Design, Synthesis and Biological Evaluation of the Quinazoline Derivatives as L858R/T790M/C797S Triple Mutant Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

Abstract

Inhibition of the epidermal growth factor receptor (EGFR) has been proved to be one of the most promising strategies for the treatment of non-small cell lung cancers. A series of 2-aryl-4-amino substituted quinazoline derivatives were designed and synthesized with the purpose to overcome L858R/T790M/C797S (CTL) triple mutant drug resistance and the biological activity for inhibition of CTL kinases and EGFR wild type (WT) were evaluated. Three compounds (20, 24 and 27) showed excellent inhibitory activities against EGFR kinases triple mutant CTL (IC₅₀ < 1 µM) and high selectivity (IC₅₀: WT/CTL >10000). Cell line evaluation showed that the most potent compound 27 was significantly potent against H1975-EGFR L858R/T790M (IC₅₀ = 3.3 µM) and H1975-EGFR L858R/T790M/C797S (IC₅₀ = 1.2 µM). Compound 27 also exhibited good microsomes stabilities in human, rat and mouse liver species, but low bioavailability. This work would be very useful for discovering new quinazoline derivatives as tyrosine kinase inhibitors targeting triple mutant L858R/T790M/C797S.

Introduction

Lung cancer is the leading cause of death for all cancer patients in the world, with an estimated 1.6 million deaths per year, and the incidence and mortality show rapid growth.¹⁾ Non-small cell lung cancer (NSCLC) is the most common type of lung cancers, approximately 85% for all lung cancer patients. Dysregulation of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) gene mutation have been shown responsible for the development and growth of many tumor cells. It has been identified that NSCLC is associated with EGFR and ErbB-2.²⁾

The epidermal growth factor receptor (EGFR) family includes four family members: EGFR/ErbB-1, HER2/ErbB-2, Her3/ErbB-3 and Her4/ErbB-4, and these receptor tyrosine kinases play key roles in cell proliferation, survival, adhesion, migration and differentiation.^3,4) Therefore, EGFR becomes a key target for developing inhibitors as potential anticancer agents.⁵⁾ Four EGFR tyrosine kinases are anchored in the cell membrane and shared similar cysteine-rich extracellular regions with unique subdomain motifs.⁶⁾ The mutation of EGFR is related to pathogenesis of NSCLC, and the common mutation sites are located in the ATP-binding region of the kinase TK-domain, including deletion mutations at positions 747 to 750 of exon 19 and L858R point mutations in exon 21.⁷⁾

Several 4-anilinoquinazolines EGFR inhibitors have been developed, such as gefitinib and erlotinib, which have significant effects in the treatment of NSCLC patients with L858R and del E746-A750 EGFR mutations,^8,9) and become the main therapeutic strategy of NSCLC. However, after treatment of 10–16 months with these inhibitors, drug resistance via an acquired secondary EGFR T790M mutation is commonly observed, approximately 50% of the NSCLC patients in western countries.¹⁰⁾ The EGFR T790M mutations increases the ATP binding affinity, prevents the reversible inhibitors in the catalytic domain binding with EGFR tyrosine kinase at higher ATP concentrations and decreases the sensitivity of gefitinib or erlotinib.¹¹⁾ In order to overcome the T790M mutation resistance, several second-generation irreversible EGFR inhibitors were developed with covalent bond to Cys797 within the EGFR active site such as covalent inhibitors of EGFR dacomitinib and afatinib.^12,13) Imperfectly, these covalent inhibitors lack of excellent selectivity between EGFR T790M mutants and the wild-type kinase, simultaneous inhibition of wild-type EGFR eventually cause most patients to suffer from an unacceptably low maximum tolerated dose (MTD).¹⁴⁾ Considering the accurate toxicity, medicinal chemists have done great efforts to develop new generation of irreversible covalent inhibitors maintaining potent inhibitory effects on the EGFR T790M mutation, while showing better selectivity to the wild-type EGFR. These compounds including osimertinib (AZD9291),^15,16) rociletinib (CO-1686),¹⁷⁾ olmutinib (HM61713)¹⁸⁾ and PF-06459988¹⁹⁾ with potent inhibition of L858R/T790M and d746-750/T790M mutation. However, the triple C797S mutation were observed in clinical treatment of patients carrying the EGFR T790M mutation, which impairs the formation of covalent bonds with the cysteine residue at position 797 of EGFR.^20,21)

So far, there are no effective therapeutic strategies to overcome the L858R/T790M/C797S triple mutation of mediated EGFR-TKI resistance since the therapeutic antibody could not inhibit the expression of EGFR phosphorylated. Accordingly, it is particularly necessary to discovery novel EGFR inhibitors against the triple mutants involving C797S to overcome the resistance to third-generation drugs.^22,23) Several different binding modes of the fourth-generation inhibitors have been reported and described. Thiazole amide derivative A (EA1045)²⁴⁾ combination with cetuximab induced marked tumor shrinkage in engineered L858R/T790M/C797S models, that was the first reported inhibitor overcome EGFR L858R/T790M/C797S mutations, and the clinical efficacy now requires validation through clinical trials. 4-Amino pyrazolopyrimidine compound B (1a)²⁵⁾ and tri-substituted imidazoles C (31b) and D (1a) are also reported as the fourth-generation inhibitors that can overcome the EGFR triple C797S mutation^26,27) (Fig. 1).

Fig. 1. Chemical Structures of the EGFR Inhibitors That Are Selective for EGFR L858R/T790M/C797S Mutant

4-Anilinoquinazoline derivatives, such as afatinib and dacomitinib, are typical structures of second-generation irreversible EGFR inhibitors. More potent and better selective EGFR compounds inhibiting the expression of phosphorylation have been successfully developed on the modification of 4-anilinoquinazoline.^28–30) Substitutions with different amines at C-4 position were the most popular strategies, as well as alkyl substitutions at C-6 or C-7 positions. Structure–activity relationship (SAR) studies revealed that methyl group at C-2 position of quinazoline ring could significantly improve the antitumor potency of 4-anilinoquinazolines.³¹⁾ Inspired by these successful examples, in this manuscript, we designed and synthesized a series of 2-aryl-4-amino substituted quinazoline derivatives by structure-based approach to develop novel and selective EGFR inhibitors overcoming L858R/T790M/C797S triple mutant, and their biological activity in vitro and in vivo were evaluated.

Results and Discussion

Design Strategy of the New Compounds

Crystal structure analysis of quinazoline EGFR inhibitors indicates that the quinazoline moiety fits into the ATP binding pocket of the kinase domain, and the C-4 aniline moiety lies in the deep and hydrophobic pocket. Furthermore, the substitution at C-6 or C-7 positions of the quinazoline ring affects pharmacokinetic profile and the physical properties significantly. N-1 and N-3 positions of the quinazoline ring interact with the backbone NH of Met-769 and Thr-766 side chain via hydrogen bonds, separately.

With the aim to develop new potent and selective EGFR inhibitors overcoming EGFR L858R/T790M/C797S mutant, the structure of the second-generation EGFR inhibitor Afatinib E (BIBW2992) was modified at quinazoline moiety, C-2 moiety (R¹) and C-4 aniline moiety (R²), and a series 2-aryl-4-aminoquinazoline were designed and synthesized (Chart 1), and the biological activity for inhibition of EGFR L858R/T790M/C797S kinases and EGFR-wild type (WT) were evaluated, and the SAR was discussed.

Chart 1. Design Strategy of the New Compounds Based on 4-Aminoquinazoline Scaffold

Synthesis

The synthetic routes of target compounds were illustrated in Chart 2. Compounds 5–18 and 20–31 were prepared by using commercially available 2-bromo-5-methoxybenzaldehyde 1 or intermediate 2, which was prepared from 1 by cyanation with cuprous cyanide. Compounds 1 or 2 reacted with 2-aminobenzamide to generate the key intermediates 3a and 3b. Halogenization of 3a and 3b with phosphorus oxychloride to give compound 4a and 4b, which was then substituted by different NHR₂ groups to offer the target compounds 5–18. Demethoxylation of 4a and 4b using boron tribromide yield hydroxyl substituted 19a and 19b, and the target compounds of 20–31 were prepared by further amination with various amino fragments NH₂R².

Chart 2. Synthetic Route for Target Compounds 5a–18 and 20–31

Reagents and conditions: (a) DMF, CuCN, reflux, 14 h, 66.8%; (b) 2-aminobenzamide, I₂, EtOH, reflux, 56.8–70.0%; (c) phosphorus oxychloride, reflux, 76.5–91.7% ; (d) NH₂R², THF, DIPEA, 45–60°C, 38.6–94.5%; (e) CH₂Cl₂, BBr₃, 0–5°C, 76.0–85.8%; (f) NH₂R², THF, DIPEA/DMAP, −5–60°C, 44.7–92.6%.

Biological Activity

The kinase inhibitory activities of the target compounds were evaluated via a well-established homogeneous time resolved fluorescence (HTRF) method based assay³²⁾ against different types of EGFR kinases. The EGFR tyrosine kinase assay results for compounds 5–18 and 20–31 were summarized in Table 1.

Table 1. Chemical Structures of the Target Compounds and Their Inhibitory Activities against Different Types of EGFR^a

N.D.: Not determined (>10000 µM). ^aThe values are means ± standard deviation (S.D.) from three independent experiments. ^bIC₅₀ determinations for EGFR L858R/T790M/C797S were performed with the HTRF (Homogenous Time-Resolved Fluorescence) KinEASE-TK assay. Each reaction was performed in duplicate, and at least three independent determinations of each IC₅₀. ^cCTL, EGFR L858R/T790M/C797S triple mutant. ^dWT, wild type. ^eWT/CTL, wild type/EGFR L858R/T790M/C797S triple mutant.

It could be seen that compounds with methoxy groups (5 to 18) on the aromatic ring at quinazoline 2-position showed no inhibitory activities for the triple mutant L858R/T790M/C797S EGFR tyrosine kinase, no matter R¹ was CN or Br substituents. Some of compounds (20, 22, 24, 27 and 28) with hydroxyl group on the aromatic ring at quinazoline 2-position exhibited good inhibitory activity, perhaps due to that they have the ability to form hydrogen bonds with the amino acid residues in the ATP binding site (see “Theoretical Docking Analysis”). When R¹ was bromine, for aliphatic ring substitution of R², it was found that compound with morpholinyl (22, 1.50 µM) had strong inhibition while piperidinyl (23) and piperazinyl (26) had no inhibition. For aromatic ring substitution of R², compounds with phenyl (21) or pyridinyl (25) had no inhibition, while compounds of pyrimidinyl (20) and pyrazinyl (24) were active (0.44 and 0.69 µM, respectively). As can be seen, substituents R² had great influence on the inhibition of the triple mutant L858R/T790M/C797S EGFR tyrosine kinase. On the other hand, when R¹ was cyano, for aliphatic ring substitution of R², we found that compounds with morpholinyl (29), piperidinyl (30) and piperazinyl (31) had no inhibition. For aromatic substitution of R², compound with 1,3-diazole (27, 0.63 µM) exhibited higher activity than phenyl (28, 43.22 µM). Finally, all the active compounds showed high selectivity to the wild type kinases (200–10000 folds). In summary, an active compound 27 with high selectivity was obtained for further development.

Evaluation of Compounds against Various Cell Lines

Several compounds were further evaluated against human non-small cell lung cancer (NSCLC) H1975 (harboring EGFR T790M/L858R (TL) double mutations and L858R/T790M/C797S (CTL) triple mutations). The cellular activities were showed in Table 2. Compounds 20, 22, 24 and 27 showed good activities against all the cancer cell lines. Especially the most potent compound 27 inhibited the two cancer cell lines H1975 (TL) and H1975 (CTL) in one digital micromolar scale.

Table 2. Cell Viabilities of Representative Compounds

Compd.	Cancer cell lines (IC50, µM) a
	H1975 (TLb)	H1975 (CTLc)
14	ND	112.0
15	ND	28.4
20	20.3	12.6
22	25.6	27.4
24	20.0	10.1
27	3.3	1.2
28	>25	>25

^a All experiments were repeated at least three times. ^b TL, T790M/L858R double mutant. ^c CTL, EGFR L858R/T790M/C797S triple mutant.

Stabilities in Liver Microsomes of Various Species

The metabolic stability of the promising compound 27 was determined with various species of liver microsomes, such as human, mouse and rat liver microsomes (data were shown in Table 3), and the marketed Testosterone was selected as the standard. The half-life (t_1/2) and intrinsic clearance (CL_int) parameters were used to evaluate their metabolic stabilities that could give good indications of the in vivo hepatic clearance. The results revealed that compound 27 displayed good metabolic stabilities and all the half-lives of compound 27 in human, rat and mouse liver microsomes were more than 30 min, which indicated that this compound could be developed as an oral drug candidate in our further researches.

Table 3. Stabilities of Compound 27 in Liver Microsomes of Various Species

Liver microsomes	Compound 27		Testosterone
	t1/2 (min)	CLint (mL/min/kg)	t1/2 (min)	CLint (mL/min/kg)
Human	66.6	20.8	16.1	86.2
Mouse	35.4	39.2	3.1	451
Rat	34.1	40.6	0.4	3519

In Vivo Pharmacokinetic Parameters

With these encouraging in vitro data, candidate compound 27 was further investigated by profiling iv and ig pharmacokinetics in male Sprague-Dawley (SD) rats. The results were illustrated in Table 4. After oral administration in male SD rats, compound 27 showed short half life time (t_1/2: 0.9 h), partially because of its high systemic clearance (CL: 14700 mL/min/kg). Furthermore, the ig exposure of this compound showed moderate value (area under the curve (AUC)_0–inf: 353 h.ng/mL). It also showed a low oral bioavailability (18.6%). So our further work will focus on the improvement of bioavailability based on this compound.

Table 4. In Vivo Pharmacokinetic Parameters for Compound 27

Parameters	Compound 27
Dose (mg/kg)	1.0 (i.v.)	5.0 (i.g.)
t1/2 (h)	0.6	0.9
Tmax (h)	0.1	0.7
Cmax (ng/mL)	535	199
AUC0–t (h.ng/mL)	378	351
AUC0–inf (h.ng/mL)	379	353
Vz	2377	19685
CL (mL/min/kg)	2648	14700
MRT0–t (h)	0.7	1.4
F (%)	/	18.6%

Theoretical Docking Analysis

The most active compound 27 was docked into the pocket of EGFR L858R/T790M/C797S and the results were showed in Fig. 2. It indicated that the 4-aminomethyl pyrimidine moiety protruded into the hydrophobic pocket of EGFR, where the nitro atoms of substituted pyrimidine could readily interact with (within 2.5 Å) the sulfur atom of the Met790 residue of EGFR where they are ready for nucleophilic attack. On the other hand, the benzonitrile moiety could insert into the ATP-binding pocket, accompanied by the one π–alkyl interaction with Val 726. The results of docking analysis indicated that the potent compound 27 was accommodated in the ATP-binding site of the EGFR L858R/T790M/C797S mutant, which was contributed to the inhibitory activity. In addition, the quinazoline scaffold and 1,3-diazole fragments formed obvious non-bonded interactions (four p–π and one π–alkyl) with residues including Met765, Leu788, Val726, Asp855, Met790, Thr854, Lys745 and Ala743 of EGFR L858R/T790M/C797S. Briefly, the molecular docking predicted the inhibitory activity of compound 27 against EGFR L858R/T790M/C797S.

Fig. 2. Putative Binding Mode of Compound 27 within the ATP-Binding Pocket of EGFR L858R/T790M/C797S (PDB ID: 5ZWJ)

Experimental

Biological Assay

EGFR Kinase Inhibition Assay

The cell culture reagents RPMI1640, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were purchased from Invitrogen (Life Technologies, U.S.A.). The EGFR proteins were purchased from Carna Bioscience. The HTRF KinEASE-TK kit (Cat# 62TK0PEC) was from Cisbio. The Cell Titer-Glo Luminescent Cell Viability Assay kit (Cat# G7573) was purchased from Promega (U.S.A.). All the other chemicals were from Sigma. The assay and cell culture plates were purchased from Corning. The EGFR kinase assays were performed in 384-well plate (Corning 3676, low volume, black, NBS), using Cisbio HTRF KinEASE-TK kit.

IC₅₀ determinations for EGFR (C797S/T790M/L858R) (Invitrogen) were performed with the HTRF (Homogenous Time-Resolved Fluorescence) KinEASE-TK assay from Cisbio according to the manufacturer’s instructions. Compounds were screened at serial diluted concentration in the presence of 1% dimethyl sulfoxide (DMSO) with a 5 min pre-incubation of kinase and compounds. All reactions were started by the addition of ATP and TK-subtrate-biotin, incubated at room temperature for 60 min and quenched with the stop buffer containing 62.5 nM Strep-XL665 and TK antibody (Ab)-Cryptate. The plates were incubated for 1 h before being read on ClARIOstar Microplate Reader (BMG LABTECH) using standard HTRF settings. And IC₅₀ values were determined using the GraphPad Prism 5.0 software. Each reaction was performed in duplicate, and at least three independent determinations were made. The data was analyzed using Graphpad Prism.

Cellular Phosphorylation Assay

NCI-H1975 (human non-small cell lung cancer) and MDA-MB-231 (human breast cancer) were purchased from ATC C and taken as experimental cell line. All cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, U.S.A.), supplemented with 10% FBS (Invitrogen), cells were connected to 96-well plates at a density of 3000 cells/well using complete medium. A gradient-diluted compound was added to the cells to dilute 9 concentrations from a final concentration of 20 or 50 µM, 3 replicates per concentration. Cells were maintained at 37°C in humidified atmosphere of 5% CO₂, and exposed to compounds treatment for 72 h. Ten microliters of CCK-8 was added to the well and incubate at 37°C for 1–2 h. The absorbance of plate reader detected at 450 nM and the IC₅₀ values were calculated.

Compound Stability Test in Liver Microsome

Compounds were incubated with human, rat and mouse liver microsomes, and reactions were initiated by the addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in 0.05 M Phosphate buffer (pH = 7.4) at 37°C for 0–60 min. The reaction was quenched, and the amount of the remaining compound was analyzed using LC-MS/MS.

Determination of Pharmacokinetic Parameters in Rats

Male Sprague-Dawley rats (200–240 g) were administrated with the test compounds intravenously (i.v.) at 1 mg/kg and by oral gavage (p.o.) at 10 mg/kg. The compounds were dissolved in 2% DMSO, 20% polyethylene glycol (PEG) 400, and 78% buffered saline for intravenous tail-vein administration or a mixture of 0.5% methyl-cellulose for oral administration. Blood samples (0.2 mL) were then obtained via orbital sinus puncture at 2, 5, 15, 30 min, 1, 2, 4, 8, 12 and 24 h time points and collected into heparinized tubes. Heparinized blood samples collected for PK analyses were centrifuged at 3500 rpm for 15 min at 25°C. LC/MS/MS analysis was performed under optimized conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction monitoring mode (SRM). Plasma concentration-time data were analyzed by a non-compartment model using the software WinNonlin Enterprise, version 6.3 (Pharsight Co., Mountain View, CA, U.S.A.).

Docking Study

Structure of the compound was drawn with the chemdraw three-dimensional (3D) program, and the geometry was optimized using the MM2 method. All optimized ligands were saved with pdb format. Autodock was used to determine the molecules rotatable bonds and torsion angles, then saved with pdb.qt format for molecular docking studies after charge treatment. The protein crystal (PDB ID: 5ZWJ) was downloaded from the Protein Data Bank database as a receptor structure. Pymol and Autodock softwares were used to remove water molecules and added hydrogen, charge and force field of heteroatoms to optimize protein structure prior to docking. In addition, the active cavity was defined with the Gride Box module: the grid box centered on (19.119, 7.013, 9.282), the size was 18 × 18 × 18 and the grid point spacing is 1 Å. The prepared molecules were docked to the active site of the EGFR kinase using the AutoDock Vina molecular docking tool.

Synthesis of Intermediates and Products

Instruments

All reactions were monitored by TLC carried out using silica gel aluminum plates (60F-254) and spots were visualized with UV light or iodine. All reactions were performed in air or moisture site unless additional specification for water sensitive reactions. Melting points were determined on an YRT-3 melting point apparatus. ¹H-NMR and ¹³C-NMR spectra were recorded at room temperature on BRUKER Avance 300 or BRUKER Avance 400 spectrometers. Data of ¹H-NMR are reported as follows: chemical shift, multiplicity (s = singlet, br = broad, d = doublet, t = triplet, m = multiple), coupling constants and integration. High-resolution (HR) MS were obtained using Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS.

Materials

Unless otherwise stated, reagents and solvents were obtained from commercial suppliers and used without further purification. Absolutely dry solvents (CH₂Cl₂, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), etc.) were purchased from Aldrich packaged in Sure/Seal bottles. Yields mean chromatography detection unless otherwise stated. Compound E (BIBW2992) was provided by the laboratory preparing according to the literature method.

Chemistry

5-Cyano-2-methoxybenzaldehyde (2): 5-Bromo-2-methoxybenzaldehyde 1 (20 g, 93 mmol) was added to DMF (300 mL). copper (I) cyanide (16.7 g, 186 mmol) was added under nitrogen protection, and the resulting mixture was heated to 145°C for 14 h. The reaction mixture was cooled to room temperature and filtered through celite. The solution was poured into ice water (800 mL) with stirring, the solid is separately and the resulting residue was basified with saturated aqueous ammonia solution and extracted with ethyl acetate for three times. The organic layer was separated and dried using anhydrous sodium sulfate, concentrated to afford a yellow solid 10.0 g, yield 66.8%, mp 116–118°C; ¹H-NMR (400 MHz, CDCl₃) δ: 4.02 (s, 3H), 7.12 (d, J = 8.8 Hz, 1H), 7.77–7.85 (m, 1H), 8.06 (d, J = 2.4 Hz, 1H), 10.39 (s, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: 56.37, 104.61, 112.95, 118.02, 125.14, 132.80, 139.04, 164.21, 187.60; HRMS (electrospray ionization time-of-flight (ESITOF)) m/z: Calcd. for C₉H₇NO₂ [M + H]⁺ 162.0555, Found 162.0564.

2-(5-Bromo-2-methoxyphenyl)-4-hydroxyquinazoline (3a): Intermediate 1 (20.0 g, 93.0 mmol) was dissolved in ethanol (60 mL), 2-aminobenzamide (12.7 g, 93.0 mmol) was added followed by iodine (26.0 g, 102 mmol). The reaction mixture stirred at reflux for 4 h. The reaction mixture was cooled to room temperature and quenched by 5% sodium thiosulfate solution, and extracted with dichloromethane for three times, the organic layer was separated and dried using anhydrous sodium sulfate, concentrated in vacuum, and the crude product was recrystallized from ethyl acetate to afford white solid 21.6 g, yield 70.0%, mp 174–176°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.86 (s, 3H), 7.17 (d, J = 8.8 Hz, 1H), 7.53–7.56 (m, 1H), 7.69–7.74 (m, 2H), 7.82–7.86 (m, 2H), 8.16 (d, J = 7.6 Hz, 1H), 12.19 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 56.67, 112.13, 114.78, 121.60, 125.20, 126.26, 127.29, 127.945, 133.01, 134.94, 149.29, 151.37, 156.97, 161.65; HRMS (ESITOF) m/z: Calcd. for C₁₅H₁₁BrN₂O₂ [M + H]⁺ 331.0082, Found 331.0082.

3-(4-Hydroxyquinazolin-2-yl)-4-methoxybenzonitrile (3b): Intermediate 2 (4.4 g, 27.3 mmol) was dissolved in ethanol (60 mL), 2-aminobenzamide (3.8 g, 27.3 mmol) was added followed by iodine (7.6 g, 29.8 mmol). The reaction mixture stirred at reflux for 4 h. The reaction mixture was cooled to room temperature and quenched by 5% sodium thiosulfate solution, and extracted with dichloromethane for three times, the organic layer was separated and dried using anhydrous sodium sulfate, concentrated in vacuum, the crude product was recrystallized from ethyl acetate afforded white solid 4.3 g, yield 56.8%, mp 210–212°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.94 (s, 3H), 7.38 (d, J = 8.8 Hz, 1H), 7.54–7.56 (m, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.83–7.87 (m, 1H), 8.00–8.03 (m, 1H), 8.08 (d, J = 2.4 Hz, 1H), 8.16–8.18 (m, 1H), 12.34 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 57.00, 103.37, 113.63, 118.96, 121.68, 124.72, 126.29, 127.45, 127.95, 134.89, 136.81, 149.23, 151.10, 160.94, 161.69; HRMS (ESITOF) m/z: Calcd. for C₁₆H₁₁N₃O₂ [M + H]⁺ 278.0930, Found 278.0920.

2-(5-Bromo-2-methoxyphenyl)-4-chloroquinazoline (4a): Compound 3a (3.3 g, 10 mmol) was added to phosphorus oxychloride (10 mL), and then the reaction system was refluxed for 2 h, and then cooled to below 60°C. The phosphorus oxychloride was distilled under reduced pressure, and the mixture was cooled to room temperature. Finally, water (10 mL) was added, and the pH was adjusted to 6–7 with 5% saturated sodium carbonate solution, extracted with dichloromethane for three times. After drying over anhydrous sodium sulfate, purification by column chromatography gave white solid 3.2 g, yield 91.7%, mp 139–142°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.86 (s, 3H), 7.20 (d, J = 8.8 Hz, 1H), 7.57–7.61 (m, 1H), 7.73–7.78 (m, 2H), 7.83–7.84 (m, 2H), 7.86–7.88 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 56.75, 112.06, 114.88, 121.39, 126.43, 127.68, 133.13, 135.26, 135.37, 151.93, 157.00, 161.45; HRMS (ESITOF) m/z: Calcd. for C₁₅H₁₀BrClN₂O [M + H]⁺ 348.9743, Found 348.9751.

3-(4-Chloroquinazolin-2-yl)-4-methoxybenzonitrile (4b): Compound 4b was prepared from 3b according to similar procedure of 4a, gaving white solid, yield 76.5%, mp 210–213°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.91 (s, 3H), 7.42 (d, J = 8.8 Hz, 1H), 7.92–7.96 (m, 1H), 8.02–8.04 (m, 1H), 8.09 (d, J = 2.4 Hz, 1H), 8.14–8.20 (m, 2H), 8.34–8.35 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 57.12, 103.35, 113.77, 118.85, 121.46, 123.52, 126.45, 126.83, 127.82, 135.10, 135.29, 137.23, 147.65, 151.61, 160.99, 161.41; HRMS (ESITOF) m/z: Calcd. for C₁₆H₁₀ClN₃O₂ [M + H]⁺ 296.0591, Found 296.0590.

3-(4-(Ethylamino)quinazolin-2-yl)-4-methoxybenzonitrile (5): To a stirred mixture of compound 4a (0.30 g, 1 mmol) and ethylamine (0.07g, 1.2 mmol) in THF (5 mL), and N,N-diisopropylethylamine (DIPEA) (0.4 g, 3.0 mmol) was added dropwise, then stir at 45–60°C for 4 h. The organic layer was washed with EtOAc (3 mL). After drying over anhydrous sodium sulfate, purification by column chromatography to yield off white solid 250.6 mg, yield 82.4%, mp 248°C (dec); ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.23–1.27 (m, 3H), 3.54–3.59 (m, 2H), 3.87 (s, 3H), 7.32 (d, J = 8.4 Hz, 1H), 7.52–7.56 (m, 1H), 7.71–7.73 (m, 1H), 7.77–7.81 (m, 1H), 7.90–7.96 (m, 2H), 8.28 (d, J = 8.0 Hz, 1H), 8.47 (t, J = 5.2 Hz, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 14.64, 36.01, 56.62, 102.89, 113.69, 113.91, 119.47, 123.13, 126.24, 127.67, 131.43, 133.21, 134.71, 134.99, 149.38, 159.79, 160.04, 161.17; HRMS (ESITOF) m/z: Calcd. for C₁₈H₁₆N₄O [M + H]⁺ 305.1402, Found 305.1401.

Compounds 6–18 Were Prepared from 4a and 4b According to Similar Procedure of 5

3-(4-(Benzylamino) quinazolin-2-yl)-4-methoxybenzonitrile (6): Pale yellow solid, yield 94.5%, mp 187–190°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.80 (s, 3H), 4.78 (d, J = 6.0 Hz, 2H), 7.25–7.58 (m, 4H), 7.40–7.42 (m, 2H), 7.55–7.56 (m, 1H), 7.72–7.90 (m, 4H), 8.33 (d, J = 7.6 Hz, 1H), 8.95 (s, 1H, adding D₂O disappear); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 44.18, 56.54, 102.79, 113.67, 113.90, 119.45, 123.07, 126.36, 127.25,127.97, 128.19, 128.71, 131.54, 133.26, 134.86, 134.88, 140.12, 150.13, 159.87, 160.11, 161.18; HRMS (ESITOF) m/z: Calcd. for C₂₃H₁₈N₄O [M + H]⁺ 367.1559, Found 367.1555.

4-Methoxy-3-(4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl) benzonitrile (7): Pale yellow solid, yield 80.3%, mp 189–192°C (dec); ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.81 (s, 3H), 4.78(d, J = 5.6 Hz, 2H), 7.29–7.31 (m, 1H), 7.35–7.38 (m, 1H), 7.55–7.59 (m, 1H), 7.74–7.91 (m, 5H), 8.30 (d, J = 8.0 Hz, 1H), 8.47 (d, J = 0.8 Hz, 1H), 8.64 (s, 1H). 8.98–8.99 (m, 1H, adding D₂O disappear); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 41.99, 56.58, 102.85, 113.67, 113.87, 123.04, 123.89, 126.45, 128.23, 133.34, 134.78, 134.93, 135.54, 135.84, 148.57, 149.68, 159.80, 160.11, 161.19; HRMS (ESITOF) m/z: Calcd. for C₂₂H₁₇N₅O [M + H]⁺ 368.1511, Found 368.1507.

4-Methoxy-3-(4-((pyrimidin-2-ylmethyl)amino)quinazolin-2-yl)benzonitrile (8): Yellow solid, yield 79.9%, mp 169–171°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.72 (s, 3H), 4.94 (d, J = 5.6 Hz, 2H), 7.23 (d, J = 8.8 Hz, 1H), 7.38–7.41 (m, 1H), 7.57–7.61 (m, 2H), 7.73–7.76 (m, 1H), 7.80–7.86 (m, 2H), 8.38 (d, J = 7.6 Hz, 1H), 8.75–8.77 (m, 2H), 9.07 (t, J = 6.0 Hz, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 47.00, 56.55, 102.71, 113.73, 113.99, 119.39, 120.15, 123.33, 126.42, 128.11, 131.11, 133.31, 134.85, 134.96, 149.90, 153.82, 157.74, 159.61, 160.09, 161.19, 167.93; HRMS (ESITOF) m/z: Calcd. for C₂₁H₁₆N₆O [M + H]⁺ 369.1464, Found 369.1461.

4-Methoxy-3-(4-(piperazin-1-yl)quinazolin-2-yl)benzonitrile (9): Pale yellow solid, yield 38.6%, mp 166–168°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.22 (s, 1H), 2.96–2.99 (m, 4H), 3.72–3.75 (m, 4H), 3.87 (s, 3H), 7.32–7.34 (m, 1H), 7.54–7.60 (m, 1H), 7.83–7.86 (m, 2H), 7.91–7.93 (m, 1H), 8.02–8.04 (m, 2H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 45.51, 50.37, 56.68, 102.97, 113.82, 114.77, 119.43, 125.69, 126.15, 128.68, 130.94, 133.30, 134.98, 135.12, 152.31,158.73, 161.27, 164.17; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₉N₅O [M + H]⁺ 346.1668, Found 346.1662.

4-Methoxy-3-(4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)benzonitrile (10): Yellow solid, yield 67.8%, mp 198–201°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.75 (s, 3H), 4.89 (d, J = 5.6 Hz, 2H), 7.27 (d, J = 8.8 Hz, 1H), 7.57–7.61 (m, 1H), 7.75–7.78 (m, 2H), 7.80–7.89 (m, 2H), 8.35 (d, J = 8.0 Hz, 1H), 8.53–8.54 (m, 1H), 8.59–8.60 (m, 1H), 8.71 (d, J = 1.2 Hz, 1H), 9.10 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 45.51, 50.37, 56.68, 102.97, 113.82, 114.77, 119.43, 125.69, 126.15, 128.68, 130.94, 133.30, 134.98, 135.12, 152.31,158.73, 161.27, 164.17; HRMS (ESITOF) m/z: Calcd. for C₂₁H₁₆N₆O [M + H]⁺ 369.1464, Found 369.1464.

4-Methoxy-3-(4-((2-morpholinoethyl)amino)quinazolin-2-yl)benzonitrile (11): Yellow solid, yield 60.9%, mp 185–187°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.48–2.51 (m, 3H), 2.65 (m, 2 H), 3.55–3.58 (m, 4H), 3.65–3.70 (m, 3H), 3.87 (s, 3H), 7.31 (d, J = 8.4 Hz, 1H), 7.51–7.56 (m, 1H), 7.71–7.80 (m, 2H), 7.88–7.91 (m, 1H), 7.97 (d, J = 2.0 Hz, 1H), 8.24–8.26 (m, 1H), 8.33–8.35 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 29.90, 31.99, 53.80, 55.36, 56.62, 57.24, 66.58, 102.84, 113.68, 113.96, 119.47, 123.04, 126.18, 128.15, 131.71, 133.11, 134.80, 134.84, 150.04, 159.91, 160.14, 161.22; HRMS (ESITOF) m/z: Calcd. for C₂₂H₂₃N₅O₂ [M + H]⁺ 390.1930, Found 390.1928.

2-(5-Bromo-2-methoxyphenyl)-N-ethylquinazolin-4-amine (12): Pale yellow solid, yield 86.0%, mp 220–223°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.25 (t, J = 7.2 Hz, 3H), 3.56 (q, J = 7.2 Hz, 5.6, 2H), 3.77 (s, 3H), 7.10 (d, J = 8.8 Hz, 1H), 7.49–7.59 (m, 2H), 7.66–7.69 (m, 2H), 7.71–7.79 (m, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.34–8.35 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 14.69, 35.92, 56.56, 111.90, 113.91, 115.28, 123.02, 125.97, 128.03, 132.61, 132.98, 133.00, 133.20, 149.94, 157.13, 159.70, 160.68; HRMS (ESITOF) m/z: Calcd. for C₁₇H₁₆BrN₃O [M + H]⁺ 358.0555, Found 358.0551.

N-Benzyl-2-(5-bromo-2-methoxyphenyl) quinazolin-4-amine (13): Yellow solid, yield 88.6%, mp 188–190°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.67 (s, 3H), 4.72(d, J = 5.6 Hz, 2H), 7.04–7.06 (m, 1H), 7.21–7.37 (m, 5H), 7.51–7.55 (m, 3H), 7.73–7.81 (m, 2H), 8.21 (d, J = 8.4 Hz, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 44.16, 56.54, 111.90, 113.83, 115.34, 123.03, 126.19, 127.24, 127.99, 128.17, 128.71, 132.71, 133.17, 133.40, 140.15, 150.18, 157.19, 159.84, 160.52; HRMS (ESITOF) m/z: Calcd. for C₂₂H₁₈BrN₃O [M + H]⁺ 420.0711, Found 420.0710.

2-(5-Bromo-2-methoxyphenyl)-N-(pyridin-3-ylmethyl)quinazolin-4-amine (14): Pale yellow solid; yield 79.9%, mp 181–184°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.72 (s, 3H), 4.78 (d, J = 5.6, 2H), 7.08–7.10 (m, 1H), 7.34–7.37 (m, 1H), 7.53–7.57 (m, 3H), 7.73–7.82 (m, 3H), 8.28–8.30 (m, 1H), 8.46–8.47 (m, 1H), 8.65 (d, J = 1.6 Hz, 1H). 8.90 (t, J = 6.0 Hz, 1 H, adding D₂O disappear); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 41.95, 56.50, 111.88, 113.80, 115.22, 123.00, 123.89, 126.29, 128.23, 132.65, 132.75, 133.25, 133.35, 135.59, 135.84, 148.55, 149.66, 150.20, 157.16, 159.74, 160.52; HRMS (ESITOF) m/z: Calcd. for C₂₁H₁₇BrN₄O [M + H]⁺ 421.0664, Found 421.0666.

2-(5-Bromo-2-methoxyphenyl)-N-(pyrimidin-2-ylmethyl) quinazolin-4-amine (15): Yellow solid, yield 73.4%, mp 148–151°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.63 (s, 3H), 4.94 (d, J = 5.6 Hz, 2H), 7.03 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 2.4 Hz, 1H), 7.40 (t, J = 4.8 Hz, 1H), 7.50–7.53 (m, 1H), 7.57–7.61 (m, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.80–7.84 (m, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.76 (d, J = 4.8 Hz, 2H), 9.06–9.08 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 32.01, 46.99, 56.57, 111.85, 113.88, 115.39, 120.16, 123.23, 126.33, 127.90, 132.10, 132.86, 133.31, 133.45, 149.65, 157.16, 157.76, 159.91, 160.08, 167.87; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₆BrN₅O [M + H]⁺ 422.0616, Found 422.0613.

2-(5-Bromo-2-methoxyphenyl)-4-(piperazin-1-yl)quinazoline (16): Pale yellow solid, yield 69.8%, mp 123–125°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.92–2.94 (m, 4H), 3.68–3.71 (m, 4H), 3.78–3.79 (m, 4H), 7.12 (d, J = 8.8 Hz, 1H), 7.54–7.61 (m, 2H), 7.75 (d, J = 2.4 Hz, 1H), 7.81–7.83 (m, 2H), 8.02 (d, J = 8.4 Hz, 2H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 32.01, 45.88, 50.89, 56.66, 112.00, 114.71, 115.46, 125.67, 125.93, 128.67, 132.18, 132.97, 133.16, 133.40, 152.37, 157.26, 159.19, 164.16; HRMS (ESITOF) m/z: Calcd. for C₁₉H₁₉BrN₄O [M + H]⁺ 399.0820, Found 399.0820.

2-(5-Bromo-2-methoxyphenyl)-N-(pyrazin-2-ylmethyl) quinazolin-4-amine (17): Pale yellow solid, yield 82.3%, mp 179–182°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.66 (s, 3H), 4.89 (d, J = 5.6 Hz, 2H), 7.05 (d, J = 8.8 Hz, 1H), 7.50–7.59 (m, 3H), 7.74–7.80 (m, 1H), 7.81–7.83 (m, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.53–8.54 (m, 1H), 8.59–8.60 (m, 1H), 8.71 (d, J = 1.2 Hz, 1H), 9.04–9.07 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 44.52, 56.50, 111.86, 113.83, 115.21, 123.13, 126.38, 128.22, 132.47, 132.77, 133.31, 133.36, 143.55, 144.34, 150.13, 154.95, 157.13, 159.81, 160.32; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₆BrN₅O [M + H]⁺ 422.0616, Found 422.0611.

2-(5-Bromo-2-methoxyphenyl)-N-(2-morpholinoethyl) quinazolin-4-amine (18): Yellow solid, yield 51.3%, mp 136–139°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.46–2.51 (m, 4H), 2.61–2.65 (m, 2H), 3.55–3.57 (m, 4H), 3.64–3.69 (m, 2H), 3.78 (s, 3H), 7.10 (d, J = 8.8 Hz, 1H), 7.50–7.58 (m, 2H), 7.70–7.72 (m, 2H), 7.75–7.79 (m, 1H), 8.22–8.24 (m, 1H), 8.27–8.29 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 38.24, 53.88, 56.58, 57.32, 66.66, 111.88, 113.88, 115.27, 122.98, 126.03, 128.17, 132.66, 132.87, 133.03, 133.33, 150.10, 157.19, 159.85, 160.56; HRMS (ESITOF) m/z: Calcd. for C₂₁H₂₃BrN₄O₂ [M + H]⁺ 443.1083, Found 443.1077.

4-Bromo-2-(4-chloroquinazolin-2-yl)phenol (19a): 2-(5-Bromo-2-methoxyphenyl)-4-chloroquinazoline 4a (5.0 g, 14.3 mmol) was added to dichloromethane (25 mL) at 0–5°C, then boron tribromide (3.8 g, 15.2 mmol) was added dropwise, the mixture was stirred at room temperature for 12 h. After evaporating at reduced pressure, the organic phase was added to 15 mL water, and adjusted to pH 6–7 with saturated sodium carbonate solution. The aqueous phase was extracted with dichloromethane (20 mL × 3). The organic layer was combined, dried over anhydrous sodium sulfate, and then purified by column chromatography to give off-white solid 4.12 g, yield 85.8%, mp 219–221°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.05 (d, J = 8.4 Hz, 1H), 7.63–7.66 (m, 1H), 7.90–7.94 (m, 1H), 8.18–8.21 (m, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.51 (s, 1H), 13.24 (s, 1H); HRMS (ESITOF) m/z: Calcd. for C₁₄H₈BrClN₂O [M + H]⁺ 334.9587, Found 334.9588.

3-(4-Chloroquinazolin-2-yl)-4-hydroxybenzonitrile (19b): 3-(4-Chloroquinazolin-2-yl)-4-methoxybenzonitrile 4b (0.3 g, 1.0 mmol) was added to dichloromethane (15 mL) at 0–5°C, then boron tribromide (1.3 g, 5.0 mmol) was added dropwise, the mixture was stirred at room temperature for 7–8 h. After evaporating at reduced pressure, the organic phase was added to water (10 mL), and adjusted to pH 6–7 with saturated sodium carbonate solution. The aqueous phase was extracted with dichloromethane (15 mL × 3). The organic layer was combined, dried over anhydrous sodium sulfate, and then purified by column chromatography to give off-white solid with the yield of 76.0%, mp 161–164°C (dec). ¹H-NMR (400 MHz, DMSO-d₆) δ: 8.71 (d, J = 6.1 Hz, 1H), 8.31 (dt, J = 21.8, 10.8 Hz, 1H), 8.22–8.13 (m, 1H), 7.92 (d, J = 6.9 Hz, 1H), 7.84 (dd, J = 26.4, 8.8 Hz, 2H), 7.59 (t, J = 7.5 Hz, 1H), 7.18 (dd, J = 23.0, 8.7 Hz, 1H); HRMS (ESITOF) m/z: Calcd. for C₁₅H₈ClN₃O₂ [M + H]⁺ 282.0434, Found 282.0443.

Compounds 20–31 Were Prepared from 19a and 19b According to the Similar Procedure of Compound 5

4-Bromo-2-(4-((pyrimidin-2-ylmethyl)amino)quinazolin-2-yl) phenol (20): Yellow solid, yield 82.4%, mp 201–203°C (dec); ¹H-NMR (400 MHz, DMSO-d₆) δ: 5.00 (d, J = 5.6 Hz, 2H), 6.87 (d, J = 8.8 Hz, 1H), 7.44–7.47 (m, 1H), 7.61–7.64 (m, 1H), 7.80–7.89 (m, 2H), 8.24 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.53–8.54 (m, 1H), 8.63 (m, 1H), 8.78 (m, 1H), 9.50–9.52 (m, 1H), 14.66 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 45.17, 109.83, 114.07, 120.08, 121.23, 123.64, 126.65, 127.01, 131.42, 134.37, 135.27, 143.67, 144.00, 144.49, 146.84, 154.65, 159.70, 159.85, 160.35; HRMS (ESITOF) m/z: Calcd. for C₁₉H₁₄BrN₅O [M + H]⁺ 408.0460, Found 408.0466.

2-(4-(Benzylamino)quinazolin-2-yl)-4-bromophenol (21): Pale yellow solid, yield 89.7%, mp 208–210°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.87 (d, J = 5.6 Hz, 2H), 6.88 (d, J = 8.8 Hz, 1H), 7.26–7.27 (m, 1H), 7.34–7.37 (m, 2H), 7.46–7.49 (m, 3H), 7.57–7.61 (m, 1H), 7.78–7.85 (m, 2H), 8.37–8.44 (m, 2H), 9.38 (s, 1H), 14.74 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 44.90, 109.81, 114.06, 120.11, 121.40, 123.52, 126.67, 126.87, 127.44, 127.77, 128.90, 131.51, 134.24, 135.20, 139.54, 146.95, 159.57, 159.96, 160.41; HRMS (ESITOF) m/z: Calcd. for C₂₁H₁₆BrN₃O [M + H]⁺ 406.0555, Found 406.0552.

4-Bromo-2-(4-((2-morpholinoethyl) amino)quinazolin-2-yl)phenol (22): Yellow solid, yield 76.5%, mp 195–196°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.51–2.56 (m, 4H), 2.66–2.70 (m, 2H), 3.60–3.63 (m, 4H), 3.78–3.80 (m, 2H), 6.91 (d, J = 8.8 Hz, 1 H), 7.49–7.59 (m, 2 H), 7.77–7.86 (m, 2H), 8.27 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 2.8 Hz, 1H), 8.76–8.78 (m, 1H), 14.85 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 38.70, 53.98, 56.87, 66.71, 109.77, 114.07, 120.19, 121.47, 123.44, 126.64, 126.75, 131.37, 134.16, 135.20, 146.86, 159.65, 160.02, 160.52; HRMS (ESITOF) m/z: Calcd. for C₂₀H₂₁BrN₄O₂ [M + H]⁺ 429.0926, Found 429.0930.

4-Bromo-2-(4-(piperidin-3-ylamino)quinazolin-2-yl)phenol (23): Pale yellow solid, yield 45.2%, mp 170–172°C (dec); ¹H-NMR (400 MHz, DMSO-d₆) δ: 14.50 (s, 1H), 8.43 (s, 1H), 8.00 (s, 1H), 7.85 (s, 2H), 7.52 (d, J = 13.0 Hz, 2H), 6.91 (d, J = 7.7 Hz, 1H), 3.89 (s, 5H), 2.27 (s, 3H), 1.34 -1.14 (m, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 162.27, 160.28, 158.48, 149.29, 135.41, 134.21, 131.20, 127.03, 126.38, 121.13, 120.23, 120.21, 114.55, 109.97, 54.80, 49.44, 46.01; HRMS (ESITOF) m/z: Calcd. for C₁₉H₁₉BrN₄O [M + H]⁺ 399.0820, Found 399.0817.

4-Bromo-2-(4-((pyrazin-2-ylmethyl)amino)quinazolin-2-yl)phenol (24): Pale yellow solid, yield 79.4%, mp 193–195°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 5.00 (d, J = 5.2 Hz, 2H), 6.86 (d, J = 8.8 Hz, 1H), 7.46 (t, J = 2.4 Hz, 1H), 7.62 (t, J = 7.2, 7.6 Hz, 1H), 7.80–7.82 (m, 1H), 7.85–7.87 (m, 1H), 8.25 (d, J = 1.6 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.54 (s, 1H), 8.63 (s, 1H), 8.77 (s, 1H), 9.49 (s, 1H), 14.64 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 45.17, 109.83, 114.08, 120.07, 121.25, 123.64, 126.65, 127.00, 131.43, 134.36, 135.26, 143.67, 144.00, 144.48, 146.87, 154.63, 159.72, 159.86, 160.36; HRMS (ESITOF) m/z: Calcd. for C₁₉H₁₄BrN₅O [M + H]⁺ 408.0460, Found 408.0464.

4-Bromo-2-(4-((pyridin-3-ylmethyl)amino)quinazolin-2-yl)phenol (25): Pale yellow solid, yield 75.6%, mp 202–204°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.91 (d, J = 5.2 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 7.36–7.39 (m, 1H), 7.47–7.50 (m, 1H), 7.60 (t, J = 7.2, 7.6 Hz, 1H), 7.80–7.82 (m, 1H), 7.85–7.87 (m, 2H), 8.35–8.37 (m, 1H), 8.42–8.43 (m, 1H), 8.47–8.48 (m, 1H), 8.71 (s, 1H), 9.35–9.37 (m, 1H), 14.72 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 42.64, 109.84, 114.05, 120.15, 121.35, 123.54, 124.03, 126.67, 126.94, 131.43, 134.33, 134.98, 135.27, 135.61, 146.92, 148.76, 149.33, 159.61, 159.93, 160.43; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₅BrN₄O [M + H]⁺ 407.0507, Found 407.0515.

4-Bromo-2-(4-(4-methylpiperazin-1-yl)quinazolin-2-yl)phenol (26): Pale yellow solid, yield 61.1%, mp 163–165°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 14.51 (s, 1H), 8.43 (d, J = 2.8 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.91–7.77 (m, 2H), 7.59–7.52 (m, 1H), 7.50 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 3.88 (s, 4H), 2.57 (s, 4H), 2.27 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 163.44, 160.30, 158.49, 149.30, 135.41, 134.21, 131.19, 127.04, 126.38, 126.26, 121.14, 120.23, 114.54, 109.97, 54.80, 49.45, 46.01; HRMS (ESITOF) m/z: Calcd. for C₁₉H₁₉BrN₄O [M + H]⁺ 399.0820, Found 399.0820.

4-Hydroxy-3-(4-((pyrimidin-2-ylmethyl)amino) quinazolin-2-yl) benzonitrile (27): Yellow solid, yield 79.9%, mp 219–220°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 5.05 (d, J = 5.6, 2H), 7.02 (d, J = 8.4 Hz, 1H), 7.59–7.60 (m, 1H), 7.62–7.64 (m, 1H), 7.71–7.74 (m, 1H), 7.79–7.88 (m, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.51–8.55 (m, 2H), 8.61–8.62 (m, 1H), 8.81 (d, J = 1.2 Hz, 1H), 9.54 (t, J = 5.6 Hz, 1H), 15.72 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 45.07, 101.00, 114.14, 119.33, 119.70, 120.00, 123.68, 126.40, 127.21, 134.05, 134.46, 136.07, 143.73, 144.21, 144.40, 146.26, 154.63, 159.45, 159.77, 165.13; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₄N₆O [M + H]⁺ 355.1307, Found 355.1311.

4-Hydroxy-3-(4-((benzyl)amino)quinazolin-2-yl)benzonitrile (28): Yellow solid, yield 92.6%, mp 278–280°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.93 (d, J = 5.6 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.26–7.28 (m, 1H), 7.34–7.38 (m, 2H), 7.48 (d, J = 6.8 Hz, 2H), 7.60–7.64 (m, 1H), 7.74–7.77 (m, 1H), 7.81–7.87 (m, 2H), 8.39–8.41 (m, 1H), 8.66 (d, J = 2.0 Hz, 1H), 9.45 (m, 1H), 15.83 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 44.93, 100.98, 114.17, 119.41, 119.75, 120.17, 123.62, 126.44, 127.14, 127.46, 127.82, 128.87, 134.14, 134.42, 136.08, 139.50, 146.37, 159.60, 159.69, 165.25; HRMS (ESITOF) m/z: Calcd. for C₂₂H₁₆N₄O [M + H]⁺ 353.1402, Found 353.1405.

4-Hydroxy-3-(4-((2-morpholinoethyl)amino)quinazolin-2-yl)benzonitrile (29): Pale yellow solid, yield 78.4%, mp 206–208°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.68–2.70 (m, 4H), 2.71–2.74 (m, 2H), 3.60–3.62 (m, 4H), 3.80–3.84 (m, 2H), 7.08 (d, J = 8.8 Hz, 1H), 7.56–7.62 (m, 1H), 7.77–7.86 (m, 3H), 8.28 (d, J = 8.0 Hz, 1H), 8.76–8.8.77 (m, 1H), 8.81–8.84 (m, 1H), 15.94 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 53.94, 56.86, 66.66, 100.90, 114.16, 119.47, 119.81, 120.19, 123.53, 126.39, 127.01, 134.06, 134.31, 136.01, 146.24, 159.61, 159.70, 165.36; HRMS (ESITOF) m/z: Calcd. for C₂₁H₂₁N₅O₂ [M + H]⁺ 376.1773, Found 376.1770.

4-Hydroxy-3-(4-(piperidin-3-ylamino)quinazolin-2-yl)benzonitrile (30): Pale yellow solid, yield 44.7%, mp 143–146°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.58–1.63 (m, 1H), 1.72–1.79 (m, 1H), 1.95–1.98 (m, 1H), 2.06–2.07 (m, 1H), 3.27–3.31 (m, 2H), 3.43–3.51 (m, 3H), 4.22–4.25 (m, 1H), 4.42–4.45 (m, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.58–7.62 (m, 1H), 7.78–7.79 (m, 1H), 7.81–7.90 (m, 2H), 8.08 (d, J = 8.0 Hz, 1H), 8.75 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 23.33, 47.55, 50.36, 101.28, 114.71, 119.51, 119.79, 120.02, 126.55, 126.63, 126.78, 133.90, 134.44, 136.33, 148.83, 158.21, 163.79, 165.08; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₉N₅O [M + H]⁺ 346.1668, Found 346.1671.

4-Hydroxy-3-(4-(4-methylpiperazin-1-yl)quinazolin-2-yl)benzonitrile (31): Yellow solid, yield 58.8%, mp 185–187°C; ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.28 (s, 3H), 2.58 (s, 4H), 3.98 (s, 4H), 7.11 (d, J = 8.4 Hz, 1H), 7.57–7.61 (m, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.86–7.92 (m, 2H), 8.07 (d, J = 8.0 Hz, 1H), 8.74 (s, 1H), 15.57 (s, 1H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 30.31, 35.06, 46.02, 49.44, 54.90, 101.26, 114.56, 119.46, 119.79, 120.01, 126.42, 126.58, 126.82, 133.92, 134.36, 136.27, 148.87, 158.18, 163.34, 165.05; HRMS (ESITOF) m/z: Calcd. for C₂₀H₁₉N₅O [M + H]⁺ 346.1668, Found 346.1676.

Conclusion

In summary, a series of novel 2-aryl-4-amino substitute quinazoline derivatives as inhibitors against triple mutant L858R/T790M/C797S kinase were designed, synthesized and biologically evaluated. The enzymatically biological results indicated that compounds with 4-methoxybenzonitril and 1-bromo-4-methoxybenzene at 2-position of quinazoline ring exhibited no inhibition against EGFR L858R/T790M/C797S kinases, while 4-hydroxyl substituted compounds showed moderate to good inhibition against triple mutant L858R/T790M/C797S. All the active compounds showed excellent selectivity to the EGFR WT. Cellular evaluations with NSCLC H1975 harboring both EGFR L858R/T790M double mutations and L858R/T790M/C797S triple mutations indicated that the most active compound 27 could inhibit the proliferation of two cell lines in one digital micromolar scale. The microsomal stabilities of compound 27 against human, rat and mice species displayed good metabolic stabilities and could be developed as an oral candidate. However, the pharmacokinetic parameters of 27 in rat was moderate and the oral bioavailability was too low. So our future work will focus on the optimization of this compound to improve the bioavailability.

Acknowledgments

The present study was supported by Natural National Science Foundation of China (No.31470592).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials. Synthesis of the intermediates and products, ¹H-NMR, ¹³C-NMR and HRMS spectra of the target compounds 2–31 can be found at supplementary materials.

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