Design, synthesis and biological evaluation of aminopyrimidine derivatives bearing dihydroquinoxalinone as novel EGFRL858R/T790M kinase inhibitors against non-small-cell lung cancer

The clinical responses to the EGFR kinase inhibitors in non-small cell lung cancer (NSCLC) patients are always followed by drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFRT790M are also limited by the toxicity due to the concurrent inhibition of wide-type (WT) EGFR. Here we employed splicing principle to design and synthesize a series of aminopyrimidine derivatives bearing dihydroquinoxalinone (8–15) as novel third-generation inhibitors against double mutant L858R/T790M in EGFR. It is worth noting that compound 10 presented remarkable inhibitory activity against EGFRL858R/T790M (IC50 = 15 ± 1.8 nM) and anti-proliferative effect against H1975 cells (IC50 = 166.43 ± 14.45 nM). Moreover, the obvious down-regulation effect of EGFR-mediated signaling pathways and the promotion of apoptosis in H1975 cells confirmed its potent efficacy. These results demonstrated that compound 10 deserves the further exploration.


Introduction
Non-small cell lung cancer (NSCLC) accounts for a large proportion of lung cancer, which is a type of cancer with high morbidity and mortality worldwide [1,2]. Epidermal growth factor receptor (EGFR), a trans-membrane glycoprotein, belongs to the ErbB receptor family and acts as a crucial mediator in signaling pathways of the cell proliferation, survival and apoptosis [3,4]. Pathologically, the aberrant expression of EGFR is closely associated with tumorigenesis, while activating mutations (L858R and exon 19 deletion mutations) in the kinase domain of EGFR have been identified as the primary oncogenic drivers in NSCLC [5][6][7]. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) could effectively inhibit tumor cell proliferation and have been recommended as the first-line treatment in patients with EGFR-positive NSCLC [8,9].
The first-generation EGFR-TKIs are represented by Gefitinib, which are developed for the therapy of NSCLC patients harboring activating mutations in the EGFR kinase domain. They are reversible ATP-competitive inhibitors featured with quinazoline structure [10][11][12]. However, the clinical efficacy of the first-generation EGFR-TKIs is ultimately limited by acquired drug resistance due to mutation of the gatekeeper T790M [13,14]. The binding assays have demonstrated that the presence of T790M increased the affinity for ATP, which reduced the affinity between EGFR-TKIs and EGFR tyrosine kinase [15,16]. Second-generation EGFR-TKIs with irreversible targeting Cys797 moieties have been developed to overcome EGFR T790M mutation mediated drug resistance, such as Afatinib and Dacomitinib [17][18][19][20]. Nevertheless, the application of second-generation EGFR-TKIs was restricted as a result of toxicity caused by concurrent inhibition of wide-type (WT) EGFR [21]. With respect to EGFR WTdriven toxicities, several selective covalent EGFR T790M inhibitors as third-generation EGFR-TKIs have been developed. These third-generation EGFR inhibitors, such as Osimertinib and WZ4002, not only have good activities against activating mutations and the T790M resistance mutation, but also have excellent selectivity to EGFR WT ( Fig. 1) [22,23].
Osimertinib with promising efficacy and low toxicity were granted accelerated approval to treat EGFR T790M -positive NSCLC patients by the US FDA in 2015. However, the risk of irreversible binding to proteins other than the target motivated the optimization of Osimertinib [24,25]. Quinoxalinone rings have been utilized as important structural motifs in drug discovery due to their ubiquitous biological activities including antitumor, antibacterial and antiallergy (Fig. 3) [26,27]. Thus, the "splicing principle" has been employed to replace indole ring with quinoxalinone motif (P 1 ), which was speculative to form new hydrogen bonds with corresponding amino acid residues. The X-ray crystal structure of Osimertinib and mutant EGFR T790M has confirmed that the U-shaped configuration and the important interaction of pyrimidine core which formed two-dentate hydrogen bonds with the hinge residue Met793 [28,29]. Therefore, the scaffold of pyrimidine has been retained and fused with various electron-withdrawing or  electron-donating groups (P 2 ) to explore the interactions with EGFR. P 3 is the moiety of side chain, which could penetrate into the solvent zone. Thus, side chains bearing different aliphatic amine were introduced to modulate the physicochemical properties of molecules, exploring the most suitable group. P 4 is the electrophilic Michael acceptor of which was able to form a covalent bond with the sulfhydryl of Cys797. We sought to replace the alkene with less reactive electrophilic warhead like chloroethyl to reduce the toxicity burden creating by nonspecific covalent binding (Fig. 2) [30,31] . In our current study, 8 novel compounds have been synthesized based on the above design strategy and carried out a series of biological evaluations.

Chemistry
The synthesis routs of target compounds 8-15 were summarized in Scheme 1. The nucleophilic substitution reaction was carried out between the commercially available substituted 2, 4-dichloropyrimidine (1a-d) and phenylenediamine or N-methylbenzene-1, 2-diamine in the presence of Et 3 N to give intermediates 2a-d, and further reacted with chloroacetyl chloride to afford compounds 3a-d. Then the intramolecular cyclization reaction of 3a-d was proceeded to get intermediates 4a-d. The key intermediates 5a-d were prepared by reacting 4a-d with 4-fluoro-2-methoxy-5nitroaniline at high temperature. Intermediates 6a-h could be obtained by the nucleophilic substitution reactions between 5a-d and secondary amines with DIPEA. After that, the reduction reaction was proceeded in the presence of Fe powder and NH 4 Cl to get amines 7a-h. Finally, the amines 7a-h were treated with chloroacetyl chloride to furnish the target compounds 8-15 shown in Table 1.

In vitro anti-proliferative activities
Among the Osimertinib derivatives with good inhibitory activities against EGFR L858R/T790M , analogues (8-10 and 14) were selected for further evaluation of their anti-proliferative activities against H1975 cells expressing EGFR L858R/T790M and A549 cells harboring EGFR WT with Osimertinib as the positive control by CCK8 assay. As shown in Table 2, all of the tested compounds exhibited moderate cytotoxicity activity against H1975 cell line with IC 50 values ranging from 166.43 to 398.03 nM, which produced a decrease compared with the positive control Osimertinib. It was speculated that the replacement of acrylamides strongly affected the interaction with Cys797. Meanwhile, the selected analogues displayed selective anti-proliferative

Apoptosis induction assay
With the comprehensive consideration of inhibitory activities against kinases and tumor cells, compound 10 was chosen to investigate whether it could induce apoptosis of

Inhibitory effects of EGFR and downstream signaling pathways
Due to the good inhibitory potency of compound 10 in H1975 cells, we further assessed the effect of compound 10 on the phosphorylation of EGFR and the downstream signaling pathway by Western-blot analysis. As illustrated in Fig. 5, the phosphorylation of EGFR and the downstream signaling AKT level were obviously reduced in a dosedependent manner after treated with compound 10 for 2 h.

Binding mode analysis
To have better explanation for the inhibitory activity of compound 10, the docking model was established with the mutated EGFR kinase of T790M (PDB: 6jx0, Cocrystalized with Osimertinib) by Glide (Schrödinger version 11.8). The predicted binding mode was depicted in Fig. 6. The N atom in the dihydroquinoxalinone ring could generate hydrogen bond with the Asn842, which was speculated for contributing to the affinity with kinase. In addition, the N atom at the terminal of sidechain was able to form a hydrogen bond with Asp800. The two-dentate hydrogen bonds were formed between the aminopyrimidine of compound 10 and Met793 residue, which was same to the Osimertinib.

Conclusion
In this study, a series of aminopyrimidine derivatives bearing dihydroquinoxalinone were designed, synthesized and evaluated as EGFR inhibitors to reduce the toxicity caused by nonspecific covalent binding. Several compounds exhibited excellent inhibitory activities against L858R/ T790M mutated EGFR kinase with IC 50 at nanomolar range. The anti-proliferation activities of the selected compounds (8-10 and 14) against H1975 and A549 cell lines were also evaluated and most of them showed moderate efficacy. Compound 10 showed lower cytotoxic activity against MCR-5 cell (IC 50 > 10000 nM) compared with Osimertinib, which suggested it may have a high safety profile. Compound 10 displayed remarkable inhibitory activities in vitro against mutated EGFR L858R/T790M and H1975 cells with IC 50 values of 15 nM and 166.43 nM, respectively and high selectivity, which was selected to further perform the apoptosis induction assay and Westernblot analysis on H1975 cell lines. The analysis of flow cytometric suggested that compound 10 could effectively induce cell apoptosis in a concentration-dependent manner. Besides, compound 10 significantly inhibited the phosphorylation of EGFR and its downstream signaling pathway in a dose-dependent manner. Further optimizations to this series of derivatives are still underway, which will be reported in the future.

Chemistry
The commercially reagents and solvents were used without further purification unless stated otherwise. Column chromatography was performed using 200-300 mesh silica gel (Haiyang, Qingdao). All yields are unoptimized and generally represent the result of a single experiment. The NMR spectra were recorded for 1 H NMR at 500/400 MHz and for 13 C NMR at 125/100 MHz. For 1 H NMR, CDCl 3 (δ = 7.26) and DMSO (δ = 2.50) were served as internal standard and data were reported as follows: chemical shift, multiplicity, coupling constant in Hz and integration. For 13 C NMR, CDCl 3 (δ = 77.23) and DMSO (δ = 39.51) were served as internal standard and spectra were obtained with complete proton decoupling. HRMS data were obtained on Agilent 1290 HPLC-6224 Time of Flight Mass Spectrometer.

General procedure for the synthesis of 2a-2d
The materials 1a-1d (5 mmol) were dissolved and stirred in n-BuOH (20 ml) under the protection of nitrogen. Phenylenediamine or N-methylbenzene-1, 2-diamine (5 mmol) and Et 3 N (10 mmol) were added in the mixture and heated to 110°C in oil bath, then stirred for 3 h. The process of reaction was monitored by TLC (petroleum ether: ethyl acetate = 3: 1) until finished and cooled to room temperature. The solvent was evaporated and the mixture was diluted with 0.1 M hydrochloric acid (10 ml) stirring for 0.5 h. When the solids were precipitated completely, they were filtered off to obtain crude products, which were used directly for next step without further purification.   General procedure for the synthesis of 3a-3d Corresponding intermediates 2a-2d (1 mmol) and anhydrous K 2 CO 3 (138.2 mg, 1 mmol) were dissolved in DCM (25 ml) and stirred at 0°C. Then chloroacetyl chloride (100 μl) was added stirring for 1-2 h at 0°C. Upon completion, the solvent was evaporated under reduced pressure. The resulting solids were washed with water and dried in a vacuum oven to give crude products 3a-3d.

General procedure for the synthesis of 4a-4d
To solution of intermediates 3a-3d (1 mmol) in DMF was added K 2 CO 3 (138.2 mg, 1 mmol). The reaction mixture was stirred at 100°C for 1-2 h. TLC analysis showed that 3a-3d were consumed and the reaction system was then cooled to room temperature. The residue was poured to ice water (20 ml). The solid precipitates of the mixture were filtered and then washed with water until neutral and dried to yield the title compounds 4a-4d. General procedure for the synthesis of 5a-5d The material 4-fluoro-2-methoxy-5-nitroaniline (185 mg, 1 mmol) was added into the mixture of 4a-4d and ptoluenesulfonic acid monohydrate (0.12 mmol) in n-BuOH (5 ml) and then stirred at 105°C for 2.5 h. The processes of reactions were monitored by TLC until finished and cooled to room temperature. The resulting solids were filtered and washed with petroleum ether to obtain crude intermediates 5a-5d for next step. General procedure for the synthesis of 6a-6h Under the protection of nitrogen, corresponding intermediates 5a-5d (1 mmol) were added into the mixture of amines (2 mmol) and DIPEA (0.6 ml, 3 mmol) in 2,2,2-trifluoroethanol (12 ml) and then stirred at 120°C for 24 h. After cooling to room temperature, the mixture was concentrated under vacuum. The crude products were purified by silica gel chromatography to furnish pure intermediates 6a-6h. General procedure for the synthesis of 7a-7h The intermediates 6a-6h (1 mmol) were dissolved in EtOH (12 ml) and water (4 ml) and then iron powder (280 mg, 5 mmol) and NH 4 Cl (161 mg, 3 mmol) were added in the reaction system. The mixture was heated to 85°C. After completion of the reaction, the mixture was filtered through celite and the filtrate was extracted with DCM. The organic phase was evaporated under reduced pressure. The crude products 7a-7h were used directly in the next step without purification.
General procedure for the synthesis of target compounds 8-15 The materials 7a-7h (1 mmol) and DIPEA (1.2 mmol) were dissolved in dry DCM (5 ml) under the protection of nitrogen. Acryloyl chloride (181 mg, 1 mmol in DCM) was added dropwise to the mixture at −10°C. The resulting suspension was stirred at −10°C until finished. Then the mixture was quenched with water, extracted with DCM (10 ml × 3) and washed by brine (10 ml × 3). The organic phase was separated, dried over anhydrous Na 2 SO 4 and evaporated in vacuo. The crude products were further purified by silica gel chromatography to afford target compounds 8-15.  13

Cell proliferation assay
In vitro cytotoxicity activities of target compounds against H1975 and A549 cell lines were evaluated by CCK8 assays. H1975 and A549 cells (ATCC, Rockville, MD, USA) were seeded in 96-well plates (1 × 10 4 cells/well) and exposed to different concentrations of tested compounds for 72 h. Then CCK8 (Beyotime, Shanghai, China) was added to each well of the plate and incubated with cells at 37°C for 4 h. The absorbance was measured at 450 nm by multifunction microplate reader (Bio Tek Synergy LX). The growth inhibitory ratio was calculated as follows: Growth inhibitory ratio = (A control − A sample )/A control . IC 50 values were obtained from a nonlinear regression model (curve fit) on the base of sigmoidal dose response curve (variable slope) and computed using GraphPad Prism version 5.0 (GraphPad Software).

Apoptosis assay
The apoptosis of H1975 cells was determined by an Annexin V-FITC/PI assay. H1975 cells (ATCC, Rockville, MD, USA) were seeded in 6-well plate (3 × 10 4 cells/well) and treated with vehicle, Osimertinib (MedChemExpress, Shanghai, China) or various concentrations of 10 at 37°C for 24 h. After incubation, H1975 cells were harvested and incubated with 5 μl of FITC-conjugated Annexin V (Key-GEN Biotech, NanJing, China, KGA108). Then PI solution was added to the medium for another incubation of 10 min. The cells were screened by flow cytometry (Beckman DxFlex). The percentage of apoptosis cells was calculated with EXPO32 ADC Analysis software.

Western blotting
H1975 cells (ATCC, Rockville, MD, USA) were seeded in 6-well plates at a density of 5 × 10 5 cells per well. After 24 h, cells were treated with tested compounds for 2 h. After incubation, the cells were washed twice with ice-cold PBS, and then lysed in RIPA lysis buffer containing PMSF (Solon, OH, USA). The lysates were incubated at 0°C for 30 min, and vortexed every 10 min intermittently, then the total protein was harvested by centrifuging at 12,000 × g for 5 min at 4°C. After the protein concentrations were determined by a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, Illinois, USA), the protein extracts were reconstituted in loading buffer and boiled at 100°C for 10 min. An equal amount of the protein (20 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to NC membranes (Millipore, Schwalbach, Germany). After blocked with 5% non-fat dried milk in TBS containing 1% Tween-20 for 60 min at room temperature, the membrane was incubated overnight with primary antibodies against phosphor-EGFR (CST, Tyr1068, #3777T), EGFR (AB clonal, #A11351), phosphor-AKT (Protein-tech, Ser473, #66444-1-Ig), AKT (CST, #4691S) and GAPDH (Proteintech, #60004-1-Ig) at 4°C. After three washes in TBST, the membranes were incubated with the appropriate HRPconjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h. The blots were developed with enhanced chemiluminescence (Thermo Fisher Scientific, Pittsburgh, PA, USA) and were detected by an Tanon 6600 (Tanon, China).

Molecular docking study
The co-crystal structure of T790M mutated EGFR kinase (PDB ID code: 6jx0) was used for the docking models. Prediction of the binding mode of the ligand to corresponding EGFR kinase was performed by Glide (Schrödinger version 11.8). For the preparation of protein, the hydrogen atoms were added by using Protein Preparation Wizard module of Maestro, and the OPLS3 force field was employed to minimize the energy. For the preparation of ligands, the 3D structures were generated and their energy was minimized in LigPrep. Interactions of the docked EGFR kinase with ligand were analyzed and then the docking conformations were selected and saved based on calculated glide docking energy score.