Design, synthesis, and anti-cancer evaluation of new pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as potential EGFRWT and EGFRT790M inhibitors and apoptosis inducers

Abstract A new series of pyrido[2,3-d]pyrimidin-4(3H)-one derivatives having the essential pharmacophoric features of EGFR inhibitors has been designed and synthesised. Cell viability screening was performed for these compounds against A-549, PC-3, HCT-116, and MCF-7 cell lines at a dose of 100 μM. The highest active derivatives (8a, 8 b, 8d, 9a, and 12b) were selected for IC50 screening. Compounds 8a, 8 b, and 9a showed the highest cytotoxic activities and were further investigated for wild EGFRWT and mutant EGFRT790M inhibitory activities. Compound 8a showed the highest inhibitory activities against EGFRWT and EGFRT790M with IC50 values of 0.099 and 0.123 µM, respectively. In addition, it arrested the cell cycle at pre-G1 phase and induced a significant apoptotic effect in PC-3 cells. Furthermore, compound 8a induced a 5.3-fold increase in the level of caspase-3 in PC-3 cells. Finally, docking studies were carried out to examine the binding mode of the synthesised compounds against both EGFRWT and EGFRT790M.


Introduction
According to WHO, cancer was the direct cause of 10 million deaths in 2020 and the cost of cancer treatment globally was US$1.16 trillion in 2010 1 . Several internal and external factors can cause cancer. The most well-known factors are hormonal disorders, genetic mutations, radiations, smoking tobacco, metals, polluted food, chemicals, and infectious organisms [2][3][4] . Resistance against anticancer drugs is considered one of the most serious problems in cancer management 5 . Due to the high residence of many cancer types, the discovery of new anticancer agents with high effect, less resistance, and fewer side effects is an urgent need.
Protein kinases (PKs) are a group of enzymes that are responsible for the transference of phosphate from ATP molecule to tyrosine, serine and/or threonine amino acids in protein substrates 6,7 . Furthermore, PKs promote cellular signalling processes such as cell growth regulation, differentiation, migration, and metabolism 8 . PKs have been found to be overexpressed in a variety of human malignancies 9 . Accordingly, the inhibition of PKs has emerged as a selective method for killing cancer cells 10 . Receptor tyrosine kinases (RTKs) are vital category protein kinases. About 20 different RTKs have been discovered that have similar structures 11 .
The epidermal growth factor receptor (EGFR) belongs to the RTKs family that stimulates differentiation and proliferation of cells after the binding of its specific active ligand 12 . EGFR structure has an extracellular part (at the surface of the cells) and an intracellular part. The activation of the outer part leads to an activation of the intracellular region of the receptor and a phosphorylation of the intracellular substrates 13 . This step facilitates cell growth, synthesis of DNA, and the expression of oncogenes 14 . It was reported that EGFR is over-expressed and implicated in the pathogenesis and progression of various human carcinomas 15 . In many patients, resistance against cancer therapy arises from an acquired mutation in the EGFR kinase domain (T790M). Such mutant EGFR is called EGFR T790M16 . Thus, EGFRs (wild and mutant types) are interesting biological targets for the discovery of new anticancer agents 17,18 .
The ATP binding site of EGFR consists of five regions; an adenine-binding pocket, a sugar region (ribose binding pocket), a hydrophobic region I, a hydrophobic region II, and a phosphatebinding region [19][20][21] . Most of the reported EGFR inhibitors are ATPcompetitive inhibitor small molecules that have specific moieties to occupy the adenine-binding pocket, the hydrophobic region I, and the hydrophobic region II 10 (Figure 1).
EGFR inhibitors have a specific Y-shaped structure 23 . In addition, the structure of EGFR inhibitors should comprise many essential pharmacophoric features 24 . Each feature binds at a specific region in the ATP binding site. For example, a flat hetero aromatic system is an essential feature of EGFR inhibitor to occupy the adenine binding pocket of the ATP binding site. Such hetero structure can form hydrogen bonds with some amino acids as Met769, Thr790, and Thr854 25 . Also, a terminal hydrophobic head of the EGFR inhibitor can occupy the hydrophobic region I forming many hydrophobic interactions 24 . Finally, a hydrophobic tail be buried in the hydrophobic region II producing high affinity 19,26 .
Till now, three generations of EGFR inhibitors were approved by the FDA (Figure 2). Erlotinib I 27 and gefitinib II 28 are examples of the first generation. The generated mutation in EGFR led to the acquired drug resistance and reduced efficacy in cancer treatment 29 . The mutant form of protein (EGFR T790M ) resists the affinity of ATP-competitive inhibitors 30 . The second-generation of EGFR inhibitors was approved to overcome the drug resistance that was induced by EGFR T790M . These inhibitors can form covalent interactions with Cys797 at the ATP binding site [31][32][33] . Pelitinib III 34 is a well-known example of this class. Unfortunately, low maximal-tolerated-dose, the major drawback of this class, led to poor clinical outcomes 35,36 . Osimertinib 5 37 , an example of the third-generation EGFR inhibitors, exhibited greater activities against mutant form (EGFR T790M ) than the wild form (EGFR WT ). Recently, toxic epidermal necrolysis was reported upon the administration of olmutinib 38 . Hence, many efforts are still required to reach more potent and less toxic EGFR inhibitors.

Rationale of molecular design
For years, our team synthesised several EGFR inhibitors which showed promising anticancer activities. In 2018, a series of 1H-pyrazolo [3,4-d]pyrimidine derivatives were synthesised and evaluated for their inhibitory activities against EGFR WT and EGFR T790M . compound V potently inhibited the two EGFR types with a good apoptotic effect and arrested the cell cycle at the G 2 /M phase. Such compounds comprise two hetero-aromatic rings (1H-pyrazolo [3,4-d]pyrimidine) to occupy the adenine binding pocket 22 .
In 2019, we designed and synthesised a series of thieno [2,3d]pyrimidine derivatives as EGFR and HER2 tyrosine kinase inhibitors. Compound VI was the most active member producing significant apoptosis. This compound contains two hetero-aromatic rings (thieno [3,2-d]pyrimidine) to occupy the adenine binding pocket 54 .
In 2020, our team designed and synthesised a new series of pyrimidine-5-carbonitrile derivatives as EGFR inhibitors. Compound VII showed high inhibitory activities against EGFR WT and EGFR T790M . In addition, it arrested the cell cycle at the G2/M phase and induced a significant apoptotic effect in HCT-116, HepG-2, MCF-7cells. This compound contains one hetero-aromatic ring (pyrimidine) to occupy the adenine binding pocket 55 .
In the current work, we used the previously reported active candidates (V, VI, and VII) 22,54,55 as lead compounds in the design of the new derivatives. The rationale of our molecular design depended on the modification of such compounds to get new EGFR inhibitors. The modification was carried out at three features following the essential features of EGFR inhibitors. Concerning the terminal hydrophobic head and the hydrophobic tail, different substituted benzene rings were used to study the SAR of the synthesised compounds. Regarding the flat hetero-aromatic system, we used three different systems. The first one is pyrido [2,3-d]pyrimidin-4(3H)-one moiety which comprises two hetero-aromatic rings (compounds 9a-e). The second one is pyrido [2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one moiety which composes three heteroaromatic rings (compounds 10a-d, 11a-e, and 12a-d). The third one is 5H-pyrido[2 0 ,3 0 :4,5]pyrimido[2,1-b]quinazoline-5,7(12H)dione moiety which constitutes four hetero-aromatic rings (compounds 8a-d; Figure 3).

Chemistry
In continuation of the previous work 59 , the starting precursor 2thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one derivatives 7a-e were afforded via the reaction of the appropriate chalcones 6a-e with 6-aminothiouracil 3. The target compounds were synthesised in acceptable yield as reported 59 . Here in, the structure of the new 2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one derivative 7a was proved by elemental and spectral analyses. 1 H NMR spectrum showed two D 2 O exchangeable singlet signals at d 12.51, 13.23 ppm correspond to the two protons of each NH groups. Also, a singlet signal was recorded at d 7.97 ppm, corresponding to the proton at C6 of pyridopyrimidine ring. The 13 C NMR spectrum of 7a analogue displayed two characteristic signals at d 162. 29,175.61 corresponding to carbons of C¼O and C¼S groups, respectively.
The 5H-pyrido[2 0 ,3 0 :4,5]pyrimido[2,1-b]quinazoline-5,7(12H)dione analogues 8a-d were synthesised through the reaction of compounds 7b-e with anthranilic acid in the presence of catalytic amount of sodium ethoxide under reflux condition 60 . Their chemical structures were confirmed by elemental and spectral data for example the 1 H NMR of compound 8d revealed an increase in the integration of aromatic region at d 6.76-8.15 ppm, and the presence of D 2 O exchangeable singlet signal assigned for one proton of NH group at d 11.64 ppm. The 13 C NMR spectrum showed the characteristic two signals for the two carbons of C¼O signals at d 161. 45 and 169.46 ppm. The mass spectrum for 8d revealed the expected molecular ion peak at m/z of 520. Finally, IR spectrum of 8 b displayed absorption bands at 1693, 1750 and 3410 cm À1 corresponding to two C¼O and one NH groups, respectively.
The 2-hydrazinopyrido[2,3-d]pyrimidin-4(3H)-one derivatives 9a-e were depicted through the nucleophilic attack of hydrazine hydrate upon the key derivatives 7a-e following the reported method 60 . The newly hydrazinyl derivative 9a was proved by spectral data. The 1 H NMR spectrum showed two singlet signals at d 8.23, 9.12 ppm assigned for three protons of hydrazinyl group NHNH 2 .
Cyclo-condensation of the 2-hydrazinyl derivative 9a-e with ethyl chloroformate in dry pyridine produced pyrido a]pyrimidine-3,5-dione derivatives 10a-d. The IR spectrum of compound 10d revealed the presence of three absorption bands at 1708, 3437, and 3425 cm À1 assigned for two carbonyl and two NH groups, respectively. The 1 H NMR spectrum for the same compound showed two D 2 O exchangeable signals at d 9.26, 11.07 ppm assigned for two NH groups. Mass spectrum of compound 10c showed molecular ion peak at m/z of 479 and its isotope at m/z of 481.
The 3-phenylpyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)one analogues 12a-e were obtained via the reaction of hydrazinyl derivatives 9a-e with benzoyl chloride in pyridine under reflux conditions. Analytical and spectroscopic measurements confirmed the structures of compounds 12a-d. The IR spectrum of 12b displayed two absorption bands at 1720, 3414 cm À1 corresponds to C¼O and NH groups, respectively. The 1 H NMR spectrum of the same series gave an increase in aromatic integration due to the presence of an extra phenyl ring. The mass spectrum of 12b revealed a molecular ion peak at m/z of 459 (Schemes 1 and 2).
All compounds were barely active against breast cancer (MCF-7) cell line at 100 lM (% of inhibition ranging from 5 to 68% (Table 1). By focussing on the prostatic cell line (PC-3), the anticancer profile of the tested compounds was significantly improved especially the tetracyclic derivatives 8a (IC 50 ¼ 7.98 mM) and 8d (IC 50 ¼ 7.12 mM) that exhibited about 1.5 times more active than erlotinib (11.05 mM). In addition, compound 9a showed a strong activity against PC-3 line with an IC 50 value of 9.26 mM. For compound 8d, it showed a strong anti-proliferative activity against A-549 with an IC 50 value of 7.23 mM which is comparable to erlotinib (IC 50 ¼ 6.53 mM). Compound 8b revealed a moderate inhibitory activity against PC-3 cell line with an IC 50 value of 18.01 mM. Generally, no cytotoxic activity was observed against the colon cancer cell line (HCT-116), but compounds 8a, 8b, 8d, 12b revealed mild cytotoxic activity.

Structural-activity relationship
The synthetic pathway of the target compounds was depicted in two schemes starting with thioxo-precursors 7a-e to afford tetracyclic derivatives 8a-d, hydrazinyl derivatives 9a-e, and triazolyl derivatives 10a-d, 11a-e, and 12a-d ( Figure 4).

EGFR WT kinase inhibitory assay
The promising antiproliferative compounds (8a, 8d, and 9a) were further examined for their EGFR WT kinase inhibitory activities using Homogeneous time resolved fluorescence (HTRF) assay 64 . Erlotinib was used as a reference molecule (Table 3). The tested derivatives 8a, 8d, and 9a showed promising inhibitory activities against EGFR WT with IC 50 values of 0.099, 0.419, and 0.594 mM, respectively. compounds 8a showed a good activity compared to erlotinib (IC 50 ¼ 0.043 mM). Whereas compounds 8d and 9a showed moderate act nlp0m kinase inhibitory assay To evaluate the potential activity of the synthesised compounds against the mutant form of EGFR, the most active cytotoxic compounds (8a, 8d and 9a) were tested for their inhibitory effect against EGFR T790M . Erlotinib was used as a positive control.

Cell cycle analysis
Based on the above-mentioned biological testing, the most promising candidate 8a was subjected to flow cytometry analysis to investigate its effect on the cell cycle distribution in the most sensitive cell line (PC-3). The reported protocol described by Wand et al. 65 was applied in this test. PC-3 cells were incubated with compound 8a for 24 h in a concentration equal to its IC 50 against such cell line (7.98 mM). After that, the different phases of the cell cycle were analysed.
Compound 8a showed different effects on the cell cycle distribution. Compared to the control cells (Cont. (PC-3)), the cell population increased at the phases of pre-G 1 and %S by 22 and 1.3 folds, respectively. For the Pre-G1phase, the cell increased from 1.78% (in cont. cells) to 41.06% (at the treated cells). In the S phase, the cell increased from 41.03% (in cont. cells) to 53.69% (at the treated cells). On the other hand, the cell population decreased in both the G0-G1 and the G2-M phases. Such results obviously reveal that compound 8a can arrest the PC-3 cell line at pre-G 1 of the cell cycle ( Figure 5 and Supplementary data).

Annexin V-FITC apoptosis assay
To analyse the apoptotic effect of the most active compound 8a, Annexin V and PI double staining assay with FITC was applied 66 . In this test, PC-3 cells were incubated with compound 8a at a concentration of 7.98 mM for 24 h. The results were depicted in ( Figure 6 and Supplementary data).
Investigating the results of Annexin V and PI double staining assay, revealed that compound 8a produced a significant increase in the early apoptosis ratio from 0.43 to 13.92% (32-fold). Also, it exerted an increase in the late apoptosis ratio from 0.15 to Scheme 2. General procedure for the synthesis of the target compound 10a-d, 11a-e and 12a-d. 22.49% (150-fold). Such findings indicate that compound 8a has a significant apoptotic effect against PC-3 cells.

Caspase-3 determination
The ability of a drug to induce apoptosis determines the sensitivity of the cancer cells against it. There are many signalling pathways that control apoptosis induction. Caspases family are considered as one of the most apoptotic regulators 67 . Activation of caspases especially (caspase-3) produces cell death 68 . In addition, it was reported that EGFR inhibitors exhibit significant   apoptotic effects through the caspase pathway 69,70 . Here, the effect of the most active EGFR inhibitor 8a on caspase-3 was examined in PC-3 cells. Compound 8a was applied on PC-3 cells at a concentration of 3.04 mM for 24 h. The results revealed that such a compound generated a marked increase in the level of caspase-3 (452.3 pg/mL, 5.3-fold) compared to the control cells (84.24 pg/mL). In addition, the tested compound showed a comparable effect with the reference compound; staurosporine (413.1 pg/mL; Table 4 and Supplementary data).

Docking studies
To confirm our rationale of design, the binding modes of the synthesised compounds were investigated against the proposed targets using a docking approach. The used biological targets in docking studies were EGFR-TK Wild-type (EGFR WT , PDB: 4HJO) 71 and EGFR-TK mutant type (EGFR T790M , PDB: 3W2O) 72 using MOE 14.0 software. The co-crystallised ligands were used as reference molecules. The output of docking studies showed a high affinity of the synthesised compounds against the two tested targets compared to the reference molecules (Table 5).
To validate the docking procedures, the co-crystallised ligands (Erlotinib and TAK-285) were re-docked against EGFR WT and EGFR T790M , respectively. The RMSD of docked and original ligands of erlotinib and TAK-285 were 0.88 and 1.05 Å, respectively. These values indicate the validity of the docking protocol (Figures 7  and 8).
The co-crystallised ligand (erlotinib) of EGFR WT showed a binding energy of À22.12 kcal/mol. The heterocyclic system (quinazoline moiety) was buried in the adenine pocket forming a hydrogen bond with Met769. Also, it formed four hydrophobic interactions with Lue694, Ala719, and Leu820. The ethynylphenyl moiety was oriented into the hydrophobic pocket I forming three hydrophobic interactions with Ala719, Val702, and Lys721. The 2methoxyethoxy groups occupied the hydrophobic region II forming a hydrogen bond with Cys773 ( Figure 9).
The synthesised compounds showed good binding affinities against EGFR T790M with binding free energies ranging from À11.59 to À22.39 kcal/mol ( Table 5). The co-crystallised ligand (TAK-285) exhibited a binding energy of À18.70 kcal/mol. The pyrrolo[3,2d]pyrimidine moiety was buried in the adenine pocket forming a hydrogen bond with Met793 and three hydrophobic bonds with Leu844 and Ala743. The terminal 3-(trifluoromethyl)phenoxy group occupied the hydrophobic pocket I forming a hydrogen bond with Lys745. Also, it formed seven hydrophobic interactions with Lys745, Glu762, Leu788, and Ile759. In addition, the N-ethyl-3hydroxy-3-methylbutanamide moiety occupied the hydrophobic region II forming hydrogen bond with Ser720. The phenyl moiety formed hydrophobic interactions with Met790, Val726, and Ala743 ( Figure 14).

In vitro cytotoxic activity
In vitro cytotoxicity was carried out using MTT assay protocol 63 as described in Supplementary data.

In vitro EGFR kinase assay
In vitro EGFR inhibitory activity was assessed using Homogeneous time-resolved fluorescence (HTRF) assay 64 as described in Supplementary data.

Cell cycle analysis
The effect of compound 8a on cell cycle distribution was performed using propidium iodide (PI) staining technique as described in Supplementary data 65,74,75 .

Apoptosis analysis
The effect of compound 8a on cell apoptosis was investigated as described in Supplementary data [76][77][78] .