key: cord-0063994-f81qikyt
authors: Abdelgawad, Mohamed A; Musa, Arafa; Almalki, Atiah H; Alzarea, Sami I; Mostafa, Ehab M; Hegazy, Mostafa M; Mostafa-Hedeab, Gomaa; Ghoneim, Mohammed M; Parambi, Della G T; Bakr, Rania B; Al-Muaikel, Nayef S; Alanazi, Abdullah S; Alharbi, Metab; Ahmad, Waqas; Bukhari, Syed N A; Al-Sanea, Mohammad M
title: Novel Phenolic Compounds as Potential Dual EGFR and COX-2 Inhibitors: Design, Semisynthesis, in vitro Biological Evaluation and in silico Insights
date: 2021-05-31
journal: Drug Des Devel Ther
DOI: 10.2147/dddt.s310820
sha: 12f40234919a2cfdff0ffe23f910b5686bb0eab6
doc_id: 63994
cord_uid: f81qikyt

INTRODUCTION: Epidermal growth factor receptor (EGFR) inhibition is an imperative therapeutic approach targeting various types of cancer including colorectal, lung, breast, and pancreatic cancer types. Moreover, cyclooxygenase-2 (COX-2) is frequently overexpressed in different types of cancers and has a role in the promotion of malignancy, apoptosis inhibition, and metastasis of tumor cells. Combination therapy has been emerged to improve the therapeutic benefit against cancer and curb intrinsic and acquired resistance. METHODS: Three semi-synthetic series of compounds (C1-4, P1-4, and G1-4) were prepared and evaluated biologically as potential dual epidermal growth factor receptor (EGFR) and COX-2 inhibitors. The main phenolic constituents of Amaranthus spinosus L. (p-coumaric, caffeic and gallic) acids have been isolated and subsequently subjected to diazo coupling with various amines to get novel three chemical scaffolds with potential anticancer activities. RESULTS: Compounds C4 and G4 showed superior inhibitory activity against EGFR (IC(50): 0.9 and 0.5 µM, respectively) and displayed good COX-2 inhibition (IC(50): 4.35 and 2.47 µM, respectively). Moreover, the final compounds were further evaluated for their cytotoxic activity against human colon cancer (HT-29), pancreatic cancer (PaCa-2), human malignant melanoma (A375), lung cancer (H-460), and pancreatic ductal cancer (Panc-1) cell lines. Interestingly, compounds C4 and G4 exhibited the highest cytotoxic activity with average IC(50) values of 1.5 µM and 2.8 µM against H-460 and Panc-1, respectively. The virtual docking study was conducted to gain proper understandings of the plausible-binding modes of target compounds within EGFR and COX-2 binding sites. DISCUSSION: The NMR of prepared compounds showed characteristic peaks that confirmed the structure of the target compounds. The synthesized benzoxazolyl scaffold containing compounds showed inhibitory activities for both COXs and EGFR which are consistent with the virtual docking study.

Phenolic compounds are considered as one of the most ubiquitously distributed phytochemicals; they are abundantly found in most vegetables and fruits and utilized as food supplement. 1, 2 Most of them possess various pharmacological activities and can be therapeutically indicated as antioxidants, 3, 4 anti-inflammatory, 5,6 antimicrobial, 7 antifungal, 8 antidiabetic, 9 and anticancer agents. 10 They can be classified chemically into curcuminoids (curcumin, tetrahydrocurcumin), stilbenes (resveratrol, pterostilbene), quinones (naphthoquinones, anthraquinones), lignans (sesamin, enterolactone), coumarins (umbelliferone, aesculetin), tannins (ellagitannins), flavonoids (silymarin, genistein, diosmin, quercetin) and simple phenolics (phenolic acids as caffeic and gallic acids). 11 Cancer is considered one of the most dreadful diseases that lead to fatality; it is the second key reason for mortality after cardiovascular diseases. 12, 13 The discovery of new and multitarget drugs for the treatment of cancer is of great importance due to the severity of the disease and the produced side effects of the already approved drugs. As oncogenic kinases play an important role in cell proliferation, inhibition or blocking of their actions has become an important strategy for cessation and termination of human malignancies. 14, 15 Overexpression of COXs enzymes has been detected in different types of tumor and implicated in cancer progression. [16] [17] [18] [19] Accordingly, the design and discovery of drugs possessing dual kinases and COXs enzymes inhibition are of great interest to prevent oncogenesis. 20 Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK). It has crucial role in different cell physiological processes including proliferation, differentiation and migration. The extracellular domain of EFGR has the binding site for endogenous epidermal growth factors (EGF), which consequently induces a conformational change in the tyrosine kinase domain (TKD) in the wild type from an inactive to active state. However, EGFR L858R represents one of the most common oncogenic point mutation that make EGFR kinase constitutively active. EGFR has become a potential drug target for treatment of different human malignancies. Overexpression of mutant forms of EGFR was identified in various types of human carcinomas. Erlotinib is a competitive EGFR inhibitor. For EGFR L858R , the point mutation does not affect negatively the affinity of the inhibitor to the kinase active site. [21] [22] [23] Herein, we amend the naturally occurring active phytochemicals to develop novel chemical entities as a perusal of discovering new, multi-targeted drugs through various semi-synthetic step reactions. 19, [24] [25] [26] [27] Structural modification of plants secondary metabolites can enhance the biological activity of the parent compounds or augment the scope of their pharmacological activities. ÷n continuation of our efforts to find potent anti-cancer agents, new hybrid molecules were semi synthesized based on benzimidazole/ benzoxazole moieties as they incorporated in many propitious oncogenic proteins inhibitors. 17, 23, [28] [29] [30] [31] Through azo coupling reaction, benzimidazole and benzoxazole were chemically combined with naturally isolated phenolic compounds in three chemical scaffolds, which serve our target core structures ( Figure 1 ). The COX-2 and EGFR inhibitory activities for all target compounds will be addressed. Moreover, in silico, study will be investigated for those compounds tested against EGFR tyrosine kinase using docking to elucidate a postulated model for their binding at the molecular level.

All cell lines used in this study, were purchased commercially, from the ECACC collection through local suppliers of Merck and Sigma-Aldrich and Cells were grown according to ECACC recommendations.

Five kilograms of the aerial parts of Amaranthus spinosus L. (Amaranthaceae) were thoroughly washed, shade dried, ground, defatted with n-hexane and extracted with ethyl ethanoate. The ethyl ethanoate extract (24 g) was divided on vacuum liquid chromatography (VLC) to afford five fractions (I-V). Based on the analytical high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS) chromatograms, fractions II and V were selected for isolation and purification of their phenolic constituents. 12 After a series of chromatographic development for fractions II and V, the subfractions II b and V d were separately subjected to further isolation and purification on normal silica gel and Sephadex LH-20, followed by injection on preparative HPLC to obtain the target phenolic acids. 32 The HPLC was operating with a one-hour program, starting at 10% methanol in water for 5 min. For 45 min, a gradual increase of methanol concentration from 10% to 100%. The flow rate will then be maintained at 20 mL/min for 10 min with 10% MeOH. The retention times (t R ) were 16.13, 15.47, 14. 58 min for p-coumaric acid (45 mg), caffeic acid (36 mg) and gallic acid (24 mg), respectively. Several runs have been performed per sample to obtain the required amounts for semi-synthesis of the target compounds.

To the mixture of aniline derivatives (0.01 mol) and HCl (conc, 5mL) in an ice bath, sodium nitrite solution (0.01 mol, 5mL distilled water) was added dropwise and stirred for 2h. The diazonium mixture was added to the phenolic https://doi.org/10.2147/DDDT.S310820

Drug Design, Development and Therapy 2021:15 solution (0.01 mol) in NaOH (30% 10mL). The mixture was then neutralized using CH 3 COONa (10%, H 2 O). The separated dye was collected, filtered, and dried. The obtained dye was chromatographed on normal column silica gel using a mixture of DCM:CH 3 OH solvent system. The prepared compounds were characterized as (C1-4, P1-4 and, G1-4). 

Determination of the cytotoxic effect of the target compounds was performed to assess their ability to kill the tumor cells, which reflects the anticancer activity of the tested compounds. 33 

To assess the mechanism of action for the tested compounds as potential EGFR and BRAF inhibitors, all target compounds and Erlotinib were subjected experimentally to the kinase screening protocol according to the previously reported procedure (Table 1) 

Ellman's photometric assay was followed to assess secretory PLA2 activity. 37 

A commercially available kit was used for the determination of azo-compounds activities on COX-1 and COX-2, which assessed the quantity of prostaglandin E2 (PGE2). The obtained data were shown in contrast to the control (solvent-treated sample). The test was performed in 

The assay methods for IL-6 and TNF-α were adapted according to reported methods (Table 3) . 41 

ANOVA (One-way analysis of variance) was performed to investigate all the statistics. P < 0.05 was considered statistically significant.

The crystal structures of the COX-2 isoform and EGFR were obtained from the PDB (COX-2 ID: 1CX2 and EGFR ID: 5UGB). Identification of key amino acids required for inhibitory activity of enzymes' ligands were identified. Molecular Operating Environment (MOE, Version 2010) was applied for protein preparation and docking procedure of test compounds (Table 4 ). All energy minimizations were carried out with MOE until an RMSD gradient of 0.05 kcal•mol −1 Å −1 with MMFF94x force field and the partial charges were automatically calculated. For docking preparation, the water molecules in the proteins' crystal structures were removed and followed by the protonation using Protonate 3D protocol in MOE using the Triangle Matcher placement method and London dG scoring function. The co-crystallized ligands were used to define the binding site for docking and the essential binding modes. 35, 42, 43 Docking Validation Docking protocols were validated for both receptors through re-docking of the co-crystallized ligands and reproducing all original binding modes between the cocrystallized ligands and the two receptors' pockets in the vicinity of the binding sites. For COX-2, a docking pose with an energy score (S) = −11.99 kcal/mol and an RMSD of 0.574 Å from the co-crystallized ligand pose. For EGFR, a docking pose with an energy score (S)= −13.874 kcal/mol and an RMSD of 0.774 Å from the co-crystallized ligand pose were observed.

The first set of target compounds (C1-2, G1-2 and P1-2) were synthesized starting from 3 (and 4)-aminoacetophenones which are commercially available (Scheme 1). However, the second set of target compounds (C3-4, G3-4 and P3-4) were synthesized from amino-imidazole (B1) and amino-oxazole (B2) which were prepared via condensation reaction of o-phenylene diamine (and 2-aminophenol) with 3-aminobenzoic acid using polyphosphoric acid as dehydrating agent (Scheme 2). All target compounds were finally prepared through diazotization and coupling reaction using sodium nitrite in presence of HCl to afford diazonium salts, which simultaneously added to a basic solution of phenolic acids to produce the target compounds C, P and G. The structure elucidation of newly synthesized compounds P1-4, C1-4, G1-4 was done using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) in addition to the elemental analysis. 1 

MTT cytotoxic assay was conducted on tested compounds for human colon cancer (HT-29), pancreatic cancer (PaCa-2), Human malignant melanoma (A375), Lung cancer (H-460), and Pancreatic ductal cancer (Panc-1) cell lines. The IC 50 for all compounds was expressed in µM (Table 5) . Compounds C1-3, G1-3 displayed moderate activity on all tested cell lines, while C4 and G4 exhibited a potent effect on all tested cell lines, particularly H-460 (1.7 and 2.7 µM, respectively) and Panc-1 (1.3 and 3 µM, respectively) ( Table 5 ). The potent cytotoxic activity derived because of diazo coupling between benzimidazole and/or benzoxazole with caffeic and/or gallic acids which are new scaffolds amended in this experiment. Protein Kinase Inhibition Activity

The targets were evaluated for their kinase inhibitory effect on BRAF and EGFR to explore their cytotoxic mechanism on the tested cell lines. Compounds C4 and G4 exhibited potent kinase inhibitory activity against BRAF (5.8±1.3 and 5.4±0.6 µM, respectively) and EGFR (0.9±0.3 and 0.5±0.2 µM, respectively), in comparison to the standard Erlotinib drug (IC 50 = 0.07 and 0.05 µM, respectively) ( Table 1) .

Ellman's method-based assay was applied to assess the in vitro inhibitory activity against sPLA2 of the preparation at doses ranging from 1.25 to 20 µg/mL and the results are shown in Table 2 . Dexamethasone was a positive control, and the inhibitory activities of targets azo-compounds against sPLA2-V observed IC 50 : were in the range of 43.91 (P2) to 5.68 uM (G4). The µM with SI of 0.084). The other compounds showed moderate to weak activity against COX-2 despite the SI of the compounds is being high. 17, 44 Inhibition of Interleukin 6 (IL-6) and Tumor Necrosis Factor-Alpha (TNF-α)

In the current study, the inhibitory activity of G2-G4 against LPS-triggered TNF-α and IL-6 release was evaluated in mouse macrophages (RAW264.7). To 10 µM of compounds G2 to G4, the macrophages were added and subjected to incubation for 2h. To each well, LPS (0.5 µg/ mL) was added and followed by 22h incubation. The amount of TNF-α and IL-6 was estimated by applying an enzyme-linked immunosorbent assay (ELISA). Table 2 shows the results of the anti-inflammatory test and all tested compounds were able to inhibit LPS-triggered IL-6 and TNF-α expression to various levels, exhibiting the maximum inhibition (32-61%) and (29-56) against LPStriggered expression of TNF-α and IL-6, respectively. G4 displayed the highest inhibition percentage of LPS-stimulated IL-6 and TNF-α (56 and 61%), respectively, as compared to LPS control.

Molecular modelling studies were applied to check the plausible-binding interactions between the newly synthesized compounds and their potential targets. At first, the docking protocols were validated to get more reliable data.

The validation based on re-docking the co-crystallized ligands imitated the same binding modes of the co-crystallized ligands with root-mean-square deviation (RMSD) values of 1.2 and 0.58 for EGFR and COX-2 receptors, respectively. Figure 2 depicts that much similarity exists between the co-crystallized and docked conformer of cocrystallized ligand.

The docking results showed that C4 has an energy score of −13.8376 Kcal/mol (less than ligand 8BM energy score of −11.250 Kcal/mol) and binds with EGFR (5UGB) through HB with Met793 amino acid, which is the same amino acid binder of the ligand 8BM. 45, 46 However, the docking of G4 with COX-2 protein (1CX2) demonstrated five interactions; four of them are HBs and one arenecation interaction. The HBs of G4 with 1CX2 formed between amino acid residue His90, Tyr355, Ser530, Ser530 and Arg513 with N-imidazole, C=O, 3-OH, and 4-OH, respectively (Figures 3 and 4) . 12, 19 Compared to the ligand bromocelecoxib, G4 formed five interactions; one H bond with His90 amino acid (a main amino acid in the DovePress pocket) with an energy score of −24.1500 Kcal/mol, however, the ligand displays only one HB with an energy score of −13.8924 Kcal/mol. The highest COX-2 inhibitory activity of the benzoxazole-gallic acid scaffold is attributed to the benzoxazole moiety's large hydrophobic volume (Table 4 ).

Most of the target semi-synthetic products showed moderate to potent dual inhibitory activity against EGFR and COX-2 inhibitory activities. The coupling between benzoxazole moiety with caffeic acid in one scaffold displayed potent cytotoxic and kinase inhibitory activity for both EGFR and BRAF, while the scaffold of benzoxazole with gallic acid demonstrated COXs inhibitory actions and showed anti-inflammatory activity. Docking studies, which aligned with the biological evaluations, displayed that the energy scores are lower than for both EGFR and COX-2 enzymes also, the G4 demonstrated more interactions than the bromocelecoxib. The Nitrogen of benzoxazole and benzimidazole moieties has important role in binding with both COX 2 and EGFR enzymes through HB.

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The authors extend their appreciation to the Deanship of Scientific Research at Jouf University for funding this work through research grant No (DSR-2021-01-0103). The authors would like to extend their sincere appreciation to Taif University for funding this project through the Researchers Supporting program, (Project number TURSP-2020/208), Taif University, Taif, Saudi Arabia. Also, the authors thank AlMarrefa, University, Ad Diriyah, 13713, Saudi Arabia for support and Dr Ahmed Khames for his evert in reviewing and editing manuscript.

The authors reported no conflicts of interest for this work.

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