Synthesis and preliminary structure-activity relationship study of 3-methylquinazolinone derivatives as EGFR inhibitors with enhanced antiproliferative activities against tumour cells

Abstract In this paper, a set of 3-methylquniazolinone derivatives were designed, synthesised, and studied the preliminary structure-activity relationship for antiproliferative activities. All target compounds performed significantly inhibitory effects against wild type epidermal growth factor receptor tyrosine kinase (EGFRwt-TK) and tumour cells (A431, A549, MCF-7, and NCI-H1975). In particular, compound 4d 3-fluoro-N-(4-((3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)methoxy)phenyl)benzamide showed higher antiproliferative activities against all tumour cells than Gefitinib (IC50 of 3.48, 2.55, 0.87 and 6.42 μM, respectively). Furthermore, compound 4d could induce apoptosis of MCF-7 cells and arrest in G2/M phase at the tested concentration. Molecular docking and ADMET studies showed that compound 4d could closely form many hydrogen bonds with EGFRwt-TK. Therefore, compound 4d is potential to develop as novel anti-cancer drug.


Introduction
Epidermal growth factor receptor (EGFR) tyrosine kinase (TK), a receptor tyrosine kinase, plays an important role in the process of survival, proliferation, angiogenesis, tumour micro environment, and adhesion of several malignant tumors 1 . Overexpression or mutation of EGFR has been observed in many human tumour cells, such as nonsmall-cell lung cancer (NSCLC), breast cancer, and brain cancer 2 . Due to its crucial status related to cancer development, EGFR has been considered a potential drug target for many years [3][4][5] . Currently, a series of EGFR inhibitors were developed and approved into clinical phases, such as Gefitinib, Erlotinib, Icotinib, Afatinib, and Osimertinib 6 . However, with the continuous emergence of drug resistance in EGFR inhibitors in clinic, design and synthesis of novel EGFR inhibitors have become an urgent work for anti-tumour drug discovery 7 .
Quinazolone is the main skeleton of many natural products 8,9 , for example Allicin C, Cyclosporin F, Rutaecarpine, and (þ)-Changshanine ( Figure 1). These compounds have a wide range of biological activities, including antitumor, antibacterial, antiviral, and anti-inflammatory [10][11][12] . Therefore, many groups have developed new methods to synthesise quinazolone derivatives 13 . Patel et al. reported 2-phenylquinazolone derivatives as EGFR inhibitors in 2017 14 and recently inspired from natural alkaloid L-norephedrine, Ghorab et al. published a 3-substituted quniazolinones for EGFR inhibitors 15 . These results indicate that the development of novel quinazolone EGFR inhibitors has attracted extensive attentions.
Our group has been focussed on the research of developing new anti-tumour drugs for several years [16][17][18][19] . We have synthesised lots of antiproliferative compounds such as benzodiazepines, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles 20 , and pyrimidines 21 . Based on the broad activities of quinazolone and imperative need for EGFR inhibitors, we reported a series of 3-methylquinazolone derivatives for EGFR inhibitors in last year 22 . As shown in Figure 1, the IC 50 value of compound A against EGFR wt reached to 47 nm. However, further studies showed that compound A was not stable in mice plasma ( Figure S1). The result prompted us to seek more valuable 3-methylquniazolinone derivatives. To overcome the metabolic stability in our previous research, we herein describe the design, synthesis, and studies the preliminary structure-activity relationship (SAR) of new 3-methylquinazolinone derivatives as EGFR inhibitors.

Experimental section
All reagents were commercially available in Sigma-Aldrich with analytical purity. Melting points were tested in digital melting point analyser with micro-display window (uncorrected, Shanghai Microelectronics Technology Co. Ltd.). The 1 H and 13 C NMR spectra were recorded on Bruker (Avance) 400 MHz and JEOL (Japan) 500 MHz NMR instrument with chemical reported as d in CDCl 3 and DMSO-d 6 , tetramethylsilane (TMS) as the internal standard. The high-resolution mass spectrometer (HRMS) was tested in TSQ 8000 high-resolution mass spectrometer and AB SCIEX X500R QTOF.

Synthesis
The synthetic route of compounds 4a-4g was shown in Scheme 1. The experimental methods and details were described as follows.
2.1.1. 2-(Chloromethyl)-3-methylquinazolin-4(3H)-one (2) 2-Amino-N-methylbenzamide (1.5 g, 10 mmol) was dissolved in acetic acid (50 ml), and then 2-chloroacetyl chloride (3.36 g, 30 mmol) was added. The mixture was stirred for 6 h under refluxing. Then, the solution was evaporated in vacuo and neutralised with aqueous NaHCO 3 . The crude product was filtered and purified with recrystallisation from ethanol. The product was dried in vacuum to give a white solid.  (20 ml), and then anhydrous K 2 CO 3 (759 mg, 5.5 mmol) was added. After stirred for 15 min at room temperature, the mixture was added compound 2 (1.04 g, 5 mmol) and KI (913 mg, 5.5 mmol). The solution was stirred for the other 2 h, and poured into water (40 ml). The crude product was filtered and purified with recrystallisation from ethanol. The product was dried in vacuum to give a light-yellow solid. 1 g; 68% yield; 1 (588 mg, 2 mmol) were dissolved in EtOH (20 ml), and then 2 mol/l NaOH (1 ml) was added. The mixture was stirred for 6 h at room temperature. Then, the solution was neutralised with 2 mol/l HCl (1 ml). The crude product was filtered and purified by chromatography using PE/EtOAc ¼ 1/1 as the fluent solvent. The product was dried in vacuum to give a white solid. 348 mg; 42% yield; m.p. 208-209 C; 1 (20 ml), and then anhydrous K 2 CO 3 (759 mg, 5.5 mmol) was added. After stirred for 15 min at room temperature, the mixture was added compound 2 (1.04 g, 5 mmol) and KI (913 mg, 5.5 mmol). The solution was stirred for the other 2 h, and poured into water (40 ml). The crude product was filtered and purified with recrystallisation from ethanol. The product was dried in vacuum to give a white solid. 1

In vitro EGFR wt -TK assay
Enzymatic inhibition of synthesised compounds against wild type EGFR tyrosine kinase was determined with enzyme-linked immunosorbent assays (ELISAs) as our previous methods. The recombinant EGFR wt -TK and Antiphosphotyrosine mouse mAb were purchased from PTM Bio. The IC 50 value was determined from a sigmoid dose À response curve using Graph-Pad Prism (GraphPad Software, San Diego, CA).

Cytotoxicity evaluation (MTT assay)
In vitro cytotoxicity of compounds 4a-4g against four cancer cell lines (A431, A549, MCF-7, and NCI-H1975) and normal rat kidney cells (NRK 52E) was determined by MTT assay as our previous report. Gefitinib were used as positive controls. The IC 50 value was determined from a sigmoid dose À response curve using Graph-Pad Prism (GraphPad Software, San Diego, CA).

Cell apoptosis analysis
The apoptosis of tumour cells MCF-7 treated by different concentrations of compound 4d, was measured with Annexin V -FITC/PI apoptosis detection kit (Solarbio, Beijing, China), according to instructions of kit, and detected by BD Accuri C6 flow cytometry (American BD Corporation Shanghai Co. Ltd.)

Cell cycle analysis
The distribution of cell cycle for MCF-7 treated by different concentrations of compound 4d, was measured with Annexin V -FITC/PI cell cycle detection kit (Solarbio, Beijing, China), according to instructions of kit, and detected by BD Accuri C6 flow cytometry (American BD Corporation Shanghai Co. Ltd.)

Molecular docking
X-ray crystal structures of EGFR in both "active" (PDB entry 1M17) and "inactive" (PDB entry 4HJO) states were used for identifying candidate binding modes. The possible binding modes of compound A, compounds 4a-4g, Gefitinib with EGFR were predicted by molecular docking with Sybyl X-2.0 software from Tripos Inc, St. Louis, MI.

Admet studies
The absorption, distribution, metabolism, elimination, and toxicity (ADMET) parameters of compounds 4a-4g and Gefitinib were calculated in and calculated in CHARMM Force Field of Discovery Studio 2.5 Software (Accelrys Inc., San Diego, CA). The data of ADMET included Solubility, Absorption, Cytochrome P450 (CYP2D6), Hepatotoxiciy, Plasma protein binding (PPB), Blood brain barrier permeability (BBB), and water partition coefficients for the unionised species (AlogP98) 26 .

Chemistry
All target compounds (4a-4g) were synthesised and confirmed based on 1 H-NMR, 13 C-NMR and HRMS. The synthetic route was shown in Scheme 1. At first, compound 2 and 3 were prepared under the described conditions in literature 23 . And then, compound 3 experienced in aldol-condensation with 3-fluorophenylethanone to give the product 4a. Subsequently, compound 3 reacted with ethyl 4-hydroxybenzoate under the condition of K 2 CO 3 /KI/DMF to give the compound 5 with 64% yield. After hydrolysis in the presence of NaOH, compound 6 was obtained with 58% yield and formed amide 4 b with 3-fluorobenzenamine. Next, compound 9 was prepared with the reported method and reacted with compound 2 to give styrylquniazolinone 4c. At last, treatment 2 with 4-nitrophenol and reduce the nitro-group to give the intermediate 11. Compound 11 reacted with corresponding acid or isocyanate to obtain the final targets 4d-4g. The synthetic details were described in experimental section and the spectra can be found in the supplementary material.

In vitro EGFR kinase inhibitory activity and antitumor activity of target quinazolinone derivatives
With the compounds 4a-4g in hand, the activity against EGFR wt -TK was tested with ELISA assay 27 . As shown in Table 1, when the enamine bond of compound A (IC 50 of 0.047 lM) was substituted with ketene group (  1, 4b and 4d), the IC 50 values of compounds 4a-d to EGFR wt -TK were 2.71 lM, 0.2 lM, 1.63 lM and 0.053, respectively. Fortunately, compound 4d reached in the similar activity with compound A. Compared to 4d, 3,4-difluoro substitution on the phenyl ring (Table 1, 4e) decreased the activity. Meanwhile, the extended amide bond (Table 1, 4f) and urea bond ( Table 1, 4g) also significantly weaken the activity.
To investigate the anticancer activity of the synthesised compounds, four EGFR over-expressed human tumour cell lines derived from human epidermoid cancer (A431), non-small cell lung cancer (A549), breast cancer (MCF-7), and non-small cell lung cancer (NCI-H1975) were used to evaluate the antiproliferative activities by methyl thiazolyl tetrazolium colorimetric assay (MTT). Gefitinib, a clinical drug targeted EGFR was employed as positive control. As shown in Table 2 Based on the result of EGFR wt -TK and four tumour cell assays, we found that the similar structure-activity relationship (SAR) was observed in cell lines and EGFR-TKs. The amide group (4d and 4e) instead of enamine was more potent than the ketene group (4a), vinyl group (4c), urea group (4 g) and other groups (4 b and 4f). Besides, compound A showed lower inhibitor activities against four tumour cell lines than the compounds 4a-4g. The reason may be due to the lower stability of compound A in cells. Generally, compound 4d was the best in the synthesised compounds and particularly showed much higher inhibitor activities than Gefitinib against four tumour cells. These data indicate that 4d was worth to further investigation.

In vitro cytotoxicity of 3-methyl quinazolinone derivatives on normal cells
The cytotoxicity study of compounds 4a-4g was first evaluated by MTT colorimetric assays to normal rat kidney cell line (NRK-52E) at different concentrations. There was no inhibition of all compounds including Gefitinib at 10 lM. Therefore, the concentration was accelerated to 100 lM. As shown in Figure 2, all compounds were similar with Gefitinib and not more than 25% against NRK-52E at 100 lM. This indicate that the synthesised compounds 4a-4g were low cytotoxicity and potent for development for drugs.

Effects of compound 4d on cell apoptosis of MCF-7 cell line
Apoptosis of tumour cell is the basic mechanism of tissue homeostasis, which is regarded as the efficient method to eliminate the excess cells 28 . Most of the anti-cancer drugs perform as a well inducing apoptosis for cancer cells 29 . As shown in Figure 3, compound 4d was used to investigate the mechanism of inhibition of MCF-7 cell proliferation. Flow cytometry analysis of MCF-7 treated with 4d at 5 and 10 mM for 48 h demonstrated a prominent increase in apoptotic cells with a dose-dependent fashion. The trend of compound 4d showed that early apoptosis rate was priority over late apoptosis. Compared compound 4d with Gefitinib, the results in Figure 3(B) showed that 4d significantly performed better ability to induce early apoptosis at the same concentration. Particularly, the ratio of apoptotic cells for compound 4d reached 28.14% (early) and 41.36% (late) even at 5 mM.

Effects of compound 4d on cell cycle of MCF-7 cell line
Next, we wished to determine whether 4d-induced decrease of EGFR phosphorylation would result in cell cycle arrest in MCF-7 cell lines, compared with Gefitinib. As shown in Figure 4, cell cycle analysis was tested in flow cytometry. MCF-7 cell lines were treated with compound 4d at 5 and 10 mM. Against MCF-7 cells at 5 mM, the control consisted of 6.06% G2 phase cells, compound 4d increased to 10.38% and Gefitinib also reached to 11.58%. However, at 10 lM of tested samples, G2/M phase decreased and S phase increased, this might be that compound 4d and Gefitinib undergo significant apoptosis. These results indicate that compound 4d and Gefitinib could arrest MCF-7 cells in G2/M phase at the appropriate concentration.

Molecular docking studies
To explain the structural activity relationship of EGFR with our compounds, the possible binding modes were performed in molecular docking studies 30 . Former researches showed that the "active" or "inactive" state of EGFR-TK has different preference of ligand binding profile, so X-ray crystal structures of EGFR in both "active" (PDB entry 1M17) and "inactive" (PDB entry 4HJO) states were used for identifying candidate binding modes 31 .
As shown in Tables S1 and S2, the predicted score of all compounds 4a-4g with inactive state of EGFR (PDB: 4HJO) is much  higher than the score of active state of EGFR (PDB: 1M7), which suggests that the compounds 4a-4g are likely to bind with EGFR in inactive state. By contrast, the predicted binding model for Gefitinib in two states are similar, which is consistent with our previous work. More interestingly, as shown in Figure 5(A,C), all the compounds 4a-4g were employed in the same binding configuration with EGFR active state or inactive state. Take compound 4d as example, in Figure 5(B) for active state, simply formed one hydrogen bond with Met769 during the docking situation. However, in Figure 5(D) for inactive state, quinazolinone ring of compound 4d formed stable hydrogen bonds with Lys692 and Lys704. Meanwhile, amide group and fluorine atom of 4d also formed hydrogen bonds with Thr766, Thr830, and Lys721 respectively. These results indicate that the synthesised compounds 4a-4g are particularly likely to be potential EGFR inhibitors against inactive state.

Admet and stability studies
The ADMET are essential aspects for drug candidates 32 . Computer aided ADMET studies have been employed in the early stage of drug discovery. Therefore, the ADMET properties of compounds 4a-4g were calculated through Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA). As shown in Table 3, the levels of solubility and absorption were the same with compounds 4a-4g and Gefitinib. CYP2D6 is non-inhibitor of cytochrome P450 enzyme for predicting drug toxicity. The compounds 4a, 4f and 4 g were less toxic than Gefitinib, and the others were similar with Gefitinib. Meanwhile, the calculated log p values of compound 4 b, 4d, 4e and 4 g were less than Gefitinib (4.20). Particularly, compound 4d reached the lowest log p (3.52) and performed highly oral bioavailability. These data are consistent with the structure-activity relationships.
At last, we investigated the stability of compound 4d in mice plasma. The results exhibited that compound 4d was stable with purity higher than 98% after incubation with Sprague Dawley (SD) mice plasma ( Figure S2). All the data suggest that compound 4d was a good candidate for developing new anti-cancer drug.

Conclusions
To solve the issues of metabolic stability in our previous research of EGFR inhibitors, seven 3-methylquinazolinone derivatives were designed and synthesised. After evaluated activities of these compounds in enzyme and cell level, we found that compounds 4d and 4e were performed well antiproliferative activities. Furthermore, the best compound 4d displayed IC 50 value of 53 nM against EGFR wt -TK activity and IC 50 value of 0.87 lM against MCF-7 cells. Analysis of apoptosis and cell cycle for MCF-7 cells  indicate that compound 4d could induce apoptosis and arrest in G2/M phase at tested concentration. At last, molecular docking, ADMET, and stability studies suggested that compound 4d was closely formed hydrogen bonds with EGFR wt -TK and potential to develop new anti-cancer drug.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was financially supported by GZU (Guizhou University) Found for Newly Enrolled Talent