Erlotinib

New pyrimidine and pyrazole-based compounds as potential EGFR inhibitors: Synthesis, anticancer, antimicrobial evaluation and computational studies

Ismail M.M. Othman a, Zahra M. Alamshany b, Nada Y. Tashkandi b, Mohamed A.M. Gad-Elkareem a, Manal M. Anwar c,*, Eman S. Nossier d
a Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt
b Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 21551, P.O. Box 42805, Saudi Arabia
c Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
d Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt

A B S T R A C T

This study was focused on the synthesis of new pyrimidines 4a,b, 5a,b and pyrazoles 6a, b as ATP mimicking tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR). The new compounds were assessed as cytotoXic candidates against human breast cancer cells (MCF-7) and hepatocellular carcinoma cells (HepG-2). All the new compounds appeared as more potent cytotoXic agents than erlotinib, while only compound 4a exhibited
more potency than 5-flourouracil and 4b analogue was equipotent to it. Accordingly, the kinase suppression effect of 4a and 4b was further evaluated against EGFRWT, EGFRL858R and EGFRT790M. Both pyrimidine ana- logues 4a and 4b displayed outstanding inhibitory activity against EGFRWT and its two mutated isoforms EGFRL858R and EGFRT790M in comparing to erlotinib and osimertinib as reference drugs. Additionally, all the new analogues were subjected to antimicrobial assay. Interestingly, both 4a and 4b represented the most promising activity of wide spectrum antimicrobial effect against the examined microbes in comparison to gentamycin and ketoconazole as standard drugs. Moreover, docking results proved the good binding interactions of the com- pounds 4a and 4b with EGFRWT and EGFRT790M which were in accordance with the results of the in vitro enzyme assay. Additional in silico ADMET studies were performed for the new derivatives which represented their good oral absorption, good drug-likeness properties and low toXicity risks in human.

Keywords: Pyrimidines Pyrazoles CytotoXic EGFR inhibition Antibacterial Antifungal Molecular docking ADMET prediction MCF-7 HepG-2

1. Introduction

Epidermal growth factor receptor (EGFR) is a receptor tyrosine ki- nase (RTK), belongs to ErbB family and performs a central role in signaling pathways of the cell such as; cell division, growth, differenti- ation, metabolism, adhesion, motility and death [1,2]. It is upregulated in multiple human malignancies, including non-small cell lung cancer (NSCLC), hepatocellular, colorectal, head and neck squamous cell car- cinoma and breast cancers [3–5]. Different studies in the chemothera- peutic field showed that suppression of EGFR-TK introduces a rational basis for the development of a class of targeted therapies, EGFR-TK in- hibitors (EGFR-TKIs). They act by competing with ATP for ATP-binding site on EGFR, thus blocking the signaling pathway leading to significant resistance to cancer cells, as well as angiogenesis, in addition to suppression of EGFR downstream PI3K/mTOR signaling pathway, leading to increase the lack of oXygen to improve the sensitivity of the cancer cells to radiation [6–8].
Despite the significant benefits of the first-generation EGFR in- hibitors (gefitinib [9] and erlotinib [10]) in cancer treatment, the mutation in the ATP binding pocket of EGFR exon 20 leading to a threonine-to-methionine substitution at the amino acid position 790 (T790M) led to predominant drug resistance in 50% of cancer patients. At the same time, L858R mutation at exon 20 was detected as another mechanism of resistance to the first-generation EGFR-TKIs [8,11–13]. The second generation, irreversible TKIs including afatinib and daco- mitinb containing a Michael acceptor functional group were developed to overcome these resistant tumor clones. However, due to low kinase selectivity between EGFRT790M mutation and EGFRWT (wild type EGFR), serious undesirable effects had occurred such as; skin rash and gastro- intestinal toXicity which limited the clinical use of these inhibitors [14–17]. Accordingly, the third-generation EGFR inhibitors with py- rimidine core structures such as WZ4002, rociletinib and osimertinib (AZD9291) were created [18–21] (Fig. 1). Osimertinib is the only US
FDA approved third-generation inhibitor, exhibiting a significant selectivity toward the drug-resistant mutant EGFRT790M over the wild- type EGFR (EGFRWT), rendering it an important medical regimen for patients with EGFRT790M mutation [22]. Despite the excellent characteristics of osimertinib, it exhibited several adverse effects in clinical application, including diarrhea, rash, decreased appetite and car- diotoXicity [23–25]. Accordingly, there is still a great demand to develop new EGFR inhibitors with high selectivity against the EGFRT790M and EGFRL858R mutation to overcome the undesirable drawbacks and drug resistance.
The ATP binding active site of EGFR-TK is composed of five major regions (i) an adenine binding pocket which bears the key amino acids that create the H-bonding with the adenine ring, (ii) a sugar region which constitutes the hydrophilic pocket, (iii) hydrophobic region I, which is not exploited by ATP but plays a crucial role in inhibitor selectivity and binding affinity, (iv) hydrophobic region II which is not used by ATP and may be exploited for inhibitor specificity, and (v) a phosphate binding pocket which is largely solvent exposed and can be utilized for improving the pharmacokinetics of the inhibitor [26,27] (Fig. 2).
Different structure-activity relationship studies (SAR) represented that there are four common pharmacophoric features are shared by EGFR-TKIs: (i) a flat heterocyclic-aromatic ring system which interact with the adenine binding site and participates in H-bonding interactions with the corresponding amino acids, (ii) a terminal hydrophobic head for insertion in the hydrophobic pocket I, (iii) NH-linker that creates H- bonding with amino acid residues in the linker area, (iv) a hydrophobic tail which inserts into the hydrophobic area II [28–31] (Fig. 3).
Various literatures investigated that pyrimidine scaffold is an important building block in many antineoplastic agents via multi- suppression of different protein kinases including EGFR-TKs [4,32–34]. In addition, pyrazole and fused pyrazole systems were investigated as promising motifs in many antiproliferative agents against a broad range of cancers targeting the kinase inhibition activity of EGFR [35–37]. Various molecular simulation studies indicated that many pyrazole analogues fitted perfectly in the active pocket of EGFR kinase resulting in potent anticancer activity [38]. Accordingly, the strategy of this work was focused on design and synthesis of new de- rivatives bearing pyrimidine-2-one(thione) or pyrazole motif having the essential pharmacophoric features of EGFR-TKIs. The first position was pyrimidine or pyrazole moieties (hetero-aromatic system) to occupy the adenine binding pocket. The second position was the terminal 2-methoX- yphenyl ring (hydrophobic head). NH linker was utilized in the third position as a hydrogen bond donor. The fourth position was either phenyl or 4-tolyl rings incorporated at pyrimidine-C6 or pyrazole-C5 to occupy the hydrophobic region II of the ATP binding pocket (Fig. 3). its biological isostere pyrazole
All the newly synthesized compounds were evaluated as anticancer agents against human breast cancer cells (MCF-7) and human liver carcinoma cell line (HepG2). The compounds exhibited the most promising cytotoXic activity were evaluated for their ability to suppress the wild-type EGFRWT and the mutated isoforms EGFRL858R and EGFRT790M. Furthermore, molecular docking study was performed for the promising derivatives to rationalize and emphasize their modes of action as EGFR-TKIs. In addition, in silico ADMET prediction was carried out for the new derivatives to explore their drug-likeness properties.
It is known that different types of cancers are essentially caused by various factors including certain types of infections [39]. Study of leading to the formation of the key intermediate 2-((2-methoXyphenyl) amino) acetonitrile (2). The latter compound 2 was treated with different aromatic aldehydes namely; benzaldehyde and/or 4-methyl- benzaldehyde in glacial acetic acid to produce the corresponding acry- lonitrile derivatives 3a, b, which were subsequently reacted with urea and/or thiourea in refluXing ethanol containing a catalytic amount of HCl to afford the pyrimidin-2-ones 4a, b and pyrimidine-2-thiones 5a, b respectively. On the other hand, the treatment of 3a, b with hydrazine hydrate in refluXing ethanol for 8 h accomplished the target pyrazole derivatives 6a, b. The molecular structures of the new compounds were confirmed depending on their elemental analyses, and spectral data. IR spectra of the target pyrimidine derivatives 4a, b and 5a, b showed absorption stretching bands at the regions 3489–3356 cm—1 represent- ing NH2, NH groups, at 1676, 1684 cm—1 due to C–O groups of com- pounds 4a, b and at 1176, 1159 cm—1 representing C–S groups of compounds 5a, b. On the other hand, 1H NMR chemical shifts represented the protons of the new pyrimidines 4a, b, 5a, b at the expected regions. Singlet signals were presented at δ 3.76–3.85 ppm due to the three protons of –OCH3 functionalities, δ 2.02, 2.15 ppm due to CH3 protons of compounds 4b, 5b, δ 8.36–10.42 representing NH, NH2 protons as well as the signals of the aromatic protons appeared at theirvarious antibiotic cytotoXic pharmaceutics including Actinomycin, expected regions δ 7.04–8.10 ppm. Also, 13C NMR spectra of the com- Adriamycin/DoXorubicin and some other candidates revealed that in addition to DNA intercalating or DNA damaging effects, the stimulation of the existing host defense mechanism is considered as one of the ap- proaches that these chemotherapeutics exert their antimicrobial effects [40]. Based on these evidences, in comparison to gentamycin and ke- toconazole as standard drugs, all the new pyrimidine and pyrazole de- rivatives were also evaluated as antimicrobial agents against various gram-positive, gram-negative bacterial and fungal strains.

2. Results and discussion

2.1. Chemistry

The new pyrimidine and pyrazole derivatives were efficiently syn- thesized as outlined in Scheme 1 starting with compound 2-methoXyani- line (1) which was allowed to react with chloroacetonitrile in ethano pounds 4a, b, 5a, b exhibited singlet signals at the range δ 55.94–58.12 ppm contributing to OCH3 carbons, δ 17.84, 20.95 ppm due to CH3 carbons of compounds 4b, 5b, as well as at δ 160.27–179.21 ppm for C–O and C–S carbons. Different signals appeared at δ 106.18–153.77 ppm due to the aromatic carbons. Additionally, 1H NMR spectra of the target pyrazole derivatives 6a, b represented the methoXy group protons at δ 3.82, 3.78 ppm, the D2O exchangeable protons of NH2 and NH at δ 5.76, 6.38, 9.71–12.16 ppm as well as the aromatic protons at δ 7.04–8.08 ppm. Also, 13C NMR spectra of the compounds 6a, b dis- played signals at δ 59.3, 58.15 ppm for OCH3 carbons, δ 21.88 ppm referring to CH3 carbon of 6b and at δ 107.56–147.91 ppm contributing to the aromatic carbons.

2.2. Biological activity

2.2.1. In vitro cytotoxic activity

The in vitro growth inhibitory potency of the newly prepared com- pounds was scrutinized against breast (MCF-7) and hepatocellular (HepG-2) cancer cell lines as well as lung fibroblast (WI38) normal cells in comparing with 5-flourouracil and erlotinib as two standard drugs using the colorimetric MTT assay [41,42]. The concentrations of the compounds that induced 50% inhibition of cell viability (IC50, μM) were detected and tabulated in Table 1.
Interestingly, all of the tested derivatives displayed significant cytotoXic activity against both types MCF-7 and HepG2 cancer cell lines of IC50 range 0.01–0.35 µM that was much more potent than that ob- tained by the reference drug erlotinib of IC50; 0.42, 0.82 µM, respec- tively. On the other hand, while comparing the resultant activities of the compounds with 5-flourouracil, compound 6-phenylpyrimidin-2-one 4a represented 2 and 3 folds more potent activity than that of the reference drug against MCF-7 and HepG-2 cancer cell lines, respectively (IC50; 0.01 0.03 µM, IC50, 5-FU; 0.02 0.01, 0.03 0.01 µM, respectively). A mild decrease in the activity was determined by the analogue 6-(p-tolyl) pyrimidin-2-one 4b to be approXimately equipotent to 5-FU of IC50; 0.02 ± 0.01 µM. Further remarkable reduction in the cytotoXic activity was detected by the pyrimidin-2-thione analogues 5a, b by 2–4 folds against both cancer cell types of IC50 values ranging from 0.04 to 0.08 µM. This result could be explained due to the tendency of the pyrimidine-2-oXygen atom to form additional H-bonds with different amino acid residues of the target protein stronger than the sulfur atom [43], thus representing more potent inhibitory effect. Also, the cytotoXic potency was weakened by 4.5–8 folds upon replacement of the pyrim- idine moiety with the five-membered pyrazole core as compounds 6a b exhibiting IC50 values ranging from 0.09 0.03–0.16 0.03 µM. Although the key intermediates 2 and 3a, b represented more potent cytotoXic activity against the tested cell lines comparing to erlotinib as a reference drug, they showed 12–17 folds less cytotoXic activity while comparing to 5-flourouracil of IC50 values ranging from 0.24 ± 0.02 to 0.35 ± 0.03 µM. Thus, cyclization of 2 and 3a, b to establish their py- rimidine and the pyrazole analogues 4a, b–6a, b is essential to enhance the cytotoXic potency.
Referring to the results of cytotoXicity against WI38 normal cells (Table 1), the nine tested derivatives showed lower cytotoXicity against WI38 (IC50; 39.26–68.01 μM) than 5-FU (IC50; 10 0.056 μM). They exhibited promising safety profile to be safer antitumor agents than the reference drug 5-FU.

2.2.2. Kinase inhibitory assessment

In the present work, the analogues that represented the most potent cytotoXic efficacy were subjected to Kinase inhibitory assessment to investigate their potential mechanism of action. Based on the cytotoXic data, compounds 4a and 4b were selected to study their EGFRWT, EGFRL858R and EGFRT790M inhibitory activity [44], utilizing erlotinib and osimertinib as standard drugs and the results were expressed as IC50 (µM) (Table 2).
Both compounds 4a and 4b demonstrated significant EGFRWT inhibitory activity (IC50; 0.087 ± 0.013 and 0.110 ± 0.014 µM) that was approXimately equal to that obtained by erlotinib (IC50 = 0.096 ± 0.021 µM) and about 5.8 and 4.6 times, respectively higher than that obtained by the reference drug osimertinib (IC50 0.510 0.011 μM). Further- more, both compounds 4a and 4b employed potent inhibitory activity against EGFRL858R that was close to that obtained by erlotinib (IC50 = 0.044 0.15, 0.058 0.12 µM, respectively, IC50, Erlotinib 0.041 0.020), but slightly less than the potency obtained by osimertinib by 2–2.5 folds. Also, 21 and 14 times more significant activity against EGFRT790M was gained by 4a and 4b, respectively in comparing to erlotinib (IC50 = 0.026 ± 0.15, 0.038 ± 0.12 µM, respectively, IC50, Erlotinib = 0.550 ± 0.10) and approXimate equal activity while comparing to osimertinib (IC50, Erlotinib 0.028 0.05). Fortunately, both com- pounds 4a and 4b exhibited more potent inhibitory activity against the mutant forms of EGFR over the wild-type form. Taking together, compounds 4a and 4b could be considered more advantageous than other EGFR-TKIs since they exhibited promising multi-kinase inhibiting ac- tivity (with the highest selectivity toward the mutants EGFRT790M and EGFRL858R) that might overcome EGFR-TKIs drug resistance by interfering with intracellular signaling pathways or by suppression the mutated binding sites or genes. Thus, both compounds 4a and 4b could be used as promising basic templates for generation of new drugs used for the treatment of different cancer types harboring EGFRT790M and EGFRL858R mutations.

2.2.3. Antimicrobial activity

It has been employed that the pyrimidine and pyrazole motifs are versatile lead molecules in the field of antimicrobial discovery and development [45–47], so it was of interest to assess the antimicrobial activity of the newly prepared compounds alongside their cytotoXic evaluation aiming to gain new pyrimidine and pyrazole candidates of dual potent anticancer and antimicrobial activities. The antimicrobial potency of the tested compounds was in vitro screened versus two pathogenic gram-positive bacteria viz. Streptococcus pneumoniae RCMB 010010 and Staphylococcus epidermidis RCMB010024, two pathogenic gram-negative bacteria viz. Proteus vulgaris RCMB 010085, E. Coli RCMB 010052 and two fungal strains viz. Aspergillus fumigatus RCMB 02568 and Syncephalastrum racemosum RCMB 05922. Gentamycin and ketocona- zole were utilized as reference drugs for antibacterial and antifungal activity, respectively [48].
The antimicrobial activity of all synthesized compounds was carried out using agar well diffusion method [49] and the obtained results were recorded for each tested compound as the average diameter of the inhibition zones in mm for the bacterial or fungal growth around the discs

On the basis of the MIC values in Table 4, it was exhibited that there is a wide variability in the antimicrobial potency of the tested de- rivatives. It has been detected that the pyrimidine-2-one derivatives 4a and 4b were 8–6 folds more potent antibacterial candidates against Streptococcus pneumoniae (MIC; 0.03, 0.06 μg/mL) than gentamicin (MIC; 0.24 μg/mL). Also, they were equipotent to the reference drug gentamicin against Staphylococcus epidermidis exhibiting MIC values; 0.06 μg/mL. Furthermore, both 4a and 4b represented approximate equal potency to that obtained by gentamicin against both tested gm- negative bacteria Proteus vulgaris and E. Coli (MIC values; 0.98, 0.06, 0.03 μg/mL) (MIC Gentamycin; 1.95, 0.03 μg/mL). Compound 4b was 4 folds less potent than gentamicin against E. Coli of MIC value; 7.81 μg/ mL. On the other hand, the antifungal activity of the 4a and 4b was slightly less than that of the reference drug Ketoconazole representing MIC values ranging from 0.12 to 0.98 μg/mL, MIC Ketoconazole; 0.12, 0.49 μg/mL.
Conversely, the pyrimidine-2-thione derivatives 5a and 5b appeared to be less potent as antibacterial agents against the tested gm-positive of MIC values range 0.49–1.95 μg/mL except compound 5a which repre- sented more potent activity against Streptococcus pneumoniae RCMB 010010 of MIC value 0.12 μg/mL, while comparing to gentamicin. Un- fortunately, both 5a and 5b exhibited moderate activity against the tested gm-negative bacteria of MIC range; 0.12–62.5 μg/mL and mod- erate to weak potency against the tested fungal strains of MIC range; 31.25–250 μg/mL, comparing to Ketoconazole.
Depending on the resultant data the pyrimidine-2-one is a promising scaffold in producing antibacterial activity more than pyrimidine-2- thione nucleus.

2.3. Computational studies

2.3.1. Molecular docking

To furnish a deep insight into the binding modes of the most potent new derivatives 4a and 4b within the active sites of EGFRWT and EGFRT790M enzymes, molecular docking simulation was done using Molecular Operating Environment software (MOE®) 2008.10 [52].
MOE program. Validation of the docking procedures were firstly done via re-docking of the original co-crystallized ligands, erlotinib and AZD9291 within the active sites of EGFRWT and EGFRT790M enzymes (PDB code: 1 M17 and 6JX0) and revealed energy scores 12.25 and 11.75 kcal/mol at root mean square deviation (RMDS) values equal 0.75 and 0.88 Å, respectively [53,54].
The docking results of 4a and 4b within the ATP-binding pocket of EGFRWT illustrated that they interact with EGFRWT through similar hydrogen bonding interactions with energy scores of 13.22 and 12.90 kcal/mol, respectively. The 4-aminopyrimidine-2-one ring was fitted through H-bond acceptors between the carbonyl oXygen and the backbone of the key amino acid Met769 (distance in 4a and 4b: 2.88 and 2.58 Å, respectively). The protons of the amino groups formed H-bond donors with the sidechain of Gln767 in case of 4a and 4b (distance: 1.66 and 2.21 Å, respectively) and additional H-bond donor with the side- chain of Gln766 in case of 4a (distance: 2.38 Å). Moreover, the side chain of Thr766 afforded two H-bonds with the proton of NH and the oXygen of methoXy group (Figs. 4 and 5).
On the other hand, the promising targets 4a and 4b bound nicely within the ATP-active site of EGFRT790M in a similar manner with energy scores of —12.65 and —11.88 kcal/mol, respectively. The 4- aminopyrimidine-2one scaffold was fiXed tightly via two hydrogen bond donors between N-3 and the carbonyl oXygen with the backbone of Met793. Furthermore, the proton of the amino group exhibited H-bond donors with the backbone of Gln791 in case of 4a and 4b (distance: 1.87 and 1.65 Å, respectively) (Figs. 6 and 7).
Finally, and referring to the superimposition models in Fig. 8, the two compounds 4a and 4b bearing 4-aminopyrimidine-2-one scaffold were well embedded in the active pockets of EGFRWT and EGFRT790M through various H-bond interactions. Furthermore, the illustrated binding pattern explained the excellent EGFRWT and EGFRT790M inhibitory activity of these derivatives comparing with the co-crystalized inhibitors erlotinib and AZD9291, respectively.

2.3.2. In silico ADMET prediction and drug likeness study

Based on the Lipinski’s rule (Rule of five) using molinspiration software [55,56] (Table 5), all tested compounds satisfy the “Rule of five” and meet all criteria for good permeability and bioavailability displaying rotatable bonds number in the range of 3–4, with the lowest value allowed to compound 2, therefore, obviously exhibiting small conformational flexibility. The percentage of absorption (%ABS) which is deduced from the TPSA values according to the following equation: % ABS 109–0.345 TPSA was also determined. All candidate com- pounds exhibited a great %ABS ranging from 76.90 to 93.45% sug- gesting their efficient oral absorption. Topological polar surface area (TPSA) results are < 140 Å2 for all the analyzed compounds suggesting that they are candidates for good solubility, capacity for penetrating cell membranes and intestinal absorption and are basically recognized as good indicators of drug transport in the intestines, Caco-2 monolayers penetration, and blood-brain barrier crossing [56–58]. Their nOHNH value (H-bond donors) are less than 5 giving them higher solubility in cellular membranes with also nON values (H-bond acceptors) in the range of 3–6 and molecular weight in the range of 162.19–338.44. Molecular volumes of the target compounds in this series increased as increasing MW and ranged from 155.65 to 301.35 Å3. The milog P (octanol/water partition coefficient) values for all compounds under inspection are less than 5, especially for compounds 2, 4a and 4b out- lining them a good lipophilicity character. The great lipophilicity of these compounds may protect against ROS (Reactive OXygen species) damage and is due in part to their smaller polar surface areas. 2.3.2.1. In silico toxicity potential. ToXicity risks and physicochemical properties of the newly synthesized compounds were estimated via the methodology developed by Osiris [59]. All compounds were predicted for their toXicity including mutagenicity, tumorgenicity, skin irritancy and reproductive effect. From the results displayed in Table 6, all compounds would be safe and were predicted to have no side effects. The best results were obtained by compounds 4a, 4b, 5a, 5b, 6a and 6b indicating that they contain predominantly active fragments which are frequently presented in commercial drugs [60]. Drug score is a measure of compound’s potential to have drug-conform behavior. The highest drug-score value was ascribed to compound 4a justifying its both effectiveness and potentiality as a new drug candidate. 3. Conclusions In summary, new pyrimidine-2-one(thione) and pyrazole derivatives bearing the essential pharmacophoric features of EGFR-TKIs were designed, synthesized and evaluated as cytotoXic candidates against human breast (MCF-7) and hepatocellular (HepG2) cancer cell lines, where 5-flourouracil and erlotinib were serving as standard drugs. All the new compounds revealed more potent cytotoXic activity than erlo- tinib. On the other hand, only the pyrimidine-2-one compound 4a exhibited more potent anticancer activity than 5-flourouracil, while 4b analogue was equipotent to it. The inhibitory profiles against the three isoforms EGFRWT, EGFRL858R and EGFRT790M were evaluated for the most promising compounds 4a, 4b, utilizing erlotinib and osimertinib as reference drugs. Both analogues 4a, 4b exhibited more potent sup- pression effect against the mutant forms EGFRL858R and EGFRT790M over the wild-type form EGFRWT and hence they might overcome resistance to EGFR-TKIs. Molecular docking study rationalized the potent sup- pression effect of the pyrimidine 2-ones 4a and 4b due to their good fitting with the best binding energy in the active site of EGFRWT and its mutated isoforms EGFRL858R and EGFRT790M. As a trial to gain new pyrimidine and pyrazole – based derivatives of dual anticancer and antimicrobial activity, all the new analogues were assessed as antibac- terial and antifungal agents against a number of pathogenic gram- positive, gram-negative bacterial and fungal strains using gentamycin and ketoconazole as standard drugs. The results proved that 4a and 4b among the tested derivatives were the most promising agents repre- senting a wide spectrum antimicrobial activity against the examined microbes. Furthermore, Lipinski rule of five and ADME profile suggested strongly that all the synthesized pyrazole and pyrimidine compounds were fulfilling the criteria of drug likeness approach. Accordingly, the pyrimidine scaffold is considered as a promising template in the field of development and optimization of new drugs of dual anticancer and antimicrobial activities. 4. Experimental 4.1. Chemistry The instruments used for measuring the melting points, spectral data (IR, Mass, 1H NMR and 13C NMR) and elemental analysis are provided in details in Supplementary material. 4.1.1. 2-((2-Methoxyphenyl) amino) acetonitrile (2) A miXture of 2-methoXyaniline (1) (1.2 g, 10 mmol) and chlor- oacetonitrile (0.75 g, 10 mmol) in absolute ethanol (20 mL) was refluXed for 5 h. The solid product formed after cooling was collected by filtration and recrystallized from ethanol to afford the title compound 2. 4.1.2. General procedure for the synthesis of 2-((2-methoxyphenyl) amino)-3-(substituted) acrylonitrile 3a, b A miXture of the acetonitrile derivative 2 (1.6 g, 10 mmol), appro- priate aromatic aldehydes namely; benzaldehyde and/or 4-methylben- zaldehyde (10 mmol) and anhydrous sodium acetate (10 mmol) in glacial acetic acid (15 mL) was refluXed for 12 h then allowed to cool. The solid product was collected, dried and recrystallized from dioXane. 4.1.3. General procedure for the preparation of 4-amino-5-((2-methoxy- phenyl) amino)-6-(substituted phenyl) pyrimidin-2(1H)-one (thione) derivatives 4a, b and 5a, b A miXture of the compounds 3a, b (10 mmol), urea and/ or thiourea (10 mmol) in absolute ethanol (20 mL) containing (1 mL) hydrochloric acid was heated under refluX for 7–9 h. The solid product produced on hot was collected by filtration and recrystallized from ethanol to give the target products 4a, b and 5a, b, respectively. 4.2. In vitro anticancer screening The screening was performed using MTT assay [41,42] at Regional Center for Mycology and Biotechnology, Al- Azhar University. More details were provided in Supplementary material. 4.3. EGFRWT, EGFRL858R and EGFRT790M kinase inhibitory assay Homogeneous time resolved fluorescence (HTRF) assay was applied [44]. More details were provided in Supplementary material. 4.4. Antimicrobial activity assay In vitro microbial activities were carryout at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Agar plate diffusion technique was used [49]. More details were provided in Supplementary material. 4.5. Minimal inhibitory Concentration (MIC) measurement The bacteriostatic activity of the compounds was then evaluated using the two-fold serial dilution technique [50,51]. More details were provided in Supplementary material. 4.6. 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