Synthesis and Evaluation of 2-Amine-4-oxyphosaniline Pyrimidine Derivatives as EGFR L858R/T790M/C797S Mutant Inhibitors

Abstract

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC₅₀ < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC₅₀ value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC₅₀ value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

Introduction

Lung cancer has become the leading cause of cancer death with more than 1.8 million mortalities per year,¹⁾ among which non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) accounts for about 80–90% and 10–20%, respectively.²⁾ Apart from surgical operation, chemotherapy is still the main approch for NSCLC therapy at present. However, traditional chemotherapies lack high selectivities between tumor cells and normal cells, which inevitability causes strong toxicity and side effects. Therefore, improving the accurate “targets”of chemotherapy drugs have significant influences on NSCLC clinical treatment and the quality of patients life.

Epidermal growth factor receptor (EGFR) plays a key role in cell signal transduction, proliferation, invasion and neovascularization, which was vadilated as the target for NSCLC therapy.³⁾ Currently, EGFR inhibitors for NSCLC treatment mainly include monoclonal antibody^4,5) and small molecule tyrosine kinase inhibitors (TKIs).⁶⁾ However, the disadvantages of poor stability, low therapeutic efficiency and large clinical doses restricted the effects of monoclonal antibodies in the clinical therapy. In contrast, TKIs showed excellent therapeutic efficiency with various scaffolds, and have gradually become the main chemotherapy drugs for the NSCLC.⁷⁾

To date, first and second-generation EGFR TKIs, represented by gefitinib⁸⁾ and afatinib⁹⁾ (Fig. 1) have been successfully employed to treat NSCLC patients with EGFR^L858R and EGFR^T790M mutant. However, afatinib is poorly selective for wild-type EGFR, resulting in unacceptable clinical toxicity. By further structure optimization, osimertinib (AZD9291) was discovered and developed¹⁰⁾ as the third generation EGFR inhibitor with acrylamide pyrimidine fragment, which has good selectivity and excellent inhibition to EGFR^L858R and EGFR^T790M double mutants. It was approved by USA Food and Drug Administration (FDA) in 2015 for patients with EGFR^T790M mutant following progression on previous EGFR TKI therapy. However, the tertiary mutant inevitably occurred after 10–16 months tretment of osimertinib,¹¹⁾ and the C797S mutation is the main resistance mechanism (Cysteine797 mutant to Serine797), accounting for about 20–40%, which prevented the corresponding covalent bond formation by Cys797 with Michael acceptor in third-generation EGFR-TKIs, ultimately resulted in the loss of activities.¹²⁾ Therefore, it is urgent to develop new EGFR inhibitors against C797S mutations.

Fig. 1. Chemical Structures of the Representative EGFR Inhibitors

Great efforts have been applied on developing new generation EGFR inhibitors overcoming the EGFR^C797S mutation. Reported structures mainly included thiazolamide derivaties (EAI045),^13,14) substituted imidazole derivaties,^15,16) pyrimidine derivaties (XO44),¹⁷⁾ substituted purine derivaties,^18,19) quinazoline derivaties (19, Fig. 1),^20,21) pyrazolopyrimidine derivaties,²²⁾ pyridopyrimidine derivaties,²³⁾ pyrimidopyrimidinone derivaties^24,25) and pyrimidopyridone derivaties.^26,27) However, allosteric inhibitor represented by EAI045 had week activity in vivo and had to be used with combination of cetuximab to strengthen the anti-proliferative inhibition¹⁴⁾; tri-substituted imidazole derivaties showed unsatisfied selectivities to EGFR^WT and lacked effective inhibition against Del19/T790M/C797S and del746-750/T790M/C797S mutants, therefore, the structures should be further optimized.¹⁵⁾ For pyrimidine derivatives, the activities in vivo were unsatisfied and the action mechanism were still unknown. Disappointingly, owing to inadequate clinical safety data, up to date, no clinical efficiacy EGFR^{L858R/T790M/C797S} inhibitor was available for NSCLC therapy.

Pyrimidine is the typical structure of many reported EGFR inhibitors. Brigatinib, an ALK inhibitor with pyrimidine skeleton combined with AZD9291 showed effective inhibition against EGFR^{L858R/T790M/C797S} tertiary mutation in vivo and in vitro. Docking studies indicated that brigatinib cut into the ATP-binding pocket of the L858R region without spatial squeeze on T790M or C797S.²⁸⁾ Two hydrogen bonds were formed between the pyrimidine and M793 residues backbone amide, and the phosphine oxide fragment completely occupied the triphosphate binding space in the ATP binding region, which significantly enhanced the electrostatic or Vander Waals force of all atoms in the group. Hydrophilic substituents for pyrimidine benefit the interaction of inhibitors and methionine at the kinase “gated position”,²⁹⁾ and the N atoms on the pyrimidine rings could form hydrogen bonds with Met793 and Thr854 at the kinase domains, which improved the inhibitory activity and selectivity.

Our team has discovered new pyrimidine derivaties as a third-generation EGFR inhibitors overcoming EGFR^L858R/T890M double mutant with nanomolar inhibition.³⁰⁾ Besides, a series of 2-aryl-4-aminoquinazoline derivatives were designed and synthesized by modifying the 4-amino-substituted fragment and 2-aryl-substituted fragment, several active compounds were obtained with moderate to excellent inhibitions and selectivities.³¹⁾ Therefore, in this manuscript, a series of new pyrimidine amine derivaties were designed and synthezied to discover new potent and selective EGFR inhibitors overcoming C797S resistance (Fig. 2), and the biological activity for inhibition of EGFR^{L858R/T790M/C797S} kinases and EGFR^WT were evaluated.

Fig. 2. The Design of Target Compounds

Results and Discussion

Synthesis of Target Compouds

The synthetic route is shown in Chart 1. Commercially available 2,4-dichloro-5-bromopyrimidine (1a) or 2,4-dichloro-5-methylpyrimidine (1b) condensed with 2-aminophenyldimethyl phosphine oxide in the presence of N,N-diisopropylethylamine or potassium carbonate respectively to give intermediates 2a and 2b in moderate yields (77–80%). Then, N-ethylaminomorpholine condensed with 2a and 2b respectively, and the target compounds 3a and 3b were obtained with the yield of 77 and 82% respectively.

Chart 1. Syhthesis of 2-Amine-4-oxyphosaniline Pyrimidine Compounds

Reagents and conditions: (a) 2-(Dimethylphosphinyl)benzenamine, DMF, N,N-Diisopropylethylamine, K₂CO₃, 80–85 °C, 78–80%; (b) 4-(2-Aminoethyl)morpholine, K₂CO₃, MeCN, 80 °C, 78–82%; (c) K₂CO₃, DMF, 60 °C, 88–92%; (d) Pd/C 5%, NH₂-NH₂ 80%, 55–60 °C, 78–89%; (e) K₂CO₃, MeCN, 80 °C, 38–44%.

1-Fluoro-5-methoxy-2-methyl-4-nitrobenzene (4a) or 5-fluoro-2-nitroanisole (4b) condensed with 4-morpholinopiperidine in the presence of potassium carbonate to give the intermediates 5a and 5b in satisfied yields (88–92%). Then, the key intermediates 6a and 6b were offorded by nitro reduction with hydrazine hydrate. Compounds 7a–7d were synthesized from commercially available 1-fluoro-5-methoxy-2-methyl-4-nitrobenzene (4a) or 5-fluoro-2-nitroanisole (4b). Compounds 2a and 2b reacted with 6a and 6b, the target compounds 7a–7d were prepared. The synthetic route is shown in Chart 1.

EGFR Kinase Activities Assay

The kinase inhibitory activities of the target compounds were evaluated and AZD9291 (osimertinib) was selected as the positive control. The EGFR tyrosine kinase assay results were listed in Table 1.

Table 1. The Inhibitory Activities of Target Compounds against Different Types of EGFR^a)

Compd.	R1	R2	IC50 (nMb))				Selectivity
			Lc)	TLd)	CTLe)	WTf)	(WT/TL)g)	(WT/CTL)h)
AZD9291	/	/	28.59 ± 0.78	23.16 ± 0.95	8138 ± 0.56	40.67 ± 0.76	1.76	0.005
3a	Br	/	ND	ND	>10 µM	ND	/	/
3b	CH3	/	ND	ND	>10 µM	ND	/	/
7a	Br	CH3	56.88 ± 0.99	32.70 ± 0.68	9.90 ± 0.54	152.20 ± 0.67	4.65	15.37
7b	CH3	CH3	13.06 ± 0.88	27.56 ± 0.56	12.11 ± 0.79	37.21 ± 0.90	1.35	3.07
7c	Br	H	46.18 ± 1.00	31.76 ± 0.92	23.53 ± 0.88	159.33 ± 0.79	5.02	6.77
7d	CH3	H	85.17 ± 0.66	210.72 ± 0.75	122.0 ± 0.93	309.0 ± 1.34	1.47	2.53

N.D.: Not determined. a) The values were means ± standard deviation (S.D.) from three independent experiments. b) IC₅₀ 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. c) L, L858R mutant. d) TL, EGFR L858R/T790M double mutant. e) CTL, EGFR L858R/T790M/C797S triple mutant. f) WT, wild type g) WT/TL, wild type/ EGFR L858R/T790M double mutant. h) WT/CTL, wild type/ EGFR L858R/T790M/C797S triple mutant.

It could be seen that compounds with ethylmorpholine (3a and 3b) at the pyrimidine ring 2-position showed no inhibitory activities for the all types of EGFR tyrosine kinase including L858R/T790M/C797S triple mutant, no matter whether R¹ was CH₃ or Br substituents. However, when the ethylmorpholine was replaced by arylamine group (7a–7d), all the compounds exhibited significant inhibitory activity against EGFR^L858R, EGFR^L858R/T790M secondary mutants and EGFR^{L858R/T790M/C797S} tertiary mutants, regardless of whether R¹ was CH₃ or Br group. The results suggested that arylamine substituent had great influence on the inhibition of triple mutant L858R/T790M/C797S EGFR tyrosine kinase. When R¹ was Br, it was found that the compound with CH₃ at R² (7a, 9.90 nM) had stronger inhibition than the compound with H at R² (7c, 23.53 nM), and both compounds showed good selectivies to wide types (> 6 folds). And when both R¹ and R² substitutents were CH₃, compound 7b (12.11 nM) displayed more potent inhibition than 7d (122.0 nM) with H substitution of R². Furthermore, compounds 7a, 7b and 7c inhibited EGFR^L858R and EGFR^L858R/T790M double mutant as significantly as EGFR^{L858R/T790M/C797S} tertiary mutants. All the designed compounds exhibited much better inhibitory activities against EGFR^{L858R/T790M/C797S} tertiary mutant than control compoud AZD9291. In summary, an active compound 7a with good selectivity was obtained for further development, and these results indicated that aryamide was an important group for maintaining the activity.

So far, four compounds were determined with effective inhibiton against EGFR^{L858R/T790M/C797S} triple mutant. Among them, the promising compound 7a displayed good inhibition and better selectivity. The properties comparison of compounds 7a, 7b, 7c, 7d and AZD9291 were summerized in Table 2.

Table 2. Properties of the Target Compounds and AZD9291

Compd.	MWa)	C Log Pb)	pKac)
AZD9291	500	4.60	9.56
7a	630	3.30	7.33
7b	565	2.90	7.33
7c	616	2.80	7.32
7d	551	2.40	7.32

a) Molecule weight (MW) was calculated from ChemBioDraw 20.0 and the digitals were rounded. b) C Log P was predicted by ChemBioDraw 20.0. c) pK_a was predicted by ChemBioDraw 20.0.

The data indicated that the predicted CLog P values were less than 4.0 and the predicated pK_a value were about 7.3 for all target compounds. According to the kinase inhibitory activities, compounds 7a and 7b displayed more potent inhibition against EGFR^{L858R/T790M/C797S} with larger CLog P and pK_a than compounds 7c and 7d. For MW, there was no consistence to the potencies. For the promising compound 7a, compared with AZD9291 (MW = 500, CLogV P = 4.6), 7a (MW = 630, CLog P = 3.3) displayed more potent enzyme inhibitory against EGFR^{L858R/T790M/C797S} and similar cellular activity to H1975(CTL) with larger MW and lower CLog P value.

Cellular Inhibition Activities of Compound 7a

In order to investigate the drugavailability, the promising compound 7a was selected for further test of antiproliferative activity against human non-small cell lung cancer (NSCLC) H1975 (harboring EGFR^T790M/L858R (TL) double mutations and L858R/T790M/C797S (CTL) triple mutations). AZD9291 was selected as positive control, the cellular activity data were showed in Table 3.

Table 3. Cell Viabilities of Compound 7a

Compd.	Cancer cell lines (IC50, nM)a)
	H1975 (TLb))	H1975 (CTLc))
AZD9291	589.9 ± 3.69	361.0 ± 4.88
7a	468.2 ± 1.22	330.5 ± 0.93

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

The results in Table 3 indicated that compound 7a displayed good inhibitory activities against H1975 (TL) and H1975 (CTL) cancer cell lines with the IC₅₀ values of 468.2 nM and 330.5 nM respectively. The control compound AZD9291 inhibited H1975(TL) and H1975(CTL) cell lines with the IC₅₀ values of 589.9 nM and 361.0 nM respectively. It could be seen that compound 7a showed slightly more potent inhibition than control compound AZD9291 against H1975(CTL). The cell proliferation inhibition results of compound 7a were consistent with the kinase activity against EGFR^{L858R/T790M/C797S} tertiary mutants.

Liver Microsomes Stability

The marketed Testosterone was selected as the standard, the experimental system was validated, then the metabolic stability of the promising compound 7a was determined with three species of liver microsomes, including human, mouse and rat liver microsomes, the datas were shown in Table 4.

Table 4. Stabilities of Compounds 7a in Liver Microsomes of Various Species

Liver microsomes	7a		Testosterone
	t1/2 (min)	CLint (mL/min/kg)	t1/2 (min)	CLint (mL/min/kg)
Human	>180	ND	15.8	84.5 ± 1.00
Mouse	71.0	19.7 ± 0.83	3.3	477 ± 0.99
Rat	56.4	24.8 ± 0.98	0.4	3498 ± 0.97

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 7a displayed good metabolic stabilities and all the half-lives in human, rat and mouse liver microsomes were more than 30 min. Especially, the half-life in human liver microsome were more than 180 min which showed that it almost was not metabolized by human liver microsome. These results indicated that compound 7a could be developed as an oral drug candidate in our further researches.

Theoretical Docking Analysis

To further explain the EGFR inhibitory activity of compound 7a, the protein crystal (PDB ID: 6LUD) was selected³²⁾ as template, the binding mode of the molecule with C797S mutant in the active site was predicted by the AutoDock Vina molecular docking tool. As depicted in Fig. 3, it was observed that compound 7a was favorably located into the receptor pocket. The nitro atom of aniline at the C-2 position of pyrimidine scaffold and the oxygen atom at 2-methoxy substituent formed two hydrogen bonds with Met793, respectively. The 2-aniline branch extended the solvent and the phenyl from pyrimide moiety was directed into the hydrophobic pocket of the ATP-binding domain. These could explain why compound 7a displayed potent inhibitory activity in enzyme and cell-based evaluation. In addition, the morpholin moiety could insert into the ATP-binding pocket, form an extra hydrogen bond with Lys745. The results of docking analysis indicated that the potent compound 7a was accommodated in the ATP-binding site of the EGFR L858R/T790M/C797S mutant, which contributed to the inhibitory activity.

Fig. 3. Putative Binding Mode of Compound 7a within the ATP-Binding Pocket of EGFR L858R/T790M/C797S (PDB ID: 6LUD)

Conclusion

In summary, based on the structure of marked ALK inhibitor Brigatinib, by modifying the pyrimidine scoffold at 4-amino and 2-aryl fragment, a series of novel 2-amine-4-aminoarylphosphoryloxy pyrimidine derivatives were designed, synthesized and biologically evaluated as potent and selective L858R/T790M/C797S mutant EGFR inhibitors. The enzymatically biological results indicated that compounds with ethylmorpholine showed no inhibition, while all the arylamine compounds showed significant inhibitions against EGFR^L858R, EGFR^L858R/T790M and EGFR^{L858R/T790M/C797S} mutant. The most promising compound 7a showed excellent enzymatic inhibitory activity against EGFR^{L858R/T790M/C797S} with IC₅₀ value of 9.9 nM, which was more potent than control compound AZD9291. Antiproliferation evaluation with NSCLC H1975 cell lines harboring both EGFR^L858R/T790M double mutations and EGFR^{L858R/T790M/C797S} triple mutations indicated that the most active compound 7a could inhibit the proliferation of two cell lines in less than 0.33 µM scale. Compared with Brigatinib, the promising compound 7a exhibited potent and selective inhibition against L858R/T790M/C797S mutant without conbination of AZD9291. Besides, the microsomal stabilities of compound 7a against human, rat and mice species displayed good metabolic stabilities. Nowadays, this compound was being developed as a drug candidate.

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). The EGFR proteins were purchased from Carna Bioscience. The HTRF KinEASE-TK kit (Cat#62TK0PEC) was obtained from Cisbio. The Cell Titer-Glo Luminescent Cell Viability Assay kit (Cat#G7573) was purchased from Promega. 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 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) was purchased from ATCC 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-tandem mass spectrometry (LC-MS/MS).

Docking Study

Chemical structure of the compound was drawn with the chemdraw 3D program, and the geometry was optimized by the MM2 method. All optimized ligands were saved with pdb format. The protein crystal (PDB ID: 6LUD) were downloaded from the Protein Data Bank database as the receptor structures. 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 molecule was docked to the active site of the EGFR kinase using the AutoDock Vina molecular docking tool.

Synthesis of Intermediates and Target Compouds

Instruments

Unless additional specification, all reactions were performed in air or moisture site. Reaction processes were monitored by TLC with silica gel aluminum plates (60F-254) and spots were visualized with UV light or iodine. Melting points were determined on an YRT-3 melting point instument. ¹H-NMR and ¹³C-NMR spectra were recorded at room temperature on BRUKER Avance 400 or 500 spectrometers with TMS as internal standard. 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 mass spectra were analyzed by Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS.

Materials

Unless otherwise required, all the reagents and solvents were commercially obtained with analytical grade and used without further purification. Frequently dry solvents (CH₂Cl₂, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), etc.) were anhydrous packaged from suppliers with Sure/Seal bottles. Yields mean chromatography detection unless otherwise stated.

Chemistry

2-((5-Bromo-2-chloropyrimidin-4-yl)amino)phenyl)dimethylphosphine Oxide (2a)

To a solution of 25 mL DMF and 2-(dimethylphosphinyl)benzenamine (1.50g, 8.9 mmol), 1a (2.01g, 8.9 mmol) and N,N-diisopropylethylamine (2.29g, 17.7 mmol) were added in small batches. Then, the mixture was stirred at 80–85 °C for 8 h. After completion of the reaction monitored by TLC, the solvent was evaporated in vacuo, and the residue was purified by column chromatography, title compound 2a (2.48 g, 78%) was obtained as grey solid. Melting point: 121–123 °C. ¹H-NMR (400 MHz, DMSO-d₆, ppm) δ: 1.79(s, 3 H), 1.82 (s, 3 H), 7.23–7.27 (m, 1 H), 7.59–7.67 (m, 2 H), 8.30–8.33 (m, 1 H), 8.52 (s, 1 H), 11.53 (s, 1 H). ¹³C-NMR (100 MHz, DMSO-d₆, ppm) δ: 159.12, 158.04, 157.85, 142.42 (d, J = 2 Hz), 132.57 (d, J = 2 Hz), 131.31 (d, J = 10 Hz), 124.27 (d, J = 11 Hz), 123.19, 122.54 (d, J = 7 Hz), 122.28, 105.08, 18.92, 18.21. HR–MS (electrospray ionization (ESI)): 359.9668 Calcd for C₁₂H₁₃BrClN₃PO [M + H] ⁺, Found: 359.9668.

2-((2-Chloro-5-methylpyrimidin-4-yl)amino)phenyl)dimethylphosphine Oxide (2b)

2-(Dimethylphosphinyl)benzenamine (1.50g, 8.9 mmol) and 2,4-dichloro-5-methylpyrimidine 1b (1.44g, 8.9 mmol) suspended in 20 mL DMF, and potassium carbonate (2.45g, 17.7 mmol) were added in portions slowly below 60 °C. Then, the mixture was stirred at 80–85 °C for 9 h. After completion of the reaction monitored by TLC, the solvent was evaporated in vacuo, and the residue was purified by column chromatography, title compound 2b was obtained (2.09 g, 80%) as off-white solid. Melting point: 147–149 °C. ¹H-NMR (400 MHz, DMSO-d₆, ppm) δ: 1.81(s, 3 H), 1.84 (s, 3 H), 2.17 (s, 3 H), 7.16–7.20 (m, 1 H), 7.58–7.65 (m, 2 H), 8.13 (d, J = 4.0 Hz, 2 H), 8.57–8.60 (m, 1 H), 11.41 (s, 1 H). ¹³C-NMR (100 MHz, DMSO-d₆, ppm) δ: 160.20, 157.00, 156.82, 143.79 (d, J = 3 Hz), 132.81 (d, J = 2 Hz), 131.45 (d, J = 11 Hz), 123.12 (d, J = 12 Hz), 121.33(t, J = 7 Hz), 120.43, 116.05, 19.14, 18.44, 13.64. HR–MS (ESI): 318.0539 Calcd for C₁₃H₁₆ClN₃PO [M + Na] ⁺, Found: 318.0553.

2-((5-Bromo-2-((2-morpholinoethyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine Oxide (3a)

To a solution of 10 mL acetonitrile, N-(2-aminoethyl)morpholine (0.219 g, 1.68 mmol) and 2a (0.499 g, 1.39 mmol), potassium carbonate (0.29g, 2.10 mmol) were added in portions slowly. Then, the mixture was stirred at 80 °C for 4 h. After completion of the reaction monitored by TLC, the solvent was evaporated in vacuo, and the residue was purified by column chromatography, title compound 3a (0.52 g, 82%) was obtained as grey solid. Melting point: 147–149 °C. ¹H-NMR (500 MHz, CDCl₃, ppm) δ: 8.64–8.55 (m, 1 H), 8.07 (s, 1 H), 7.49 (t, J = 7.9 Hz, 1 H), 7.31–7.22 (m, 1 H), 7.09 (t, J = 7.2 Hz, 1 H), 3.74 (s, 4 H), 3.56–3.43 (m, 2 H), 2.66 (s, 2 H), 2.56 (s, 4 H), 1.83 (s, 3 H), 1.80 (s, 3 H). ¹³C-NMR (125 MHz, CDCl₃, ppm) δ: 160.26, 157.69, 156.75, 143.96, 132.27, 129.61, 129.52, 122.65, 122.59, 122.44, 122.35, 66.54, 57.30, 53.35, 37.91, 18.78, 18.21. HR–MS (ESI): 454.1007 Calcd for C₁₈H₂₆BrN₅O₂P [M + H]⁺, Found: 454.1003.

Dimthyl(2-((5-methyl-2-((2-morpholinoethyl)amino)pyrimidin-4-yl)amino)phenyl)phosphine Oxide (3b)

By the same method, compound 2b and N-(2-aminoethyl)morpholine as starting materials, 3b was obtained as yellow solid with the yield of 78%, melting point: 61–63 °C. ¹H-NMR (500 MHz, CDCl₃, ppm) δ: 8.75 (dd, J = 8.3, 4.3 Hz, 1 H), 7.62 (s, 1 H), 7.52 (t, J = 7.9 Hz, 1 H), 7.28 (t, J = 7.6 Hz, 1 H), 7.13 (t, J = 7.2 Hz, 1 H), 3.70 (s, 4 H), 3.52 (d, J = 4.7 Hz, 2 H), 2.61 (t, J = 6.0 Hz, 2 H), 2.49 (s, 4 H), 2.15 (s, 3 H), 1.84 (s, 3 H), 1.82 (s, 3 H). ¹³C-NMR (125 MHz, CDCl₃, ppm) δ: 160.18, 143.98, 132.62, 132.61, 129.86, 129.77, 122.46, 122.41, 106.65, 66.85, 53.42, 38.34, 19.07, 18.50, 13.53. HR–MS (ESI): 390.2059 Calcd for C₁₉H₂₉N₅O₂P [M + H]⁺, Found: 390.2059.

4-(1-(5-Methoxy-2-methyl-4-nitrophenyl)piperidin-4-yl)morpholine (5a)

To a suspension of 30 mL DMF, 4-morpholinopiperidine (2.996 g, 17.6 mmol) and 4a (3.257 g, 17.6 mmol), potassium carbonate (4.526 g, 35.3 mmol) were added in portions slowly. Then, the mixture was stirred at 80 °C for 6 h. After completion of the reaction monitored by TLC, the solvent was evaporated in vacuo. Then, 20 mL water was added and the mixture was extracted with dichloromethane (20 mL × 3). Combined the organic phases, dried with anhydrous sodium sulfate, and the crude paroduct was recrystallized from 60 mL mixture of ethyl acetate/methanol (5/1), title compound 5a (5.193 g, 88%) was obtained as light yellow solid, meltingpoint: 133–135 °C. ¹H-NMR (400 MHz, DMSO-d₆, ppm) δ: 1.50–1.61 (m, 2 H), δ 1.88–1.91 (m, 2 H), 2.20–2.24 (m, 3 H), 2.28–2.34 (m, 1 H), 2.49–2.51 (m, 2 H), 2.70–2.75 (m, 2 H), 3.31–3.35 (m, 4 H), 3.56–3.60 (m, 4 H), 3.91 (s, 3 H), 6.69 (s, 1 H), 7.76 (s, 1 H). ¹³C-NMR (100 MHz, DMSO-d₆, ppm) δ: 158.33 153.15, 132.43, 128.51, 123.05, 104.18, 67.04, 61.37, 56.86, 50.61, 49.95, 49.71, 28.65, 17.75. HR–MS (ESI): 336.1923, Calcd for C₁₇H₂₆N₃O₄ [M + H]⁺, Found: 336.1924.

4-(1-(3-Methoxy-4-nitrophenyl)piperidin-4-yl)morpholine (5b)

To a suspension of 30 mL DMF, 4-morpholinopiperidine (2.996 g, 17.6 mmol) and 4b (3.010 g, 17.6 mmol), potassium carbonate (4.526 g, 35.3 mmol) were added in portions slowly. Then, the mixture was stirred at 60 °C for 4 h. After completion of the reaction monitored by TLC, the solvent was evaporated in vacuo. Then, 20 mL water was added and the mixture was extracted with dichloromethane (20 mL × 3). Combined the organic phases, dried with anhydrous sodium sulfate, and the crude paroduct was recrystallized from 60 mL mixture of ethyl acetate/methanol (5/1), title compound 5b (5.172 g, 92%) was obtained as light yellow solid, meltingpoint > 230 °C. ¹H-NMR (400 MHz, DMSO-d₆, ppm) δ: 1.36–1.46 (m, 2 H), 1.84–1.87 (m, 2 H), 2.41 (t, J = 3.8 Hz, 1 H), 2.45 (t, J = 4.7 Hz, 4 H), 2.92–2.98 (m, 2 H), 3.56 (t, J = 4.4 Hz, 4 H), 3.90 (s, 3 H), 4.02 (d, J = 13.2 Hz, 2 H), 6.48 (d, J = 2.6, 1 H), 6.57 (dd, J = 2.5, 9.5 Hz, 1 H), 7.87 (d, J = 9.4 Hz, 1 H). ¹³C-NMR (100 MHz, DMSO-d₆, ppm) δ: 156.65, 155.55, 128.79, 128.03, 105.74, 97.15, 66.97, 61.10, 56.68, 49.86, 46.42, 27.78. HR–MS (ESI): 322.1767 Calcd for C₁₆H₂₄N₃O₄ [M + H]⁺, Found: 322.1772.

2-Methoxy-5-methyl-4-(4-morpholinpiperidin-1-yl)aniline (6a)

Intermediate 5a (3.02 g, 9.0 mmol) was dissolved in 25 mL ethanol, 5% Pd-C (0.30 g, 60% water) was added to the solution, the temperature was raised to 55–60 °C under rapid stirring, then, 80% hydrazine hydrate (2.25 g, 36.0 mmol) was added dropwise. After completion of dropping, the reaction was incubated for 4 h, and the reaction process was monitored by TLC. Pd-C was filtred while the mixture hot, the filtration was evaporated in vacuo, title compound 6a (2.135 g, 78%) was obtained as purple solid, meltingpoint > 220 °C. ¹H-NMR (400 MHz, DMSO-d₆, ppm) δ: 1.96–2.02 (m, 2 H), δ 2.20–2.25 (m, 5 H), 2.73–2.79 (m, 2 H), 3.09–3.14 (m, 2 H), 3.24–3.31 (m, 3 H), 3.43–3.46 (m, 2 H), 3.86–3.87 (m, 3 H), 3.96–3.98 (m, 4 H), 4.72 (s, 2 H), 6.86 (s, 1 H), 7.26 (s, 1 H). ¹³C-NMR (100 MHz, DMSO-d₆, ppm) δ: 151.30, 126.24, 124.27, 104.80, 63.66, 62.64, 56.69, 50.73, 48.68, 43.00, 26.31, 17.27. HR–MS (ESI): 306.2182, Calcd for C₁₇H₂₈N₃O₂ [M + H]⁺, Found: 306.2178.

2-Methoxy-4-(4-morpholinpiperidin-1-yl)aniline (6b)

Intermediate 5b (2.99g, 9.3 mmol) was dissolved in 25 mL ethanol, 5% Pd-C (0.30 g, 60% water) was added to the solution, the temperature was raised to 55–60 °C under rapid stirring, then, 80% hydrazine hydrate (2.33g, 37.3 mmol) was added dropwise. After completion of dropping, the reaction was incubated for 4 h, and the reaction process was monitored by TLC. Pd-C was filtred while the mixture hot, the filtration was evaporated in vacuo, title compound 6b (2.421 g, 89%) was obtained as brown solid, meltingpoint > 230 °C. ¹H-NMR (400 MHz, CDCl₃, ppm) δ: 6.65 (d, J = 8.3 Hz, 1 H), 6.54 (d, J = 1.6 Hz, 1 H), 6.42–6.45 (m, 1 H), 3.85 (s, 3 H), 3.77 (t, J = 4.6 Hz, 4 H), 3.54 (d, J = 11.8 Hz, 2 H), 2.61–2.63 (m, 6 H), 2.29–2.37 (m, 1 H), 1.95 (d, J = 12.3 Hz, 2 H), 1.67–1.77 (m, 2 H). ¹³C-NMR (100 MHz, CDCl₃, ppm) δ: 147.97, 145.21, 130.08, 115.43, 109.80, 102.86, 67.27, 62.11, 55.47, 51.44, 49.75, 28.32. HR–MS (ESI) : 291.1947 Calcd for C₁₆H₂₆N₃O₂ [M + H]⁺, Found: 291.1924.

2-((5-Bromo-2-((2-methoxy-5-methyl-4-(4-morpholinopiperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl) Dimethyl Phosphine Oxide (7a)

Compounds 6a and 2a as starting materials, the title compound was prepared according to the synthetic procedures for 3a as pale solid with the yield of 39%, m.p: 214–216 °C. ¹H-NMR (400 MHz, CDCl₃, ppm) δ: 8.54 (dd, J = 8.4, 4.4 Hz, 1 H), 8.22 (s, 1 H), 8.05 (s, 1 H), 7.53 (t, J = 7.6 Hz, 1 H), 7.34 (t, J = 6.8 Hz, 1 H), 7.16 (t, J = 7.4 Hz, 1 H), 6.63 (s, 1 H), 3.87 (s, 3 H), 3.81–3.78 (m, 4 H), 3.17 (d, J = 11.7 Hz, 2 H), 2.65–2.63 (m, 5 H), 2.18 (s, 3 H), 1.98 (d, J = 11.5 Hz, 3 H), 1.88 (s, 3 H), 1.85(s, 3 H), 1.66–1.76 (m, 3 H). ¹³C-NMR (100 MHz, CDCl₃, ppm) δ: 158.06, 157.96 (d, J = 6.9 Hz), 156.68, 146.81, 146.26, 143.77 (d, J = 2.6 Hz), 132.63, 129.56 (d, J = 10.7 Hz), 124.4, 124.42, 124.18, 123.14, 121.59 (d, J = 5.2 Hz), 121.16, 120.21, 102.21(d, J = 9.4 Hz), 94.72, 67.35, 62.17, 55.88 (d, J = 13.8 Hz), 52.04 (d, J = 3.1 Hz), 49.98, 29.07, 18.81, 18.10, 17.27 (d, J = 6.0 Hz). HR–MS (ESI): 651.1824 Calcd for C₂₉H₃₈NaBrN₆O₃P [M + Na]⁺, Found: 651.1819.

2-((2-((2-Methoxy-5-methyl-4-(4-morpholinopiperidin-1-yl)phenyl)amino)-5-methylpyrimidin-4-yl) Amino)phenyl)dimethylphosphine Oxide (7b)

Compounds 6a and 2b as starting materials, the title compound 7b was prepared according to the synthetic procedures for 3a as dark yellow solid with the yield of 44%, meltingpoin: 219–221 °C. ¹H-NMR (400 MHz, CD₃OD, ppm) δ: 8.47 (dd, J = 8.4, 4.5 Hz, 1 H), 7.87 (s, 1 H), 7.80 (s, 1 H), 7.62 (m, 1 H), 7.53 (t, J = 7.9 Hz, 1 H), 7.24–7.26 (m, 1 H), 6.74 (s, 1 H), 5.52 (s, 1 H), 3.88 (s, 3 H), 3.77 (t, J = 4.7 Hz, 4 H), 3.16 (d, J = 11.7 Hz, 2 H), 2.69 (s, 5 H), 2.36 (t, J = 11.3 Hz, 1 H), 2.18 (s, 3 H), 2.14 (s, 3 H), 2.05 (d, J = 11.4 Hz, 2 H), 1.90 (s, 3 H), 1.86 (s, 3 H), 1.70 (dd, J = 11.8, 3.8 Hz, 1 H). ¹³C-NMR (100 MHz, CD₃OD, ppm) δ: 159.69, 158.38, 154.75, 147.78, 146.55, 143.62, 137.73, 132.69, 130.41, 124.16, 123.95, 122.62, 120.53, 109.03, 106.29, 102.25, 66.37, 62.30, 55.05, 53.41, 51.72, 49.71, 28.56, 16.89, 16.18, 12.23, 10.70. HR–MS (ESI): 565.3056 Calcd for C₃₀H₄₂N₆O₃P [M + H]⁺, Found: 565.3053.

2-((5-Bromo-2-((2-methoxy-4-(4-morpholinopiperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)di Methyl Phosphine Oxide (7c)

Compounds 6b and 2a as starting materials, the title compound 7c was prepared according to the synthetic procedures for 3a as dark yellow solid with the yield of 41%, meltingpoint: 95–98 °C. ¹H-NMR (400 MHz, CDCl₃, ppm) δ: 8.55 (dd, J = 8.4, 4.4 Hz, 1 H), 8.21 (s, 1 H), 8.11 (d, J = 8.8 Hz, 1 H), 7.53 (t, J = 7.9 Hz, 1 H), 7.38–7.30 (m, 1 H), 7.16 (t, J = 8.2 Hz, 1 H), 6.58 (d, J = 2.4 Hz, 1 H), 6.50 (dd, J = 8.8, 2.4 Hz, 1 H), 3.89 (s, 3 H), 3.82–3.73 (m, 4 H), 3.67 (d, J = 12.3 Hz, 2 H), 2.73 (t, J = 11.2 Hz, 2 H), 2.66–2.58 (m, 4 H), 1.99 (d, J = 12.1 Hz, 2 H), 1.88 (s, 3 H), 1.84 (s, 3 H), 1.73 (dd, J = 11.8, 3.4 Hz, 2 H). ¹³C-NMR (100 MHz, CDCl₃, ppm) δ: 158.15, 157.93, 156.64, 149.20, 147.43, 143.76 (d, J = 2.8 Hz), 132.43–132.10 (m), 129.44 (d, J = 10.8 Hz), 123.51 (d, J = 7.1 Hz), 122.59 (d, J = 12.1 Hz), 122.10, 121.16, 120.43, 120.20, 108.39, 101.03, 94.57, 67.32, 62.03, 55.61 (d, J = 6.1 Hz), 50.41, 49.78, 28.27 (d, J = 14.7 Hz), 18.80, 18.08. HR–MS (ESI): 615.1848 Calcd for C₂₈H₃₇BrN₆O₃P [M + H]⁺, Found: 615.1836.

2-((2-((2-(2-Methoxy-4-(4-morpholinopiperidin-1-yl)phenyl)amino)-5-methylpyrimidin-4-yl)amino) Phenyl) Dimethyl Phosphine Oxide (7d)

Compounds 6b and 2b as starting materials, the title compound 7d was prepared according to the synthetic procedures for 3a as light yellow solid with the yield of 38%, meltingpoint: 72–74 °C. ¹H-NMR (400 MHz, CDCl₃, ppm) δ: 10.39 (s, 1 H), 8.76 (dd, J = 8.5, 4.5 Hz, 1 H), 7.93 (s, 1 H), 7.44–7.58 (m, 2 H), 7.29 (s, 1 H), 7.07–7.11 (m, 1 H), 6.59 (d, J = 2.5 Hz, 1 H), 6.54 (dd, J = 8.8, 2.6 Hz, 1 H), 3.89 (s, 3 H), 3.79 (t, J = 9.3 Hz, 4 H), 3.67 (d, J = 11.9 Hz, 2 H), 2.73 (t, J = 11.4 Hz, 2 H), 2.64 (t, J = 4.6 Hz, 2 H), 2.34–2.40 (m, 1 H), 2.21 (s, 3 H), 1.99 (d, J = 11.7 Hz, 2 H), 1.87 (s, 3 H), 1.83 (s, 3 H), 1.69–1.79 (m, 2 H). ¹³C-NMR (100 MHz, CDCl₃, ppm) δ: 159.20, 158.36, 157.88, 155.83, 155.69, 149.13, 146.97, 145.60, 144.99, 132.85, 132.53, 129.50, 108.61, 108.47, 107.06, 101.36, 67.28, 62.13, 55.61, 50.66, 49.76, 31.96, 29.73, 28.20, 22.73, 18.98, 18.27, 13.64. HR–MS (ESI): 551.2900 Calcd for C₂₉H₄₀N₆O₃P [M + H]⁺, Found: 551.2890.

Acknowledgments

This project was supported in part by National Natural Science Foundation of China (81873134), the “Qinglan Project” of Young and Middle-aged Academic Leader of Jiangsu College (2020-2023).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

References

• 1) Sung H., Ferlay J., Siegel R. L., Laversanne M., Soerjomataram I., Jemal A., Bray F., CA Cancer J. Clin., 71, 209–249 (2021).
• 2) Kohno T., Tsuta K., Tsuchihara K., Nakaoku T., Yoh K., Goto K., Cancer Sci., 104, 1396–1400 (2013).
• 3) Lemmon M., Schlessinger J., Cell, 141, 1117–1134 (2010).
• 4) Frampton J. E., Drugs, 70, 1987–2010 (2010).
• 5) Maurizio S., Federico R., Alberto O., Judit A., Marta G., Javier C., Serena D., Xavier M. G., Santiago R. C., Joaquin A., José B., J Natl Cancer, 99, 628–638 (2007).
• 6) Patel H., Pawara R., Ansari A., Surana S., Eur. J. Med. Chem., 142, 32–47 (2017).
• 7) Yang G., Yao Y., Zhou J., Zhao Q., Oncol. Rep., 27, 2066–2072 (2012).
• 8) Mok T. S., Wu Y. L., Thongprasert S., et al., N. Engl. J. Med., 36, 1947–1957 (2009)
• 9) Li D., Ambrogio L., Shimamura T., Kubo S., Takahashi M., Chirieac L. R., Padera R. F., Shapiro G. I., Baum A., Himmelsbach F., Rettig W. J., Meyerson M., Solca F., Greulich H., Wong K. K., Oncogene, 27, 4702–4711 (2008).
• 10) Cross D., Ashton S., Ghiorghiu S., et al., Cancer Dis, 4, 1046–1061 (2014).
• 11) Niederst M. J., Hu H., Mulvey H. E., Lockerman E. L., Garcia A. R., Piotrowska Z., Sequist L. V., Engelman J. A., Clin. Cancer Res., 21, 3924–3933 (2015).
• 12) Lu X., Yu L., Zhang Z., Ren X., Smaill J. B., Ding K., Med. Res. Rev., 38, 1550–1581 (2018).
• 13) Jia Y., Yun C., Park E. Y., et al., Nature (London), 534, 129–132 (2016).
• 14) Wang S., Song Y., Liu D., Cancer Lett., 385, 51–54 (2017).
• 15) Günther M., Juchum M., Kelter G., Fiebig H., Laufer S., Angew. Chem. Int. Ed. Engl., 55, 10890–10894 (2016).
• 16) Günther M., Lategahn J., Juchum M., Döring E., Keul M., Engel J., Tumbrink H. L., Rauh D., Laufer S., J. Med. Chem., 60, 5613–5637 (2017).
• 17) Francesca F., Laura S., Federica V., Eur. J. Med. Chem., 225, 113786 (2021).
• 18) Zhou W., Liu X., Tu Z., Zhang L., Ku X., Bai F., Zhao Z. J., Xu Y. F., Ding K., Li H. L., J. Med. Chem., 56, 7821–7837 (2013).
• 19) Lei H., Fan S., Zhang H., Liu Y. J., Hei Y. Y., Zhang J. J., Zheng A. Q., Xin M. H., Zhang S. Q., Eur. J. Med. Chem., 186, 111888 (2019).
• 20) Park H., Jung H. Y., Mah S., Hong S., Angew. Chem. Int. Ed., 56, 7634–7638 (2017).
• 21) Li Q., Zhang T., Li S., Tong L. J., Li J., Su Z. C., Feng F., Sun D., Tong Y., Wang X., Zhao Z. J., Zhu L. L., Ding J., Li H. L., Xie H., Xu Y. F., Chem. Lett., 10, 869–873 (2019).
• 22) Engel J., Becker C., Lategahn J., Keul M., Ketzer J., Mühlenberg T., Kollipara L., Schultz-Fademrecht C., Zahedi R. P., Bauer S., Rauh D., Angew. Chem. Int. Ed., 55, 10909–10912 (2016).
• 23) Hao Y. J., Wang X., Zhang T., Sun D., Tong Y., Xu Y. Q., Chen H. Y., Tong L. J., Zhu L. L., Zhao Z. J., Chen Z., Ding J., Xie H., Xu Y. F., Li H. L., J. Med. Chem., 59, 7111–7124 (2016).
• 24) Lu X., Zhang T., Zhu S. J., Xun Q. J., Tong L. J., Hu X. L., Li Y., Chan S., Su Y., Sun Y. M., Chen Y., Ding J., Yun C. H., Xie H., Ding K., Chem. Lett., 9, 1123–1127 (2018).
• 25) Xun Q., Zhang Z., Luo J., Tong L. J., Huang M. H., Wang Z., Zou J., Liu Y. Q., Xu Y., Xie H., Tu Z. C., Lu X. Y., Ding K., J. Med. Chem., 61, 2353–2371 (2018).
• 26) Yu L., Huang M. H., Xu T. F., Tong L. J., Yan X. E., Zhang Z., Xu Y., Yun C. H., Xie H., Ding K., Lu X. Y., Eur. J. Med. Chem., 126, 1107–1117 (2016).
• 27) Shen J., Zhang T., Zhu S. J., Sun M., Tong L. J., Lai M. Z., Zhang R., Xu W., Wu R. B., Ding J., Yun C. H., Xie H., Lu X. Y., Ding K., J. Med. Chem., 62, 7302–7308 (2019).
• 28) Uchibori K., Inase N., Araki M., Kamada M., Sato S., Okuno Y., Fujita N., Katayama R., Nat. Commun., 8, 14768–14768 (2017).
• 29) Pettinger J., Jones K., Cheeseman M. D., Angew. Chem. Int. Ed. Engl., 56, 15200–15209 (2017).
• 30) Zhang H. Y., Wu W. K., Feng C., Liu Z. G., Bai E. H., Wang X. Y., Lei M., Cheng H., Feng H. Y., Shi J. M., Wang J., Zhang Z., Jin T., Chen S. S., Hu S. H., Zhu Y. Q., Eur. J. Med. Chem., 135, 12–23 (2017).
• 31) Zhang M. G., Wang Y. Y., Wang J., Liu Z. G., Shi J. M., Li M. X., Zhu Y. Q., Wang S. F., Chem. Pharm. Bull., 68, 971–980 (2020).
• 32) Kashima K., Kawauchi H., Tanimura H., Tachibana Y., Chiba T., Torizawa T., Sakamoto H., Mol. Cancer Ther., 19, 2288–2297 (2020).