In silico, in vitro VEGFR-2 inhibition, and anticancer activity of a 3-(hydrazonomethyl)naphthalene-2-ol derivative

Abstract In agreement with the general features of VEGFR-2 inhibitors, a new naphthalene analog (compound 7) has been designed and synthesized. The inhibitory potential of compound 7 was indicated by the proper binding and the perfect energy of −21.10 kcal/mol compared to sorafenib (−21.22) in the molecular docking studies. Next, six MD simulation studies over 100 ns (RMSD, RMSF, SASA, RoG, hydrogen bonding, and distance between the center of mass) confirmed the accurate interaction of compound 7 with the catalytic pocket of VEGFR-2. Similarly, an MM-GBSA established proper binding showing an exact total binding energy of −36.95 ± 3.03 kcal/Mol. Additionally, the MM-GBSA experiment indicated the vital amino acids in the binding process. Types and number of interactions of compound 7 with catalytic pocket of VEGFR-2 were determined through Protein-Ligand Interaction Profiler (PLIP). As a new compound, the DFT was employed to optimize the molecular structure of compound 7. The DFT experiments also verified the interaction features of compound 7 with the VEGFR-2 active site. In silico ADMET experiments revealed the general drug-likeness of compound 7. Fascinatingly, the in vitro examinations were consistent with the in silico experiments as compound 7 inhibited the VEGFR-2 enzyme with an IC50 value of 37 nM. Captivatingly, compound 7 inhibited both MCF-7 and HCT 116 cancer cells exhibiting IC50 values of 10.56 and 7.07 µM exhibiting excellent selectivity indexes of 9.04 and 13.50, respectively. Communicated by Ramaswamy H. Sarma


Introduction
Cancer is the second most common health problem in the world behind cardiovascular diseases according to the WHO. The most common cancer type, based on the new cases that were discovered in 2020, was breast cancer with new 2.26 million cases (Cancer, fact sheet, XXXX). Appropriately, medicinal chemists are challenged to find out safe, effective, and targeted chemotherapeutic agents that inhibit cancer growth via the interaction with particular molecular targets and subsequently kill the cancer cells. As tumors develop and reproduce as a result of increased angiogenesis (El-Dash et al., 2021), anti-angiogenesis was considered one of the key potential strategies to fight cancer (Quesada et al., 2006). The vascular endothelial growth factor (VEGF) pathway is a key regulator of angiogenesis that inspired medicinal chemists to invent several powerful chemotherapeutics. The vascular endothelial growth factor receptors (VEGFR) are separated into three types; VEGFR-1, VEGFR-2, and VEGFR-3 (Olsson et al., 2006). The VEGFR-2 is one of the most effective targets in cancer management  since this receptor organizes critical stages in cell division proliferation, motility, and angiogenesis (Elrazaz et al., 2021). As a consequence, if this signaling cascade is blocked, cancer proliferation will be reduced Mohamed et al., 2019). Using the over activation of VEGFR-2 receptors in cancer cells compared to normal cells, researchers were able to develop a safe, selective drug that targets angiogenesis in tumor cells without harming normal cells (Sana et al., 2020).
The field of computational chemistry employs theoretical concepts and a set of mathematical techniques to solve chemical problems, and is employed widely in pharmaceutical industry research to determine how drugs interact with biomolecules (Cavasotto et al., 2019).
We herein combine our previous experiences in computational chemistry, design, and synthesis to present a 3-(hydrazonomethyl)naphthalene-2-ol derivative as an anticancer lead compound targeting the VEGFR-2 enzyme. A design following the general features of VEGFR-2 inhibitors was conducted. Then, the binding of the designed compound in addition to its drug likeness was proved by various computational studies; started with molecular docking, then, MD simulations (for 100 ns), MM-GBSA, PLIP, DFT, ADMET, and toxicity. Then, the target compound was prepared and in vitro VEGFR-2 inhibition, anticancer and safety studies have been carried out.

Rationale
The reported VEGFR-2 has four common features which are crucial for the maximal fitting with the VEGFR-2 ATP binding site and hence producing good activity. These features can be illustrated in Figure 1 as follows (1) Hetero aromatic ring that can occupy the hinge region in the ATP binding site; (2) Spacer group that can occupy the gatekeeper region; (3) Pharmacophore moiety consisted of at least a hydrogen bond (H-B) donor and an H-B acceptor group. The pharmacophore is the most essential feature as it occupies the DFG motif region from essential H-B interactions with Asp1044 and Glu833; (4) Terminal hydrophobic tail that can interact hydrophobically with the allosteric binding pocket of the ATP binding site Yousef et al., 2021).
As shown in Figure 1, there are different moieties that can occupy the hinge region as pyridine (sorafenib I) (Adnane et al., 2006), 2-indolinone (sunitinib II) (Peng et al., 2017), quinoline (tivozanib III) (De Luca & Normanno, 2010), and naphthalene (compound IV which was reported by our team and exerted promising antiproliferative activities and VEGFR-2 inhibitory activity (Hagras et al., 2022)). In addition, the naphthalene moiety was reported as a building block in the discovery and synthesis of new anti-proliferative compounds (Wang et al., 2018;. Taking the aforementioned facts into consideration and in continuation of our efforts to discover VEGFR-2 inhibitors Ran et al., 2021), we designed and synthesized a new naphthalene derivative as a potential compound that may exert a marked VEGFR-2 inhibitory activity and consequently inhibit the growth of tumor cells.
From a drug design point of view, The 3-(hydrazonomethyl)naphthalene-2-ol moiety was utilized in the current work to engage the hinge region. This moiety has three H-Bdonor/ acceptor atoms which can bind efficiently with Cys917 at the hinge region. In addition, the phenyl ring and carbonyl group were used as a linker. The distance between the two aromatic groups (naphthalene and central phenyl rings) was exposed to chain extension to increase the flexibility of the synthesized compound. Hence, this may lead to the increased affinity against VEGFR-2 and consequently increased activity. Furthermore, as appeared in sunitinib II, the amide moiety was used as a pharmacophore moiety to occupy the DFG region. Lastly, to occupy the allosteric pocket, a phenyl ring was used ( Figure 1). The binding mode of the synthesized compound confirmed the design as each feature occupied its target pocket in the ATP binding site.

Molecular docking against VEGFR-2
To verify the operated design, docking simulations against VEGFR-2 kinase (PDB ID: 2OH4) were performed using the Molecular Operating Environment, MOE package, and version 2014.09 software. The validity of the docking studies was first authenticated though expressing the RMSD value, s 0.66 Å, indicating that the docking process was valid ( Figure 2). Sorafenib, as a reference VEGFR-2 inhibitor, was docked and revealed an affinity value of À 21.22 (Figure 3). It was noticed that sorafenib's binding pattern inside the VEGFR-2 active site was matched with the previously published data (Dietrich et al., 2010;Liu & Gray, 2006).
Compound 7 expressed an affinity value of À 21.10 kcal/ mol and demonstrated a binding mode nearly comparable to that of sorafenib. In detail, three H-Bs, seven hydrophobic, and one electrostatic interaction in the active site of VEGFR-2 were indicated. In the hinge region, the 3-hydroxy naphthalene moiety was buried to form one H-B (2.16 Å) with the key amino acid residue (Cys917) via its hydroxyl group. Also, such moiety was engaged in three hydrophobic interactions (H-Is) with Leu1033 and Leu838. Furthermore, the linker region was occupied by the central phenyl ring (spacer moiety) forming three pi-alkyl interactions with Val914, Val897, and Lys866. Also, in this region, one pi-sulfur interaction was observed with Cys1043. In the DFG region, the pharmacophore amide moiety formed two essential H-Bs with Glu883 (1.99 Å) and Asp1044 (1.75 Å). Finally, the terminal hydrophobic phenyl formed one electrostatic and one H-I with Asp1044 and Leu887, respectively ( Figure 4).

MD simulations
To confirm the indicated binding of compound 7 in molecular docking, we measured RMSD, RMSF, SASA, RoG, and the change in the hydrogen bonds in addition to the distance between the protein's center of mass of and the considered ligand in the complex of VEGFR-2-compound-7 throughout the outputted MD trajectory. Figure 5A shows the RMSD change through the whole trajectory for compound-7, VEGFR-2, and their complex. As seen, the RMSD of the VEGFR-2 protein and the complex can be separated into two regions; the first 50 ns has an average of 2.27 Å and 2.81 Å, respectively, while the second half has an average of 3.67 Å and 4.13 Å, respectively. The RMSD of compound-7 shows a very stable trend with an average of 1.17 Å. The increase in the average RMSD values is due to the large motion of the Ala1048:Leu1066 loop as can be seen from the RMSF values ( Figure 5B). Most of the amino acids have RMSF values of less than 2 Å which shows the stability of the VEGFR-2 protein during the simulation. An exception to this is the N and C terminals with RMSF values reaching 6.8 Å and 11.0 Å, respectively. The SASA ( Figure 5C, average ¼ 17662 Å 2 ), RoG ( Figure 5D, average ¼ 20.69 Å), and the change in the hydrogen bonds ( Figure 5E, average ¼ 70 bonds) values show a very stable surface area, the radius of gyration and number of H-Bs for the VEGFR-2 protein through the simulation. This indicates the VEGFR-2 protein did not undergo folding or unfolding during the simulation. The distance between the center of masses of the VEGFR-2 and compound-7  ( Figure 5F) shows a nearly constant distance with an average of 8.94 Å which indicates the stability of the complex.
Overall, the production run shows that the complex is very stable and the protein is equilibrated.

MM-GBSA
To investigate the binding free energy between the VEGFR-2-compound-7 complex, the gmx_MMPBSA library was utilized. Figure 6 illustrates the values of different energy components calculated with MMGBSA. The binding is mostly due to the H-Is as can be seen from the Van Der Waals value in Figure 6 and the electrostatic interaction has an energy of nearly half that of the Van Der Waals. The total binding energy is À 36.95 ± 3.03 kcal/mol. Decomposition analysis was performed to see which amino acids have the most contribution to the binding. Figure 7 shows the amino acids that are around 10 Å of the bound ligand. Nine amino acids show a contribution to the interaction with a binding energy of less than À 1 kcal/mol. Leu838, Leu887, Val897, Phe916, Cys917, Leu1033, Cys1043, Asp1044, and Phe1045 have a contribution of À 1.89, À 1.32, À 1.02, À 1.33, À 1.87, À 1.38, À 3.04, À 1.24, and À 1.78 kcal/mol, respectively.
To see the conformation of compound 7, the types, and the number of interactions, the trajectory was clustered obtaining a representative frame for further analysis. Protein-Ligand Interaction Profiler (PLIP) was utilized to know the types and number of interactions in the representative frames. Table 1 shows the number and types of these interactions. In all of the representative frames, the number of H-Is is larger than the hydrogen bonds supporting the components of MMGBSA analysis. Figure 8 shows the 3 D interactions obtained from PLIP between the VEGFR-2-compound-7 complex in the representative frames.

Molecular structure optimization
The Schiff base compound under investigation was formed by N-(4-(hydrazinecarbonyl)phenyl)benzamide's nucleophilic attack on 3-hydroxy-2-naphthaldehyde via an imine bond (C19-N18). The optimized chemical structure of the  synthesized Schiff base compound is shown in Figure 9, the length of the imine bond (C19-N18) was calculated to be 1.28356 Å, and the angles (C20C19N18) and (C19N18C17) were found to be 121.55325 � and 117.01603 � , respectively, as shown in Figure 9.

Frontiers molecular orbitals, reactive parameters, and total density of state analysis
The Gaussian(R) 09 program at the 6-311þþ G (d,p) basis sets and the density functional theory (DFT) method were used to do the quantum chemistry calculations at the B3LYP level. Figure 10 shows the focusing of electronic density (ED) over the central linker and hydroxy naphthalene moieties in the lowest unoccupied molecular orbital, LUMO. While, the ED of the highest occupied molecular orbital, HOMO, is centered on the hydrazine carbonyl naphthalene system. According to the frontier molecular orbital (FMO) theory, HOMO acts as an electron donor while LUMO act as an acceptor. When employing quantum chemical calculations, HOMO and LUMO play crucial roles in the electronic studies and are crucial to contemporary molecular biology as well as biochemistry. Decreasing of the molecule's energy gap, is believed that the molecule would be softer and more chemically reactive. When a molecule has a wide energy gap, it is thought to be harder chemically and more stable (Pegu et al., 2017). Using the energy differential (E gap ) between the frontier orbitals, the FMO provides extremely considerable evidence for stability. The effectiveness of the compound 7 inhibition is correlated with chemical quantum parameters, such as the chemical potential (m), energy change (DE), maximal charge acceptance (DN max ), ionization potential (IP), global hardness (g), electronegativity (v), the global softness (r), electrophilicity index (x), and electron affinity (EA). These parameters were computed on the bases of the equations of Koopmans' theory (E gap ¼ E LUMO -E HOMO, more details in the Supplementary data). Table 2 lists the calculated total ground state energy (TE), dipole moment (Dm), and other global quantum characteristics. The outcomes relate to how well compound 7 can bind and suppress VEGFR-2. The number of occupied states per unit volume for given energy is determined for a system in equilibrium by multiplying the density of states by the probability distribution function. This value is typically used to study a range of materials' physical characteristics. Calculations and analyses of the overall density of the state have been performed. The findings supported the small energy gap of the synthesized Schiff base as seen in Figure  11. This supported the experimental results as the synthesized compound has high chemical reactivity against VEGFR-2. The effectiveness of the inhibitor rises as the E gap of the border orbitals decreases (Husein et al., 2021).

The electron density maps
Based on the electron density of the donor atoms, DFT calculations may determine the reactivity level of the chosen molecule. The electron density of the whole molecule is depicted by the total electron density (TED) map, Figure 12. The O atoms of the carbonyl and hydroxyl groups in the examined molecule are the high electronegativity chemical sites that are represented by the red patches. These active sites facilitate amino acids' electrophilic attack. Additionally, blue zones indicated the most advantageous positive areas, which receive electrons from donor atoms of amino acids. The two N-H groups are electro-positive areas with blue color, the yellow regions denote atoms with moderate electronegativity and may form H-I (Wang & Husein, 2022). The naphthyl and two phenyl groups were presented in a yellow color indicating the high possibility for H-I. These results provide an explanation for the nucleophilic interaction of amino acids on the pharmacophore (amide), hydroxyl, naphthyl, central phenyl, and terminal phenyl groups. The inhibitory direction of compound 7 on the electrophilic amino acids is shown by the electrostatic surface potential (ESP) (Figure 12) and it is the same orientation as the oxygenated groups.

In silico toxicity studies
Five toxicity (acute and chronic) parameters were investigated in accordance to the toxicity models established in Discovery studio software as outlined in Table 3. Sorafenib was employed as a reference molecule.
Compound 7 was predicted to have a mouse carcinogenic potency TD 50 (C-TD 50 ) value of 38.581 mg/kg/day. Such a value was safer than sorafenib's value (19.236 mg/kg/day). Carcinogenic potency TD 50 (CP-TD 50 ) predicts the carcinogenic dose rate of a molecule in a rodent exposure (chronic) toxicity model (Venkatapathy et al., 2009). Concerning the rat maximum tolerated dose (R-MD), compound 7 was predicted to produce a dose value of 0.486 g/kg which was higher than sorafenib (0.089 g/kg). As well, compound 7 was predicted to have rat oral LD 50 (RO-LD 50 ) and rat LOAEL (LOAEL-R) values of 1.794 and 0.360 g/kg, respectively. These values were significantly higher than those of sorafenib (0.823 and 0.005 g/kg, respectively). Moreover, compound 7 was predicted to exert accepted results against skin irritancy and ocular irritancy models.

In silico ADMET profiling
The ADMET parameters were determined computationally for compound 7 against sorafenib as a reference molecule using Discovery studio 4.0. Figure 13 shows the representation of the investigated ADMET parameters as ellipses: the blue color denotes the lipid-water partition coefficient (AlogP98). Red and green ellipses denote intestinal absorption with 95% and 99% confidence limits, respectively. Pink and turquoise ellipses denote the blood-brain barrier (BBB) of 95% and 99% confidence limits, respectively. As presented in Figure 13, the two points (sorafenib and compound 7) lie out of the turquoise and pink ellipses and inside the green and red ellipses explaining that both molecules exhibited very low BBB penetration levels and good intestinal absorption levels. Both compounds were predicted as non-inhibitor of the cytochrome P-450, CYP2D6, and to have more than 90%.binding with the plasma protein.

Chemistry
In order to verify the carried-out design as well as the promising results obtained from the conducted computational studies, compound 7 has been synthesized according to the synthetic pathway displayed in Scheme 1. Initially, methyl 4aminobenzoate 2 was prepared according to the reported procedure by heating 4-aminobenzoic acid 1 with methanol in the presence of sulfuric acid (El-Zahabi et al., 2020). The methyl 4-benzamidobenzoate 4 was then furnished by the addition of benzoyl chloride 3 in DCM to methyl 4-aminobenzoate 2 in a dropwise manner in the presence of TEA at 0 � C then the mixture was allowed to react at room temperature overnight . The produced compound 4 was then refluxed with hydrazine hydrate in ethanol to produce N-(4-(hydrazinecarbonyl) phenyl)benzamide 5 . Following the Schiff's base reaction, the hydrazinecarbonyl derivative 5 was refluxed with 3-hydroxy-2-naphthaldehyde in absolute ethanol and drops of glacial acetic acid to afford the target derivative N-(4-(2-((3-hydroxynaphthalen-2-yl)methylene)hydrazine-1-carbonyl)phenyl) benzamide 7 (Scheme 1). The proposed mechanism for the formation of azomethine linkage (HC ¼ N) in compound 7 is illustrated in Scheme 2.
The structure of compound 7 was confirmed by spectral analysis. 1 H NMR spectrum showed the presence of the downfield singlet signals at 12.86, 12.19, and 10.60 ppm attributed to the two amidic protons and the OH group. Also, the integration was augmented at the aromatic region confirming the presence of the additional aromatic protons. A characteristic 1 H NMR signal of the azomethine group (CH¼N) was observed at 9.52 ppm. Also, the IR band at a frequency of 1598 cm À 1 indicates the presence of the azomethine C ¼ N bond. Furthermore, the 13 C NMR spectrum was also consistent with the assigned structures of the synthesized compound.

VGFER-2 inhibition
To verify our design as well as the conducted computational experiments that showed the strong affinity of compound 7 to bind with and inhibit the VEGFR-2 enzyme, the in vitro prohibitory effect of compound 7 against the VEGFR-2 enzyme was estimated comparing sorafenib. The results were presented as IC 50 values which are the measure of the potency of substances in inhibiting VEGFR-2 function.
Stimulatingly, compound 7 strongly inhibited VEGFR-2 expressing an IC 50 value of 37 ± 2.5 nM that was comparable to the inhibitory value of sorafenib (35 ± 2.8 nM). The obtained in vitro result was consistent with the conducted in silico studies and confirmed the great ability of compound 7 to inhibit VEGFR-2. Values are given as mean ± SEM of triplicates.

Cytotoxicity
To examine the VEGFR-2 prohibitory potential of compound 7 against cancer, in vitro cytotoxicity examination of the anticancer potential of compound 7 against breast cancer cell line, MCF-7, and human colon cancer cell lines, HCT 116, were administered employing sorafenib as a reference. As shown in Table 4 and Figure 14, compound 7 inhibited both cell lines expressing IC 50 values of 10.56 ± 2.5 and 7.07 ± 0.55 mM, respectively. The in vitro anticancer potentialities of compound 7 were too near to those of sorafenib as it demonstrated IC 50 values of 4.32 ± 0.33 and 7.28 ± 0.62 mM against the same cell lines, respectively. Values are given as mean ± SEM of triplicates.

Safety and selectivity index
To examine the safety of compound 7 and indicate the selectivity of the examined compound against the cancer cell lines, the cytotoxic activity of compound 7 against the W138 normal human cell line was administered. Compound 7 indicated a high level of safety showing a very high IC 50 value of 95.47 mM as well as elevated selectivity indexes (SI) against MCF-7 and HCT 116 cell lines of 9.04 and 13.50, respectively.

Conclusion
A new naphthalene derivative has been designed based on the features of VEGFR-2 inhibitors. The inhibitory potentialities of the targeted compound were indicated by molecular docking studies. Interestingly, the precise binding against the catalytic pocket of VEGFR-2 was confirmed via six MD simulations, three MM-GBSA, and three DFT studies. Further, in Figure 11. The total density of state analysis of compound 7 at B3LYB/6-311þþG (d,p).   silico ADMET studies revealed the drug-likeness of the proposed compound. Accordingly, the targeted compound was synthesized and examined in vitro to exhibit VEGFR-2 inhibition with an IC 50 value of 37 nM in addition to strong inhibitory potential against MCF-7 and HCT 116 cell lines expressing IC 50 values of 10.56 and 7.07 mM combined with excellent selectivity indexes of 9.04 and 13.50, respectively.

Molecular docking studies
The molecular docking studies were conducted Parmar et al., 2021) by MOE2014 software as outlined thoroughly in Supplementary data.

MD simulation
GROMACS 2021 and CHARMM-GUI web server were utilized as an MD engine  as outlined thoroughly in Supplementary data

MM-GBSA
The Gmx_MMPBSA package was utilized as outlined thoroughly in Supplementary data

DFT
GaussSum3.0 and Gaussian 09 programs were utilized as outlined thoroughly in Supplementary data. ADMET

Toxicity studies
The toxicity profile was determined by Discovery studio 4.0 (El-Adl et al., 2021) as outlined thoroughly in Supplementary data.

General procedure for the synthesis of compound 7
In 30 mL of absolute ethanol contains glacial acetic acid (few drops), the hydrazide derivative 5 (0.001 mol) and 3hydroxy-2-naphthaldehyde (0.001 mol) were refluxed for two hours. The mixture was cooled to 27 C after the reaction was finished. The obtained precipitate was then filtered, dried, and recrystallized from ethanol to afford the target compound 7.

In vitro VEGFR-2 inhibition
Was carried out utilizing human VEGFR-2 ELISA kit as outlined thoroughly in Supplementary data (Abou-Seri et al., 2016).

In vitro anticancer activities
MTT procedure was utilized as outlined thoroughly in Supplementary data (Mosmann, 1983).

Disclosure statement
No potential conflict of interest was reported by the authors.