Synthesis, Docking Study, and Cytotoxicity Evaluation of New Hydroxy benzoic Acid Derivatives

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medicinal chemistry. A review of the literature revealed that they have a wide range of biological activities, including antiinflammatory, antibacterial, and anti-tumor properties. The current study aimed to synthesize three new hydroxy benzoic acid derivatives (compounds 8, 9, and 10) and test them as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in silico and in vitro. Traditional organic synthesis methods were applied to produce these compounds. Docking studies revealed that compound 8 had nearly equal binding energy (G) to erlotinib, the standard EGFR tyrosine kinase inhibitor. -8.56 and -8.75 kcal/mol, respectively. Using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) viability assay, the cytotoxicity of the new hydroxy benzoic acid derivatives was tested against DLD-1 colorectal adenocarcinoma cells, HeLa cervical cancer cells, and MCF-7 breast cancer cells. Compound 8 showed cytotoxicity against the three cancer cell lines tested (25.05, 23.88, and 48.36 µM, respectively), while compound 9 showed cytotoxicity against HeLa cells only (37.67 µM), and compound 10 was cytotoxic to DLD-1 and HeLa cells (27.26 and 19.19 µM), respectively. The half maximal inhibitory concentrations (IC 50 ) of compound 8 were comparable to those of the standard tyrosine kinase inhibitor drug erlotinib (13.86, 36.41, and 87.34 µM, respectively). In conclusion, the findings of the present study indicate that compound 8, through its EGFR inhibitory activity, is a promising cytotoxic agent.
A r t i c l e i n f o.

Introduction
Cancer remains a major threat with high mortality and morbidity rates due to its complicated and heterogeneous nature. There is an urgent need to create new anticancer agents to counteract the increased toxicity and resistance of existing anticancer medications. Apoptosis is a major target for the discovery of new anticancer candidates because it plays a significant role in cancer progression. As a result, anticancer drugs that target different apoptotic signaling pathways have emerged as an important mechanism in cancer treatment [1]. The epidermal growth factor receptor (EGFR) tyrosine kinase regulates a variety of cellular activities including the cell cycle, adhesion, and motility [2]. Thus, EGFR overexpression or mutations promote cell proliferation and metastasis and inhibit apoptosis, leading to a variety of epidermal carcinomas including colon, breast, and bladder cancer [3][4][5][6]. Hydroxy benzoic acids can be formed directly from intermediates early in the shikimate pathway. However, they are more commonly formed in plants by the degradation of C6-C3 cinnamic acid derivatives. Examples of these acids include p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids ( Figure  1) [7]. It was reported that 3-O-Methylgallic acid (Megal) and gallic acid (Gal), two hydroxy benzoic acids, decreased cell viability in SW-480 and Caco-2 colorectal cancer cells. This reduction was primarily due to Megal and Gal's ability to inhibit cell cycle progression during the G0/G1 phase. These two hydroxy benzoic acids also inhibited the activities of survival transcription factors known to be activated in colorectal carcinoma such as NF-kB, AP-1, STAT-1, and OCT-1. The ability of these compounds to inactivate these transcription factors might have contributed to Megal and Gal's anti-proliferative effects and induction of apoptosis [8]. Different studies have reported that dihydroxy benzoic acid (Figure 2A) and 4-hydroxybenzoic acid ( Figure 2B) inhibited activity of histone deacetylases (HDACs), enzymes which modulate the expression of genes, leading to cancer cell growth inhibition through the induction of reactive oxygen species (ROS) and apoptosis mediated by caspase-3. 4-hydroxybenzoic acid was able to successfully reverse adriamycin (ADM), a drug used to treat clinical breast cancer, resistance in human breast cancer cells by acting as histone deacetylase 6 (HDAC6) inhibitor [9-11].

Thiosemicarbazides
(hydrazine-1carbothioamide) are a class of compounds with high bioactivity. These compounds have numerous biological activities, including antifungal, anticancer, antimicrobial, sodium channel blocker, and antiviral properties [12]. In addition, thiosemicarbazide derivatives such as triapine (Figure 2C), and methisazone ( Figure 2D) have anticancer activity due to their ability to suppress ribonucleotide reductase enzyme [13].
The present study aimed to design and synthesize new hydroxy benzoic acid derivatives exhibiting anticancer activity based on their predicted EGFR tyrosine kinase inhibition from molecular docking studies. The purified compounds containing (3-hydroxy,4-methoxy) 3,4dimemethoxy or (3-butoxy,4-methoxy) benzoic acid, were characterized spectroscopically and were assessed for their anti-proliferation activities in silico, and in vitro against DLD-1, HeLa, and MCF-7 cancer cell lines.

Experimental part Material and methods
All of the chemicals and reagents used were purchased commercially from hyper chem. Limited (China ) and used as received with no further purification. 1 HNMR spectra were recorded at 400 MHz (Bruker BioSpin), and chemical shift values (ppm) were reported to TMS, as an internal reference. DMSO d6 was the solvent used . Tensor II Bruker-Optics FT-IR spectrophotometer was used to measure KBr disc FT-IR spectra.

Molecular docking studies Method of docking process
For the molecular docking process, the CDOCKER protocol was used. EGFR was kept rigid during the method, while the ligands were made flexible. Each molecule was allowed to create ten different interaction poses with EGFR tyrosine kinase. When the best-fitting poses were identified, docking scores (-CDOCKER interaction energy) were recorded. The protein data bank (https://www.rcsb.org) was used to identify molecular targets for the newly synthesized compounds (8-10), compare them to other ligands, and determine the pharmacophoric functionality that may enable binding to the critical amino acids at tyrosine kinase domain of EGFR. Following the selection of a specific protein, EGFR in the current study, some procedures were performed that provide an understanding of the molecular binding modes of the test compounds inside of the pockets of the proteins (EGFR tyrosine kinase), by using MOE 19.0901 Software. The co-crystallized ligand was used to produce the binding sites within the crystal protein (PDB codes: 4HJO) (https://www.rcsb.org). Water molecules were initially removed from the complex. The crystallographic disorders and unfilled valence atoms were then corrected using protein report, utility, and clean protein options. Protein energy was minimized by applying MMFF94 force fields to it. The essential amino acids of the protein are outlined and ready for docking. The 2D structures of the compounds tested were sketched in Chem-Bio Draw Ultra17.0 and saved in MDL-SD file format using MOE 19.0901 software. The saved file was opened, 3D structures protonated, and energy was kept to a minimum by using a .05 RMSD kcal/mol MMFF94 force field. The minimized structures were then ready for docking with the prepared ligand protocol [14]. ADMET studies Erlotinib was used as a reference drug to the newly synthesized compounds in absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies, in which the Discovery Studio 2019 Software was used.

Results and Discussion Chemistry
The general route for the synthesis of the desired compounds is summarized in Scheme (1). Using FT-IR and 1 HNMR, all chemically synthesized compounds were identified. The IR spectra for compounds 8, 9 and 10 showed bands at ύ =3301. showed signals at δ= 10.4 and 9.7 ppm assigned to (NH) sec. amine. The 1 HNMR spectrum of compound 10 showed signals at δ =10.4 and 9.8 ppm due to (NH) sec. amine. The aromatic protons appeared clearly at their respective region.

Molecular docking results
Ten poses for each compound were generated, then the best orientations were captured, and affinity scores with RMSD (root-mean-square deviation) values were collected. The binding modes and interaction energy (G, docking score) of erlotinib, and the newly synthesized hydroxy benzoic acid derivatives against human EGFR target site are presented in Table 1.

ADMET studies
Six ADMET parameters were evaluated using the Discovery Studio 2019 Software and erlotinib as a reference drug (Figure 13). The findings summarized in Table 2 show that the synthesized compounds displayed high penetration of blood-brain barrier (BBB).
The solubility level of all compounds was low except for compound 8 which showed good solubility level. Additionally, all compounds showed some in silico hepatotoxicity except for compound 10. Finally, the new hydroxy benzoic acid derivatives showed an optimal absorption value with good distribution in different organs in the human body.

Biological study The in vitro anti-proliferation activities of the novel hydroxy benzoic acid derivatives
To assess the effects of the newly synthesized compounds on cell proliferation, the cytotoxicity of the novel hydroxy benzoic acid derivatives was investigated by means of the MTT assay. The results presented in Table 3 indicate that compound 8 showed cytotoxicity against the three cancer cell lines tested, while compound 9 showed cytotoxicity against HeLa cells only, and compound 10 was cytotoxic to DLD-1 and HeLa cells. Compound 8 IC 50 concentrations were comparable to those of the standard tyrosine kinase inhibitor drug erlotinib ( Table 3).