Biological evaluation, docking studies, and in silico ADME prediction of some pyrimidine and pyridine derivatives as potential EGFRWT and EGFRT790M inhibitors

Abstract Herein, a set of pyridine and pyrimidine derivatives were assessed for their impact on the cell cycle and apoptosis. Human breast cancer (MCF7), hepatocellular carcinoma (HEPG2), larynx cancer (HEP2), lung cancer (H460), colon cancers (HCT116 and Caco2), and hypopharyngeal cancer (FADU), and normal Vero cell lines were used. Compounds 8 and 14 displayed outstanding effects on the investigated cell lines and were further tested for their antioxidant activity in MCF7, H460, FADU, HEP2, HEPG2, HCT116, Caco2, and Vero cells by measuring superoxide dismutase (SOD), malondialdehyde content (MDA), reduced glutathione (GSH), and nitric oxide (NO) content. Besides, Annexin V-FITC apoptosis detection and cell cycle DNA index using the HEPG-2 cell line were established on both compounds as well. Furthermore, compounds 8 and 14 were assessed for their EGFR kinase (Wild and T790M) inhibitory activities, revealing eligible potential. Additionally, molecular docking, ADME, and SAR studies were carried out for the investigated candidates.


Introduction
Being the second leading cause of mortality globally, cancer kills roughly 8 million people each year. Additionally, cancer incidence is expected to elevate regrettably by more than 50% in upcoming years [1][2][3] . Besides, different cancer types have developed acquired chemotherapeutic resistance over the last few decades [4][5][6] . Furthermore, chemotherapeutics utilised could induce cytotoxicity to other healthy normal cells owing to their poor selectivity. Thus, severe adverse effects may be experienced, such as anaemia, nausea, alopecia, and immunosuppression 7,8 . As a result, researchers should dedicate their efforts to ice breaking and discovering more appropriate chemotherapeutics, mainly for the most invasive tumours 9,10 .
Furthermore, cellular functions such as metabolism, survival, apoptosis, and cell proliferation could be regulated by protein kinases (PKs) 11,12 . Many diseases, including cancer, are caused by disrupting cell signalling cascades via kinase alterations, particularly hyper-activation, or mutations 13,14 .
Epidermal growth factor receptor (EGFR) is regarded as one of the most outstanding PKs, which play a critical function in cell migration and proliferation 15,16 . Molecules that may affect the control of cancer cell proliferation are targeted by moderndesigned molecular strategies. These strategies are capable of improving cancer therapy efficiency more than conventional chemotherapy. Therefore, EGFRs are regarded as outstanding targets for the design of new anti-tumour agents 17,18 .
An important factor connecting environmental toxicity to the multistage carcinogenic process is oxidative stress. Responses to both endogenous and external stimuli result in the formation of reactive oxygen species (ROS). An intrinsic antioxidant defence system exists to regulate ROS-mediated harm. However, oxidative stress emerges when oxidation surpasses the regulatory systems. Numerous macromolecular components, including DNA, lipids, and proteins, undergo harmful changes as a result of chronic and cumulative oxidative stress. The increased cellular ROS levels are mediated through an alternative strategy through antioxidant use for the sake of tumour cell depletion from ROS-induced survival signalling pathways. It was revealed that increased intracellular ROS levels may be involved in early events of cancer initiation and progression. These treatments might also have a preventative purpose 19 .
In comparison to normal healthy cells, cancer cells exhibit a higher rate of ROS generation and a different redox environment. The majority of chemotherapeutic drugs increase intracellular ROS levels and can change cancer cells' redox balance 20 .
On the other hand, pyrimidine and pyridine-related compounds are an important class of heterocycles owing to their wide chemical and biological applications. They have been employed widely in the areas of medicine, and material science [21][22][23] . They are responsible for various biological significance, such as anti-inflammatory 24 , antipyretic 25 , antihypotensive 26 , anticonvulsant 27 , antiviral 28 , antimicrobial 29 , and antidiabetic activities 30 . Among this relevance also, a literature survey revealed that a variety of fused pyrimidines have been reported to be extremely potent anticancer activity against various cell lines 31-33 . Some 2-pyridone derivatives acting as PK inhibitors could exhibit potent anticancer activity 34 . Besides, cyanopyridines revealed potent PIM-1 inhibitions and PDE3A inhibition 35,36 . Hamajima et al. reported pyrazolopyridine as having high PI3Kd inhibitory activity with eligible selectivity and oral availability in mice 37 . Additionally, the cell lines A431a, HCT116, and SNU638b were employed for assessing the in vitro antiproliferative activity of pyrido [2,3-d]pyrimidine derivatives in addition to its inhibition potential for CDK4/Cyclin D, CDK2-Cyclin A, and EGFR enzyme 38 . Orlikova et al. reported pyrazolopyridine derivatives to reflect their selective cytotoxic potential against K562 cancer cells upon comparison to normal cells 39 . Given their significant cytotoxicity against various cell lines, therefore, pyridines, and pyrimidines were incorporated into many FDA-approved anticancer drugs ( Figure 1).
Because of the previous findings and our ongoing research regarding the synthesis of pharmaceutically important pyridines and pyrimidines 40 , the impact of the most active compounds was investigated on the cell cycle and apoptosis by the tumour suppressor p53 to put eyes on their effects on cancer biology assuring the proposed mechanism of action. The current work sheds light on the utility of studied compounds as lead compounds for further investigations as anticancer agents.
PKs are one of the most important families contributing to a large number of diseases like inflammation, diabetes, and/or cancer 41 . PKs constitute one of the apparent and attractive targets for the treatment of many diseases as they regulate a lot of cellular functions, such as apoptosis, proliferation, metabolism, survival, cell cycle, and DNA damage/repair 42  Moreover, EGFR is one of the outstanding tyrosine kinase receptors. It regulates several pathways of signal transduction to regulate cell proliferation and apoptosis. Also, it is overexpressed in many cancer types, such as ovarian, colon, and breast by activating the process of angiogenesis 43 . EGFR inhibitors (EGFRIs), such as erlotinib ( Figure 3) were approved by the FDA in 2004 for clinical use as an anticancer drug 44 .
The common pharmacophoric properties of EGFRI (erlotinib) are depicted in Figure 3. The first one is the presence of a hydrophobic moiety to act as a head occupying the first hydrophobic region. The second feature is the presence of an H-bond donor in the spacer region occupying the linker region between the adenine binding region and the hydrophobic region I. The third pharmacophoric feature of EGFRIs is required to be a flat heteroaromatic moiety to be able to occupy the binding pocket of adenine (hinge segment). Moreover, a second heteroaromatic or hydrophobic moiety is required to act as a tail, occupying EGFR's second hydrophobic region [45][46][47][48] .

Rationale-based design
Relying on the basic pharmacophoric properties of EGFRIs represented in Figure 3, we decided to propose the tested pyrimidine and pyridine derivatives as potential EGFRIs.
Guided by the above-discussed pharmacophoric features of EGFRIs which are a hydrophobic moiety to act as a head occupying the first hydrophobic region, an H-bond donor in the spacer region occupying the linker region between the adenine binding region and the hydrophobic region I, a flat heteroaromatic moiety to be able to occupy the binding pocket of adenine (hinge segment), and a second heteroaromatic or hydrophobic moiety to act as a tail and staying at EGFR's second hydrophobic region 45 .
Herein, the rationale-based design was based on the presence of a pyrimidine or pyridine ring to be inserted into the binding pocket of adenine and act as a flat heteroaromatic moiety. Also, both the thiophene and furan rings were proposed to act as a head to occupy the first hydrophobic region and a tail to occupy the second hydrophobic region of EGFR, respectively. However, the second pharmacophoric feature, which is an H-bond donor in the spacer region, was observed to be either amino, hydroxy, carboxy, or protonated nitrogen atom ( Figure 4).
Finally, the alkylation of 14 with ethyl chloroacetate was carried out under alkaline conditions at room temperature, giving the ethyl acetate derivative 18. However, compound 18 was used as an intermediate for the synthesis of Schiff bases 20 and 21 firstly via its condensation with hydrazine hydrate to give the hydrazide derivative 19 and then treatment of 19 with 4-nitrobenzaldehyde and isatin in boiling ethyl alcohol, respectively, as outlined in Scheme 2. Spectral and analytical measurements were used to confirm their structures.

Biological evaluations
Cytotoxicity screening against human cancer cell lines It was revealed that all investigated cell lines were affected by the afforded pyrimidine and pyridine derivatives at different concentrations (5, 12.5, 25, and 50 lg/mL). Table 1        EGFR kinase (wild and T790M) inhibition assay EGFR plays a pivotal role in tumorigenesis. Hence, cancer treatment targeting the EGFR gene has shown great progress. However, not all cancer patients are sensitive to EGFR-tyrosine kinase inhibitors and that could be attributed to EGFR gene mutation 49 . So, it is important to reveal the efficacy of our investigated compounds against both non-mutagenic EGFR (EGFR-wild type) and mutagenic EGFR (EGFR-T790M). Consequently, the most active compounds (8 and 14) that displayed outstanding anti-proliferative activities towards the utilised cancer cell lines were employed to assess their EGFRI potential. The reagent, Kinase-Glo MAX, was used 50 , and luminescence was detected by applying the microplate reader. Erlotinib was used as a reference standard in this experiment as shown in Table 2. Accordingly, considering EGFR kinase wild, it was revealed that the investigated compounds showed less inhibitory potential than erlotinib with IC 50 values of 0.131 and 0.203 mM for compounds 8 and 14, respectively, whereas, erlotinib exhibited an IC 50 value of 0.042 mM. Hence, it was elicited that compounds 8 and 14 experienced eligible inhibitory potential against EGFR kinase (wild) as depicted in Figure  12(A). However, regarding EGFR kinase T790M, it was disclosed that the assessed compounds displayed less inhibitory potential than erlotinib with IC 50 values of 0.027 and 0.156 mM for compounds 8 and 14, respectively, whereas, erlotinib exhibited an IC 50 value of 0.009 mM. Hence, we can deduce that compounds 8 and 14 could display feasible inhibitory potential against EGFR kinase (T790M) as depicted in Figure 12(B).

In silico studies
Docking studies First, the key amino acids required for EGFR-Kinase domain interaction were identified with the aid of co-crystallised downloaded pyridinone ligand (5Q4) interactions. It was revealed that 5Q4 forms four hydrogen bonds with Glu-804, Cys-775, Gln-791, and Met-793, and two ionic bonds with Glu-804 at EGFR-Kinase ( Figure 13).
Accordingly, it was clear that the tested candidates showed diverse binding scores and modes at the EGFR-Kinase domain receptor compared to that of the co-crystallised 5Q4 inhibitor. Hence, concerning its RMSD values and interactions results, synthesised compounds 8 and 14, showed favourable results between tested compounds at the EGFR-Kinase receptor. The synthesised compounds 8 and 14 showed binding interactions nearly similar to that attained by the co-crystallised ligand. The chemically synthesised compound 8 revealed binding interaction to EGFR-Kinase domain through forming two hydrogen bonds with Gln-791 and Met-793, and one pi-H bond with Leu-718 with RMSD ¼ 1.6807, whereas, the chemically synthesised compound 14 interactions revealed its binding with Cys-775 and Met-793 through forming H-bonds, and Gly-796 by a pi-H bond with RMSD ¼ 1.1802. However, the docked 5Q4 was capable of forming three hydrogen bonds with Gln-791, Cys-775, and Met-793, and one pi-H bond with Leu-718 with RMSD ¼ 1.3242 (Table 4).
The docking results of compounds 8, 14, and 5Q4 show their interactions and positioning in 3D orientation at the EGFR-kinase domain (Tables 3 and 4). The detailed docking scores, RMSD values, interactions, and visualisation of other candidates are described in the Supplementary data as 2D and 3D interactions, and surface and maps (Tables SI 1-SI 3).
In silico physicochemical, pharmacokinetic, and ADME studies The SwissADME website of the Swiss Institute of Bioinformatics (SIB) 51 was applied for the estimation and prediction of the physicochemical and pharmacokinetic characteristics of the examined derivatives [52][53][54] (Table 5).
The aim is to confirm that the most favourable compounds in molecular modelling studies (compounds 8 and 14) are promising candidates regarding their pharmacokinetic properties. Compounds 8 and 14 exhibited predicted wlogP values of 3.65 and 3.24, respectively, with no blood-brain barrier (BBB) permeability and so no CNS side effects are predicted. Both compounds 8 and 14 showed high GIT absorption with reasonable H 2 O solubility. Moreover, compound 8 is a substrate for P-glycoprotein (PGPþ) whereas compound 14 is not a substrate for it (PGPÀ), so it is not subjected to the efflux mechanism as a drug-resistance mechanism used by many tumour cell lines. Also, compound 8 manifests inhibition for the metabolising enzymes (CYP1A2 and CYP2C19) only. Whereas compound 14 is capable of inhibiting CYP1A2, CYP2C19, CYP2C9, and CYP2D6 metabolising enzymes.

Structure-activity relationship (SAR) study
To attain deep insights and get one step closer to understanding the results of chemical modifications on the activity of studied derivatives on their activities towards the EGFR target site. We decided to conclude and analyse a SAR study based on their effects on the different cell lines used as depicted in Figure 14.
The following interesting outcomes were unveiled: It was revealed that the best activity towards EGFR was attained by pyridine derivatives. In particular, it was found that the best activity was attained by pyridine derivatives with keto and cyano substitutions at positions 2 and 3, respectively (compound 14), or by fusion with 3-hydroxy pyrazole (compound 8).
On the other hand, it is worth noting that moderate activities were attained by pyridine derivatives retaining pyrazole (compound 9) and pyridine derivatives retaining fused indole with sulphur atom incorporated in the pyridine ring (compound 11), cyano and chloro groups (compound 15), cyano and oxy ethyl acetate ester (compound 18), cyano and oxy acetohydrazide (compound 19), as well as cyano and bulk group (compound 20).

Conclusion
Briefly, a series of chemically synthesised pyridine and pyrimidine derivatives were screened for their anticancer activities via EGFR inhibition. Among the investigated compounds, both 8 and 14 displayed outstanding effects on the tested cell lines with IC 50 concentrations of 3.8 and 7 mg/mL (MCF7), 4 and 3.6 mg/mL   Besides, compound 14 induced a pro-oxidant state in H460, MCF7, HEP2, HEPG2, HCT, and VERO by significantly increasing the MDA and NO. On the other hand, a significant decrease in SOD and GSH levels was observed. Additionally, compounds 8 and 14 achieved a significant increase in apoptosis percentage (total, early, and late) compared to control. Furthermore, there was a significant decrease in the G0/G1 phase with an apparent increase in the S and G2/M phases with both compounds 8 and 14 (p ¼ 0.0001). Besides, the EGFR kinase (Wild and T790M) inhibitory potential of the most active compounds (8 and 14) assured the rational and the mode of action suggested in this current work. Moreover, the molecular docking study performed ensured the outstanding anticancer activities of the investigated compounds by declaring their binding interactions with the EGFR target receptor. Finally, eligible physicochemical/pharmacokinetics properties, and drug/lead likeness, were obtained.

Chemistry
The reactions' progress and the compounds' purity were checked using thin-layer chromatography (TLC), which were monitored using UV light at 365 and 254 nm. Also, all spectral data were recorded according to our previous study 40 .

Biological evaluations
Cytotoxicity screening against human cancer cell lines In this study, a panel of cell lines was examined for their chemosensitivity. Different concentrations of the synthesised pyridines and pyrimidine derivatives were used in this study (5, 12.     Determination of reduced glutathione (GSH) content. Supplementary data (SI 2(C)).

Determination of nitric oxide (NO) content. Supplementary data (SI 2(D)).
Cell cycle analysis and apoptosis assay Liver cancer cells (HEPG2) were seeded in RPMI-1640 media at a density of 250 X 10 3 cells/mL. Both cell cycle and apoptosis evaluations were performed at 3.8 mg/mL for both compounds 8 and 14. The detailed method is described in the Supplementary data (SI 3).

EGFR kinase (wild and T790M) inhibition assay
The most promising cytotoxic compounds (8 and 14) were finally assessed for their inhibitory potential against both EGFR Wild and EGFR T790M. The assay protocol is fully described in the Supplementary data (SI 4).

Docking studies
The MOE 2019.010255-57 was used to examine the binding affinities of the chemically synthesised derivatives on the EGFR through molecular docking. Accordingly, we could reveal the anticancer inhibitory potential of these compounds as promising EGFRIs. Also, the co-crystallised 5Q4 pyridinone inhibitor was inserted in the docking process as a reference standard.
Examined compounds preparation. The chemical structures of the examined chemically synthesised compounds were drawn by ChemDraw. Using MOE, the previous structures (7-21) were prepared for docking as described earlier [58][59][60] . The synthesised compounds under investigation and 5Q4 were saved into the same database for the docking step.
EGFR-Kinase receptor preparation. The Protein Data Bank was searched to give the crystal structure of the EGFR kinase domain (code: 5EM8) 61 . The preparation process was performed as discussed before [62][63][64] . The program default items were followed as before [65][66][67] .

5Q4
Hydrogen bonds are represented in red and H-pi ones are represented in black. reference 5Q4 ligand at the EGFR-Kinase domain was carried out. The applied methodology was performed as discussed in detail [68][69][70] . The MOE specifications were modified as previously mentioned [71][72][73] . The selected poses were based on their scores and RMSD accordingly [74][75][76] .
Furthermore, low RMSD values between the conformations of the redocked and crystal 5Q4 ligand indicated a valid performance in a validation process [77][78][79] .
Physicochemical, pharmacokinetic, and ADME studies The Swiss ADME supplied from the SIB 51 was used for the physicochemical, pharmacokinetic, and ADME studies of the target compounds 41 , 80 , 81 .

Statistical analysis
All the previously presented results are the mean ± SD of three separate experiments, which were performed in duplicates. The statistical significance of the results was analysed using one-way ANOVA followed by Tukey's multiple comparison test. A significantly different from control and p < 0.05.