of L1019 ( Figure 6B). On the other hand, docetaxel made four hydrogen bonds with V916, L1025, D1046, AND L1049, as well as hydrophobic interactions with K868, L882, G885 L889, I892, and H1026 ( Figure 6C). The stability of the M6-VEGFR2 complex was confirmed by MD simulation. The backbone RMSD of the complex showed a normal dynamic behavior with a fluctuation range between 0.1 and 0.2 Å over the 20 ns simulation time. Additionally, this complex formed up to three stable hydrogen bonds over simulation period (Figure 7).   The MD summation results suggest a stable binding of M6 within the ATP binding pocket of EGFR and VEGFR2 due to the stable hydrogen bonds formed with the proteins. These results are in line with the docking and in vitro results supporting the notion that this compound might act as a dual EGFR and VEGFR2 tyrosine kinase inhibitor.   The MD summation results suggest a stable binding of M6 within the ATP binding pocket of EGFR and VEGFR2 due to the stable hydrogen bonds formed with the proteins. These results are in line with the docking and in vitro results supporting the notion that this compound might act as a dual EGFR and VEGFR2 tyrosine kinase inhibitor. The MD summation results suggest a stable binding of M6 within the ATP binding pocket of EGFR and VEGFR2 due to the stable hydrogen bonds formed with the proteins. These results are in line with the docking and in vitro results supporting the notion that this compound might act as a dual EGFR and VEGFR2 tyrosine kinase inhibitor.

Chemistry
All melting points were determined in open capillaries and are uncorrected. Microanalyses were conducted on a Perkin-Elmer 2408 analyzer (Perkin Elmer, USA). Thin layer chromatography was performed on Merck 5 × 10 cm plates. Samples were percolated with silica gel GF254 using (EtOAc, hexane 1:10) as the solvent system and a short wavelength UV light for visualization. All fine chemicals and reagents used were purchased from (Aldrich chemical Co. U.S.A.) 1 HNMR samples were recorded on a Bruker 500 MHz spectrophotometer (Bruker AXS Inc., Switzerland). Chemical shifts are in δ (ppm) val-ues downfield from tetramethyl siloxane as an internal standard. The mass spectra were measured on Waters Micromass (Water AQUITY UPLC System LCT Premier XE Serial no: KE468, USA).

Molecular Docking
The molecular docking was performed using Autodock Vina program [38]. The crystal structures of proteins (PDB code 6S9B for EGFR and 4ASD for VEGFR2) were prepared by eliminating unwanted co-crystalized ligands and water molecules using Discovery Studio Visualizer (Accelrys, USA). AutoDock Tools was employed to prepare the input pdbqt files for proteins and ligands and to set the size and the center of the grid box. The size of EGFR active site was set at 14 × 18 × 16 Å coordinates in x, y, and z dimensions and centered to (x = −51.787, y = 21.484, z= −0.026) using 1000 Å spacing. The size of VEGFR2 active site was set at 20 × 20 × 20 Å coordinates in x, y, and z dimensions and centered to (x = −23.756, y = −1.152, z = −11.701) using 1000 Å spacing. PyMol [37] and Discovery Studio Visualizer were used to analyze the binding mode and interaction of ligands with proteins.

Molecular Dynamic Simulation
All atom 20 ns molecular dynamics (MD) simulations was performed using GRO-MACS 2018.1 software [39]. OPLS-AA/L force field was used to generate the topology of proteins [40]. The topology and parameter of ligands were generated by the Swissparam server (available at http://www.swissparam.ch/, access on 17 May 2021) [41]. The MD simulations were performed using the previously reported method [42]. Briefly, the system was solvated in cubic box with TIP3P as a water model followed by adding counter ions to neutralize the system. Periodic boundary conditions were used during MD simulation. Energy minimization of system was performed using steepest descent algorithm with tolerance value of 1000 kJ mol −1 nm −1 . The system was then equilibrated using NVT and NPT ensembles for 100 ps. Finally, 20 ns production MD was performed for the system, with trajectories generated every 2 femtoseconds (fs) and snapshots saved every 2 picoseconds (PS). Standard analysis was applied to calculate the root mean square deviation (RMSD) and hydrogen bond formation over the simulation time. Standard analysis was applied to calculate the root mean square deviation (RMSD) and hydrogen bond formation over the simulation time using gmx rms and gmx hbond, respectively.

Antitumor Screening
A primary anticancer assay was performed for two enzymes VEGFR2 and EGFR and 3 human tumor cell line panels, Hela, A549, and MDA, which are related to some neoplastic diseases, like cervical, lung, and breast carcinoma, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda, MD [29][30][31][32][33].

Conclusions
A new series of 8-methoxy-2-trimethoxyphenyl-3-substituted quinazoline-4(3H)-one was synthesized and assessed for antitumor activity against three cell line panels, HeLa, A549, and MDA, using docetaxel as a reference drug. The most potent antitumor activity was found in compound 6, assessed as a VEGFR2 and EGFR enzyme inhibitor, with (IC50, 98.1 and 106 nM, respectively) relative to docetaxel (IC50, 89.3 and 56.1 nM, respectively) as reference drug. The molecular docking and 20 ns molecular dynamics (MD) simulation results suggest a stable binding of M1-6 within the ATP binding pocket of EGFR and VEGFR2 due to the stable hydrogen bonds formed with the proteins. That makes the results in line with the docking and in vitro results, supporting the notion that this compound might act as a dual EGFR and VEGFR2 tyrosine kinase inhibitor. Strong cytotoxic activity was shown for compounds 2, 6, 10, and 8 against Hela cell line with IC50 values of, 2.13, 2.8, 3.98, and 4.94 µM, respectively compared to docetaxel (IC50, 9.65 µM). Strong cytotoxic activity was shown for compound 11 against A549 cell line (IC50, 4.03 µM) in comparison to docetaxel (IC50, 10.8 µM). A strong cytotoxic activity was shown for compounds 6 and 9 against MDA cell line (IC50, 0.79 and 3.42 µM, respectively) relative to docetaxel (IC50, 3.98 µM).