CN115073439A - Pyrimidine compound and medical application thereof - Google Patents

Abstract

The invention relates to a pyrimidine compound and medical application thereof, belonging to the technical field of pharmaceutical chemistry and pharmacotherapeutics. The compound shown in the formula I or the pharmaceutically acceptable salt thereof has good EGFR (epidermal growth factor receptor) inhibition activity and better inhibition activity on H1975 cells and PC9 cells, and can be used for preparing medicaments for treating diseases related to tumors, particularly for preparing EGFR (epidermal growth factor receptor) inhibitor medicaments.

Description

Pyrimidine compound and medical application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to a pyrimidine compound. The application of the compounds in the preparation of EGFR inhibitor medicaments. The invention also relates to a pharmaceutical composition of the compound.

Background

EGFR is a member of the ErbB family of transmembrane receptor tyrosine kinases (including EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3 and HER 4/ErbB-4). It is the 170kDa glycoprotein encoded by the gene located on chromosome 7p 12-13. ErbB receptors consist of an extracellular ligand binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. EGFR has 7 different known ligands and can induce multiple biological effects even in the same cell. Wherein Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC) are high affinity ligands. In the inactivated state, the receptor is monomeric, but upon ligand binding, undergoes conformational changes, homo-or heterodimerization with other ErbB members, mediating intermolecular autophosphorylation of specific tyrosine residues, thereby triggering downstream signaling.

Due to the mutation of the EGFR, the activity of the therapeutic drug is reduced, and the EGFR-TKIs appear, so that the problem caused by the mutation is solved. EGFR-TKIs are divided into four generations according to the mechanism of treating NSCLC. Among them, AZD9291 (ocitinib) is a third generation EGFR-TKIs drug developed by AstraZeneca Pharmaceuticals, and belongs to anilinopyrimidine compound that irreversibly targets the cysteine 797 residue of the ATP binding site of EGFR kinase through a covalent bond. This is currently the only drug approved for metastatic EGFR (T790M) positive NSCLC patients.

The mechanism of acquired resistance of EGFR-TKIs can be broadly classified as EGFR-dependent or EGFR-independent. EGFR-dependent mutations include: the mutation in C797, EGFR (C797S), means that the ATP binding site at the cysteine at codon 797 is substituted with serine, resulting in a weakening of the covalent interaction between AZD9291 and mutant EGFR. Mutations in G796, solvent front mutations in G796 residues G796R, G796S and G796D adjacent to C797 may sterically interfere with AZD9291-EGFR interactions. Mutations in G724, the G724S mutation in the P-loop of the EGFR kinase domain, have been shown by structural analysis that the G724S mutation induces a conformational change in the receptor, impairing the binding of AZD 9291. Mutation of exon 20, EGFR (S768I) constituted the rare mutation of exon 20 after treatment with EGFR-TKIs was initiated. Amplification of the EGFR gene in addition to the presence of the EGFR-ex19del allele, amplification of the wild-type EGFR allele also constitutes a new drug resistance mechanism; EGFR independent mutations include: MET amplification, acquired resistance to EGFR-TKIs, is the most common cause of activation mechanism of the alternative pathway. Sustained activation of EGFR downstream signaling pathways, such as MAPK-mediated signaling pathways, Signal Transducer and Activator of Transcription (STAT) and phosphatidylinositol 3-kinase (PI3K) -Akt, is independent of EGFR activation and signaling. RAS-MAPK pathway activation, Eberlain et al found that RAS-MAPK pathway aberrations lead to resistance in EGFR-mutated NSCLC patients. PI3K pathway activation, alternative activation of PI3K pathway may occur simultaneously through PIK3CA mutation/amplification and PTEN deletion. Oncogenic fusions, which are found in 3% to 10% of the cases of acquired infection, may have oncogenic effects resistant to seco-oxitinib, and may be amplified with EGFR (C797S), BRAF mutations and MET [64-66 ]. Histological and phenotypic transformation, the histological transformation from non-small cell lung cancer to Small Cell Lung Cancer (SCLC), which occurs in 4-15% of cases, is a known cause of resistance to first-generation EGFR-TKIs, affecting patient prognosis. Is also reported to be an important cause of resistance to ocitinib.

Cancer (malignancy) is one of the major diseases causing human death worldwide. Since scientists first discovered that cancer is associated with overexpression of tyrosine kinase, the research of tyrosine kinase-targeted antitumor drugs in academia and the pharmaceutical industry is hot, and a plurality of small-molecule tyrosine kinase-targeted inhibitors have entered clinical research and come into the market, and especially EGFR-TKIs have brought great hope for tumor treatment. However, with the use of EGFR-TKIs, a series of problems also arise, such as the development of drug resistance of patients to EGFR-TKIs. How to overcome the drug resistance brought by EGFR mutation has become a hot spot of research of pharmaceutical workers.

Disclosure of Invention

The invention aims to design a pyrimidine compound by taking an EGFR irreversible inhibitor AZD9291 as a lead compound on the basis of the prior art, wherein the pyrimidine compound has good EGFR inhibitory activity and better inhibitory activity on H1975 cells and PC9 cells.

Another object of the present invention is to provide a pharmaceutical use of the above compounds, which can be used for preparing drugs for treating diseases related to tumors, especially for preparing EGFR inhibitor drugs.

The technical scheme of the invention is as follows:

a compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

x represents hydrogen, amino, -NH-R ¹ 、-NH-CH ₂ -R ¹ 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ¹ 、-NH-CO-CH ₂ -CH ₂ -R ¹ 、-NH-R ² 、-NH-CH ₂ -R ² 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ² 、-NH-CO-CH ₂ -CH ₂ -R ² 、-NH-Boc、C ₁ -C ₄ Alkyl or nitro;

y represents oxygen, a covalent bond, -CH ₂ -、-CH ₂ -CH ₂ -、-NH-、-CO-NH-、-NH-CO-、-NH-CH ₂ -、-NH-CH ₂ -CH ₂ -or-NH-CO-CH ₂ -CH ₂ -；

Z represents hydrogen, C ₁ -C ₄ Alkyl, -NH-Boc, phenyl, -S-CH ₃ 、-S-CH ₂ -CH ₃ 、C ₁ -C ₄ Alkoxy radical, R ¹ Or R ² ；

n represents 1,2, 3 or 4;

the R is ¹ Or R ² The structural formula of (A) is as follows:

in a preferred embodiment, X represents hydrogen, amino, -NH-R ¹ 、-NH-Boc、-NH-CH ₂ -R ¹ 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ¹ or-NH-CO-CH ₂ -CH ₂ -R ¹ 。

In a more preferred embodiment, X represents hydrogen, amino, -NH-Boc or-NH-CO-CH ₂ -CH ₂ -R ¹ 。

In a preferred embodiment, Y represents oxygen, a covalent bond, -CH ₂ -、-NH-、-NH-CH ₂ -or-NH-CH ₂ -CH ₂ -。

In a more preferred embodiment, Y represents oxygen, a covalent bond, -CH ₂ -or-NH-.

In a preferred embodiment, Z represents hydrogen, methyl, ethyl, isopropyl, sec-butyl, isobutyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² 。

In a more preferred embodiment, Z represents hydrogen, isopropyl, sec-butyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² 。

In a preferred embodiment, n represents 2, 3 or 4.

In a more preferred embodiment, n represents 2 or 3.

Further, X represents hydrogen, amino, -NH-Boc or-NH-CO-CH ₂ -CH ₂ -R ¹ (ii) a Y represents oxygen, a covalent bond, -CH ₂ -or-NH-; z represents hydrogen, isopropyl, sec-butyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² (ii) a n represents 2 or 3.

Still further, a compound according to formula I or a pharmaceutically acceptable salt thereof, wherein said compound is selected from:

the intermediates or the target compounds mentioned in the present invention can be purified according to conventional isolation techniques and converted into addition salts with pharmaceutically acceptable acids as required.

Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed below:

"pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) salts with acids are obtained by reaction of the free base of the parent compound with inorganic or organic acids, including hydrochloric, hydrobromic, nitric, phosphoric, metaphosphoric, sulfuric, sulfurous, and perchloric acids, and the like, and organic acids including acetic, trifluoroacetic, propionic, acrylic, caproic, cyclopentanepropionic, glycolic, pyruvic, oxalic, (D) or (L) malic, fumaric, maleic, ascorbic, camphoric, dodecylsulfuric, gluconic, glutamic, aspartic, stearic, mandelic, succinic, glutaric, or malonic acids, and the like.

(2) The acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is complexed with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, quinine, or the like.

"pharmaceutical composition" refers to the combination of one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, with another chemical ingredient, such as a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration process to an animal.

"pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to inactive ingredients in a pharmaceutical composition that do not cause significant irritation to an organism and do not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, and the like.

"alkyl" means a saturated aliphatic radical of 1 to 20 carbon atoms, including straight and branched chain radicals (a numerical range referred to herein, e.g., "1 to 20", means that the radical, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). More preferably, the alkyl group is a medium size alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, and the like. Preferably, alkyl is lower alkyl having 1 to 8 or 1 to 6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, and the like. Alkyl groups may be substituted or unsubstituted. When substituted alkyl, the substituent is preferably one or more, more preferably 1 to 3, most preferably 1 or 2 substituents.

"alkoxy" means-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy and the like.

The invention provides a pharmaceutical composition, which takes the compound or the pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and is assisted by a pharmaceutically acceptable carrier.

The compound or the pharmaceutically acceptable salt thereof has good EGFR inhibitory activity, has good inhibitory activity on H1975 cells and PC9 cells, and can be used for preparing medicaments for treating diseases related to tumors, particularly for preparing EGFR inhibitory medicaments.

By adopting the technical scheme of the invention, the advantages are as follows:

the invention provides a pyrimidine compound which has good EGFR inhibitory activity and good inhibitory activity on H1975 cells and PC9 cells, can be used for preparing medicaments for treating diseases related to tumors, and particularly can be used for preparing EGFR inhibitory medicaments.

Drawings

FIG. 1 shows the detection of p-EGFR protein expression in H1975 cells after RX-5 compound has acted on H1975 cells 24;

FIG. 2 shows the cell cycle tissue status of a compound RX-5 after 72H of flow cytometry on H1975 cells;

FIG. 3 shows H2S fluorescent probe WSP-1, Compound RX-10 at a concentration of 50. mu.M in H ₂ S release level; in the figure, panel a is a blank of the control group, panel b is a control group, panel c is a blank of the experimental group, and panel d is an experimental group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

Synthesis of 3- (2-chloropyrimidin-4-yl) -1-methyl-1H-indole (2)

2, 4-dichloropyrimidine (10g,67.1mmol) was added to 100mL of DME solution, aluminum chloride (8.96g, 67.1mmol) was added, the mixture was stirred at room temperature for 20 minutes, 8.36mL of N-methylindole was added, the mixture was reacted at 85 ℃ for 10 hours, and the solution cooled to room temperature was poured into 500mL of stirred ice water and stirred for 30 minutes. The purple compound (13.2 g) was obtained by suction filtration in 80.9% yield.

Synthesis of N- (4-fluoro-2-methoxy-5-nitrophenyl) -4- (1-methyl-1H-indol-3-yl) pyrimidin-2-amine (3)

Compound (2) (10g, 41mmol) was dissolved in 100mL dioxane, PTSA (8.58g,41mmol), 4-fluoro-2-methoxy-5-nitroaniline (8.39g,41mmol) was added, and the reaction was carried out at 90 ℃ for 12 hours with tube sealing. The solution cooled to room temperature was poured into 500mL of stirred ice-water and stirred for 30 minutes. Filtration with suction gave 14.2g of the green compound in 88.1% yield.

Synthesis of N1- (3-chloropropyl) -5-methoxy-N1-methyl-N4- (4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) -2-nitrobenzene-1, 4-diamine (4a)

Compound (3) (1.5g,3.81mmol) was dissolved in 20mL of DMF and added (0.7g,4.85mmol) to hydrochloride salt, 0.875mL of DIPEA, 110 deg.C, purged with nitrogen, and the tube was sealed for reaction for 9 h. The solution was poured into 200mL of stirred ice water, stirred for 30 minutes and filtered to give 1.1g of an orange solid with a yield of 61.1%. ¹ H NMR(400MHz,CDCl ₃ )δ9.52(s,1H),8.36-8.35(d,J＝4.0Hz,1H),8.26(s,1H),8.16-8.14(d,J＝8.0Hz,1H),7.65(s,1H),7.4-7.38(m,1H),7.32-7.28(m,2H),7.19-7.17(d,J＝8.0Hz,1H),6.62(s,1H),3.96(s,3H),3.93(s,3H),3.65-3.61(m,2H),3.38-3.34(m,2H),2.85(s,3H),2.12-2.05(m,2H).

Synthesis of ethyl tert-butyl (2- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (4b)

Starting material (3) (2.5g,6.35mmol) was dissolved in 25mL DMF and tert-butyl (2- (methylamino) ethyl) carbamate (1.21g,6.9mmol), DIPEA (1.55mL), drum nitrogen, 70 ℃ reacted for 12 h. The solution was poured into 200mL of stirred ice water, stirred for 30 minutes, and filtered by suction to give 2.8g of an orange solid in 82% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.53(s,1H),8.38-8.36(d,J＝8.0Hz,1H),8.24(s,1H),8.17-8.14(m,1H),7.58(m,1H),7.38(m,1H),7.31-7.29(m,2H),7.19-7.17(d,J＝8.0Hz,1H),7.35(t,J＝4.0Hz,1H),7.3-7.26(m,2H),7.20(d,J＝4.0Hz,1H),6.91-6.88(m,2H),6.64(s,1H),5.09(s,1H),3.98(s,3H),3.93(s,3H),3.36-3.31(m,4H),2.84(s,3H),1.39(s,9H).

Synthesis of propyl tert-butyl (3- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (4c)

Starting material (3) (2.5g,6.35mmol) was dissolved in 25mL DMF and tert-butyl (2- (methylamino) ethyl) carbamate (1.2g,6.9mmol), DIPEA (1.55mL), drum nitrogen, 70 ℃ reacted for 12 h. The solution was poured into 200mL of stirred ice water, stirred for 30 minutes and filtered to give 2.7g of an orange solid with a yield of 75.8%. ¹ H NMR(400MHz,CDCl ₃ )δ9.55(s,1H),8.37-8.36(d,J＝4.0Hz,1H),8.25(s,1H),8.16-8.13(m,1H),7.58(m,1H),7.4-7.38(m,1H),7.32-7.28(m,2H),7.18-7.17(d,J＝4.0Hz,1H),6.58(s,1H),4.88(m,1H),3.97(s,3H),3.92(s,3H),3.19-3.16(m,4H),2.83(s,3H),1.86-1.79(m,2H)1.41(s,9H).

Synthesis of N1- (3-chloropropyl) -5-methoxy-N1-methyl-N4- (4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) benzene-1, 2, 4-triamine (5a)

Compound (4a) (0.6g,1.24mmol) was dissolved in 10mL of methanol, concentrated hydrochloric acid (0.5mL) was added, stannous chloride (1.44g,6.38mmol) was added, and the mixture was refluxed at 65 ℃ for 6 hours. And (3) adjusting the pH of the reaction solution to be alkaline under the ice bath condition, adding DCM for extraction, and evaporating to dryness to obtain a product of 0.4g, wherein the yield is 71%.

Synthesis of ethyl tert-butyl (2- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (5b)

Compound (4b) (1.8g,3.28mmol) was dissolved in 20mL of tetrahydrofuran, and palladium on carbon (0.18g,1.69mmol) and ammonium formate (1.03g,16.3mmol) were added and the reaction was blocked at 65 ℃ for 10 h. The mixture cooled to room temperature was filtered with suction and the filtrate was evaporated to dryness. DCM was added for extraction, and the solution was evaporated to dryness to give 1.2g, 70% yield.

Synthesis of propyl tert-butyl (3- ((2-amino-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (5c)

Compound (4c) (1.8g,3.2mmol) was dissolved in 20mL of tetrahydrofuran, palladium on carbon (0.18g,1.69mmol) and ammonium formate (1.03g,16.3mmol) were added, and the reaction was blocked at 65 ℃ for 10 hours. The mixture cooled to room temperature was filtered with suction and the filtrate was evaporated to dryness. DCM was added for extraction, and the solution was evaporated to dryness to give 1.1g, 64% yield.

Synthesis of N- (2- ((3-chloropropyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (6a)

Compound (5a) (0.54g,1.19mmol) was dissolved in 10mL of anhydrous DCM, DIPEA0.6mL was added, acryloyl chloride 0.145mL was dissolved in 2mL of DCM, and the reaction was added dropwise at 0 ℃ to the reaction mixture and allowed to react overnight at room temperature. The reaction mixture was washed three times with 10mL of saturated brine and dried over saturated sodium sulfate. Evaporated to dryness and purified to obtain 0.4g of product with 66.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.85(s,1H),9.05(s,1H),8.25(s,1H),8.80(s,1H),8.36(s,1H),8.05(s,1H),7.78(s,1H),7.38(s,1H),7.25(s,2H),7.2(s,1H),6.76(s,1H),6.45-6.33(m,2H),3.98(s,3H),3.88(s,3H),3.56-3.55(d,J＝4.0Hz,2H),3.04(s,2H),2.64(s,3H),1.9-1.89(d,J＝4.0Hz,2H).

Synthesis of ethyl tert-butyl (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (6b)

Compound (5b) (1.7g,3.28mmol) was dissolved in 20mL of anhydrous DCM, DIPEA (1.6mL) was added, and acryloyl chloride 0.31mL was dissolved in 2 mL. The mixture was added dropwise to DCM at 0 ℃ and reacted overnight at room temperature. The reaction solution was washed three times with 10mL of saturated saline. Dried over saturated sodium sulfate. Evaporated to dryness and purified to obtain 0.8g of product with a yield of 44.4%. ¹ H NMR(400MHz,CDCl ₃ )δ9.82(s,1H),9.05(s,1H),8.86(s,1H),8.36-8.36(d,J＝4.0Hz,1H),8.06-8.04(m,1H),7.81(s,1H),7.4-7.37(m,1H),7.3-7.26(m,2H),7.21-7.19(m,1H),6.75(s,1H),6.45-6.43(m,2H),5.75-5.72(m,1H),4.64(s,1H),3.98(s,3H),3.88(s,3H),3.23-3.29(m,2H),3.98-2.95(m,2H),2.67(s,3H),1.47-1.41(m,9H).

Synthesis of propyl tert-butyl (3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) carbamate (6c)

Compound (5c) (1.7g,3.2mmol) was dissolved in 20mL of anhydrous DCM, DIPEA (1.6mL) was added, and acryloyl chloride 0.3mL was dissolved in 2 mL. The mixture was added dropwise to DCM at 0 ℃ and reacted overnight at room temperature. The reaction solution was washed three times with 10mL of saturated brine. Dried over saturated sodium sulfate. Evaporated to dryness and purified to obtain 0.82g of product with a yield of 44.4%. ¹ H NMR(400MHz,CDCl ₃ )δ9.8(s,1H),9.17-9.07(m,2H),8.34-8.33(d,J＝4.0Hz,1H),8.05-8.02(m,1H),7.87-7.83(m,1H),7.4-7.38(m,1H),7.28-7.26(m,2H),7.2-7.19(d,J＝4.0Hz,1H),6.73(s,1H),6.6-6.56(m,1H),6.6-6.42(m,1H),6.6-6.42(m,1H),5.74-5.71(m,1H),4.47(s,1H),3.98(s,3H),3.88(s,3H),3.17-3.15(m,2H),2.95-2.91(m,2H),2.59(s,3H),1.6-1.56(m,2H),1.44(s,9H).

Synthesis of 5-p-hydroxyphenyl-3H-1, 2-dithiole-3-thione (ADT-OH)

To a single-neck flask was added 5-p-methoxyphenyl-3H-1, 2-dithiole-3-thione (0.24g,1mmol) and pyridine hydrochloride (1.20g,10mmol) and mixed well. Carrying out melt reaction for 0.5h at 210 ℃, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, adding 1mol/L diluted hydrochloric acid, carrying out suction filtration to obtain a tan solid, and carrying out vacuum drying. The yield thereof was found to be 81%.

Synthesis of ZWQ-1

To a single neck flask was added (6a) (0.5g,0.99mmol), anhydrous DMF10mL, potassium carbonate (0.27g,1.95mmol), a catalytic amount of potassium iodide, stirred for 10min, ADT-OH (0.246g,1.08mmol) was added, the reaction was monitored by TLC, after completion of the reaction, cooled to room temperature, the reaction mixture was poured into ice water, stirred for 30min, filtered, dried under vacuum, separated by silica gel column chromatography (dichloromethane: ethyl acetate (2:1)), and 1mL of methanol was added to give 0.1g of an orange solid, yield 14.7%, Mp:96.1-98 ℃.

Synthesis of 3-allyldithiopropionic acid (ACS-81)

To a three-necked flask was added diallyl disulfide (5.85g,40mmol), 3-mercaptopropionic acid (0.85g,8mmol), methanol: diethyl ether (2:1V/V) mixed solvent, then 10mol/L NaOH (0.24g,6mmol) is added, nitrogen is introduced for 30min, and reaction is carried out for 24h at 25 ℃. The reaction was monitored by TLC, after completion of the reaction, the reaction solution was evaporated to dryness, 1mol/L HCl was added, extraction was performed with diethyl ether, and the solvent was evaporated under reduced pressure to give 1.2g of an off-white oil with a yield of 84%.

Synthesis of 5-p-carboxyethoxyphenyl-3H-1, 2-dithiole-3-thione (ACS-60)

Adding ADT-OH (0.45g,2mmol), potassium carbonate (0.55g,4mmol) and ethyl bromoacetate (0.66mL,6mmol) into a three-neck flask, adding 30mL of anhydrous DMF, fully stirring and dissolving, reacting at 50 ℃ for 3h, monitoring by TLC, cooling to room temperature after the reaction is finished, extracting with ethyl acetate, washing with distilled water for 3 times, drying an ethyl acetate layer with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a tan oily substance. The oily substance (0.31g,1mmol) obtained in the previous step is directly used for the next step of reaction, 50% sulfuric acid (2.2mL,20mmol) and 20mL acetic acid are added, the reaction is carried out at 100 ℃ for 2h, the reaction is monitored by TLC, the reaction is cooled to room temperature after the reaction is finished, distilled water is added, and the deep green solid is obtained by suction filtration, and the yield is 49%. ¹ H NMR(400MHz,CDCl ₃ )δ9.83(s,1H),9.05(s,1H),8.8(s,1H),8.35(d,J＝8.0Hz,1H),8.04(t,J＝8.0Hz,1H),7.92(d,J＝12.0Hz,1H),7.57-7.54(m,2H),7.4-7.38(m,1H),7.35(t,J＝4.0Hz,1H),7.3-7.26(m,2H),7.20(d,J＝4.0Hz,1H),6.91-6.88(m,2H),6.78(s,1H),6.39(q,1H),6.22(q,1H),5.67(q,1H),4.01(t,J＝12.0Hz,2H),3.96(s,3H),3.88(s,3H),3.08(t,J＝8.0Hz,2H),2.67(s,3H),1.94(t,J＝8.0Hz,2H). ¹³ CNMR(100MHz,CDCl ₃ )δ215.22,173.11,162.51,162.31,162.24,159.55,157.94,144.49,138.35,135.09,134.72,133.66,132.41,128.73,128.33,127.86,126.43,126.04,124.34,122.02,121.14,120.41,115.50,113.72,110.23,109.52,108.20,103.90,66.16,56.25,53.79,44.35,33.22,27.43.MS(m/z):HRMS(ESI)Calcd for C ₃₆ H ₃₄ N ₆ O ₃ S ₃ ([M+H] ⁺ ):695.1933,found:695.1929.

Synthesis of ZWQ-6

(6a) (0.5g,0.99mmol), anhydrous DMF10mL, potassium carbonate (0.27g,1.95mmol), catalytic amount of potassium iodide were added to a single vial, stirred for 10min, ACS-81 was added, the reaction was monitored by TLC, after completion of the reaction, cooled to room temperature, the reaction mixture was poured into ice water, stirred for 30min, filtered under suction, dried under vacuum, separated by silica gel column chromatography (dichloromethane: methanol (80:1)), and added to give 0.1g of pale yellow solid in 14.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.83(s,1H),9.07(s,1H),8.86(s,1H),8.34(d,J＝4.0Hz,1H),8.05(q,J＝8.0Hz,1H),7.85(s,1H),7.4-7.38(m,1H),7.3-7.26(m,2H),7.20(d,J＝4.0Hz,1H),6.75(s,1H),6.44(q,2H),5.77-7.72(m,2H),5.2-5.06(m,2H),4.16(t,J＝12.0Hz,2H),3.98(s,3H),3.88(s,3H),3.30(q,J＝8.0Hz,1H),3.13-3.11(m,1H),2.95-2.88(m,3H),2.74-2.69(m,2H),2.62(d,J＝4.0Hz,3H),2.57(t,J＝16.0Hz,1H),1.81-1.74(m,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.07,162.71,162.39,159.39,157.64,144.51,138.38,135.37,134.19,133.90,132.64,128.40,127.68,126.39,126.07,121.96,121.12,120.37,117.44,113.72,110.23,109.76,108.07,103.86,62.29,56.28,53.25,44.20,34.88,34.61,33.21,27.08,25.68.MS(m/z):HRMS(ESI)Calcd for C ₃₃ H ₃₈ N ₆ O ₄ S ₂ ([M+H] ⁺ ):647.2474,found:647.2472.

Synthesis of N- (2- ((2-aminoethyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (7b)

After (6b) (0.25g,0.43mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction mixture was allowed to react at room temperature for 3 hours. And (3) evaporating the reaction solution to dryness, adding a saturated potassium carbonate solution to adjust the pH value to be alkaline, adding DCM for extraction, and evaporating to dryness to obtain a product of 0.15g with the yield of 75%.

Synthesis of N- (2- ((3-aminopropyl) (methyl) amino) -4-methoxy-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (7c)

After (6c) (0.25g,0.42mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. And (3) evaporating the reaction solution to dryness, adding a saturated potassium carbonate solution to adjust the pH value to be alkaline, adding DCM for extraction, and evaporating to dryness to obtain a product of 0.14g with the yield of 70%.

Synthesis of ZL-1

ACS-60(0.265g,0.93mmol) and DMF10mL are added into a single-mouth bottle, stirred to be dissolved, HATU (0.35g,0.92mmol) and triethylamine (0.14mL) are added, stirred and activated for 20min at room temperature, slowly added (0.4g,0.84mmol) (7b) to react for 24h at 25 ℃, TLC is used for monitoring the reaction, after the reaction is finished, the reaction liquid is poured into 100mL of ice water, stirred and filtered to obtain gray solid, and the gray solid is separated by silica gel column chromatography (dichloromethane: methanol (80:1)) to obtain 0.09g of a final light orange solid product, wherein the yield is 14.5% Mp:123.2-124.4 ℃. ¹ HNMR(400MHz,d ₆ -DMSO)δ9.14(s,1H),8.97(s,1H),8.58(s,1H),8.31-8.2(m,3H),7.88(s,1H),7.77(d,J＝8.0Hz,2H),7.67(s,1H),7.48(t,J＝8.0Hz,1H),7.19(q,J＝16.0Hz,2H),7.11(t,J＝12.0Hz,1H),7.0(d,J＝8.0Hz,2H),6.93(s,1H),6.69(q,1H),6.20(q,J＝16.0Hz,1H),5.63(d,J＝12.0Hz,1H),4.55(s,2H),3.86(s,3H),3.83(s,3H),3.3(s,2H),2.98(t,J＝16.0Hz,2H),2.64(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ215.03,172.30,167.56,162.83,162.17,159.85,159.31,157.73,144.43,138.30,134.88,134.12,132.28,128.59,127.73,127.64,126.77,125.98,125.42,122.14,121.25,120.58,115.60,113.60,110.20,110.09,108.10,103.90,67.16,56.21,55.87,44.40,38.11,33.22.MS(m/z):HRMS(ESI)Calcd for C ₃₇ H ₃₅ N ₇ O ₄ S ₃ ([M+H] ⁺ ):738.1991,found:738.1989.

Synthesis of ZL-2

Adding ACS-60(0.32g,1.12mmol) and 10mL of DMF into a single-mouth bottle, stirring to dissolve, adding HATU (0.43g,1.53mmol) and triethylamine (0.17mL), stirring and activating at room temperature for 20min, slowly adding HATU (0.4g,1.1mmol) (7c), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction solution into 100mL of ice water after the reaction is finished, stirring, carrying out suction filtration to obtain a gray solid, and carrying out silica gel column chromatography (dichloromethane: methanol (80:1)) to obtain a final light orange solid product 0.1g, wherein the yield is 12.9%, and the Mp is 125.2-127.3 ℃. ¹ H NMR(400MHz,d ₆ -DMSO)δ9.14(s,1H),8.97(s,1H),8.58(s,1H),8.31-8.2(m,3H),7.88(s,1H),7.77(d,J＝8.0Hz,2H),7.67(s,1H),7.48(t,J＝8.0Hz,1H),7.19(q,J＝16.0Hz,2H),7.11(t,J＝12.0Hz,1H),7.0(d,J＝8.0Hz,2H),6.93(s,1H),6.69(q,1H),6.20(q,J＝16.0Hz,1H),5.63(d,J＝12Hz,1H),4.55(s,2H),3.86(s,3H),3.83(s,3H),3.3(s,2H),2.98(t,J＝16.0Hz,2H),2.64(s,3H). ¹³ C NMR(100MHz,d ₆ -DMSO)δ215.36,174.17,167.67,163.40,162.21,161.61,160.42,158.13,146.87,138.49,138.20,134.85,134.28,133.05,129.44,126.69,126.63,125.87,125.24,124.74,122.54,121.99,121.48,116.28,115.66,112.97,111.02,107.61,104.80,67.50,56.51,52.69,43.27,36.27,33.51,27.36.MS(m/z):HRMS(ESI)Calcd for C ₃₈ H ₃₇ N ₇ O ₄ S ₃ ([M+H] ⁺ ):752.2147,found:752.2146.

Synthesis of Zl-3

ACS81(0.187g,1.04mmol) and 10mL of DMF were added to a single-neck flask, stirred to dissolve, HATU (0.39g,1.02mmol) and triethylamine (0.15mL) were added, stirred at room temperature for 20min, slowly added (0.45g,0.92mmol) (7b) and reacted at 25 ℃ for 24h, TLC was used to monitor the reaction, and after completion of the reaction, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, vacuum dried, and chromatographed on a silica gel column (dichloromethane: methanol (80:1)) to obtain 0.1g of a final beige solid product with a yield of 16.6% and a Mp of 72.5-75.3 ℃.

¹ H NMR(400MHz,CDCl ₃ )δ9.71(s,1H),8.95(s,1H),8.72(s,1H),8.31(d,J＝4.0Hz,1H),8.05(d,J＝8.0Hz,1H),7.83(s,1H),7.38-7.36(m,1H),7.31-7.26(m,1H),7.17(d,J＝4.0Hz,1H),6.71(s,1H),6.45(t,J＝8.0Hz,2H),5.87(t,J＝8.0Hz,2H),5.84-5.75(m,2H),5.19-5.1(m,2H),3.95(s,3H),3.87(s,3H),3.36-3.32(m,2H),3.29-3.27(m,2H),3.06(t,J＝12.0Hz,2H),2.92(t,J＝12.0Hz,2H),2.65(s,3H),2.5(t,J＝12.0Hz,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ171.44,162.81,162.26,159.54,157.85,144.67,138.30,134.89,134.40,133.29,132.42,127.61,126.73,126.01,122.02,121.12,120.47,118.87,113.68,110.32,110.19,108.13,103.88,56.20,44.17,42.02,38.08,36.06,33.83,33.20.MS(m/z):HRMS(ESI)Calcd for C ₃₂ H ₃₇ N ₇ O ₃ S ₂ ([M+H] ⁺ ):632.2478found:632.2475.

Synthesis of ZL-4

ACS81(0.43g,2.41mmol) and 10mL of DMF were added to a single-neck flask, stirred to dissolve, HATU (0.43g,1.13mmol) and triethylamine (0.17mL) were added, stirred at room temperature for 20min, 7c (0.5g,1mmol) was slowly added, the reaction was monitored by TLC for 24h, and after completion of the reaction, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, vacuum dried, and chromatographed on a silica gel column (dichloromethane: methanol (80:1)) to obtain 0.14g of a final beige solid product with a yield of 21% and Mp:73.2-76.3 ℃.

¹ H NMR(400MHz,CDCl ₃ )δ9.81(s,1H),9.01(s,H),8.29(s,1H),8.38-8.36(d,J＝4.0Hz,1H),7.88(s,1H),7.38(q,J＝8.0Hz,1H),7.3-7.25(m,2H),7.19(d,J＝8.0Hz,1H),6.73(s,1H),6.56(q,J＝16.0Hz1H),6.44(q,J＝16.0Hz,1H),5.79-5.69(m,3H),5.11-5.05(m,2H),3.97(s,3H),3.87(s,3H),3.28(q,J＝16.0Hz,2H),3.12-3.1(m,2H),2.92(t,J＝16.0Hz,2H),2.73(t,J＝12.0Hz,2H),2.59(s,3H),2.40(t,J＝16.0Hz,2H),1.62-1.57(m,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ170.95,163.00,162.17,159.49,157.78,144.43,138.20,134.94,134.25,133.26,132.60,128.12,127.14,126.26,125.90,121.90,121.02,120.34,118.71,113.52,110.27,110.12,107.96,103.70,56.13,52.99,44.24,41.91,36.80,35.80,33.92,33.10,27.62.MS(m/z):HRMS(ESI)Calcd for C ₃₃ H ₃₉ N ₇ O ₃ S ₂ ([M+H] ⁺ ):646.2634,found:646.2635.

Synthesis of tert-butyl (R) - (1- ((2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (8d)

Adding leucine (0.269g,1.16mmol) and DMF10mL into a single-neck flask, stirring for dissolution, adding HATU (0.44g,1.16mmol) and triethylamine (0.17mL), stirring and activating at room temperature for 20min, slowly adding (7b) (0.5g,1.06mmol), reacting at 25 ℃ for 24h, monitoring by TLC, after the reaction is finished, pouring the reaction solution into 100mL of ice water, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum to obtain beige solid, and separating by silica gel column chromatography (dichloromethane: methanol (100:1)) to obtain the final beige solid product 0.12g, with the yield of 16.6%。 ¹ H NMR(400MHz,CDCl ₃ )δ9.74(s,1H),9.05(s,1H),8.96(s,1H),8.75(s,1H),8.36-8.35(d,J＝4.0Hz,1H),8.07-8.06(d,J＝4.0Hz,1H),7.72(s,1H),7.39-7.37(m,1H),7.29-7.25(m,3H),7.19-7.18(d,J＝4.0Hz,2H),6.71(s,1H),6.6-6.57(m,1H),6.48-6.44(m,2H),5.76-5.73(m,1H),4.81(s,1H),3.96(s,3H),3.87(s,3H),3.45-3.42(m,1H),3.25-3.21(m,1H),3.04-3.01(t,J＝12.0Hz,2H),2.63(s,3H),1.65-1.62(m,2H),1.45-1.41(m,1H),1.38(s,9H),0.9-0.88(m,6H).

Synthesis of tert-butyl (R) - (1- ((2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) amino) -1-oxopropan-2-yl) carbamate (8e)

Alanine (0.22g,1.16mmol) and 10mL of DMF were added to a single-neck flask, stirred to dissolve, HATU (0.44g,1.16mmol) and triethylamine (0.17mL) were added, stirred at room temperature for 20min, slowly added (0.5g,1.06mmol) (7b) and reacted at 25 ℃ for 24h, TLC was used to monitor the reaction, after completion of the reaction, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and dried under vacuum to give a beige solid, which was chromatographed on a silica gel column (dichloromethane: methanol (100:1)) to give 0.09g of a final beige solid product in 13.2% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.742(s,1H),8.97(s,1H),8.96(s,1H),8.72(s,1H),8.35(s,1H),8.06(s,1H),7.72(s,1H),7.37(s,1H),7.25(s,2H),7.18(s,1H),6.71(s,1H),6.58(m,2H),6.48-6.44(m,1H),5.75(s,1H),4.91(s,1H),4.07(s,1H),3.96(s,3H),3.87(s,3H),3.41(s,1H),3.27(s,1H),3.03(s,2H),2.63(s,3H),1.38(m,12H).

Synthesis of tert-butyl (2- ((2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl ] [ methyl) amino) ethyl) amino) -2-oxoethyl) carbamate (8f)

Glycine (0.2g,1.14mmol) and DMF (10 mL) were added to a single-neck flask, stirred to dissolve, HATU (0.44g,1.16mmol) and triethylamine (0.17mL) were added, stirred at room temperature and activated for 20min, slowly added (0.5g) (7b), reacted at 25 ℃ for 24h, monitored by TLC, after the reaction was completed, the reaction solution was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and dried under vacuum to give a beige solid, which was separated by silica gel column chromatography (dichloromethane: ethyl acetate (80:1)) to give 0.1g of a final beige solid product with a yield of 15.1%. ¹ H NMR(400MHz,CDCl ₃ )δ9.67(s,1H),8.93(s,1H),8.66(s,1H),8.35-8.34(d,J＝4.0Hz,1H),8.08-8.06(m,1H),7.71(s,1H),7.39-7.37(m,1H),7.28-7.25(m,2H),7.19-7.17(m,1H),6.71(s,1H),6.55-6.46(m,3H),5.71-5.74(m,1H),5.28(s,1H),3.96(s,3H),3.87(s,3H),3.7-3.68(m,2H),3.36-3.35(m,2H),3.04(s,2H),2.64(s,3H),1.39(m,9H).

Synthesis of tert-butyl (S) - (1- ((3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl ] amino) phenyl) (methyl) amino) propyl) amino) -4-methyl-1-oxopentan-2-yl) carbamate (8g)

Leucine (0.26g,1.12mmol) and DMF (10 mL) are added into a single-neck flask, stirred to dissolve, HATU (0.43g,1.13mmol) and triethylamine (0.17mL) are added, stirred and activated for 20min at room temperature, (0.5g, 1.02mmol) (7c) are slowly added, the reaction is monitored by TLC for 24h at 25 ℃, after the reaction is finished, the reaction solution is poured into 100mL of ice water, stirred, extracted by ethyl acetate, washed by brine, dried by anhydrous sodium sulfate, the solvent is evaporated under reduced pressure and dried in vacuum to obtain beige solid, and the beige solid product is separated by silica gel column chromatography (dichloromethane: methanol (100:1)) to obtain the final beige solid product with the yield of 0.11g and 15.4%. ¹ H NMR(400MHz,CDCl ₃ )δ9.8(s,1H),9.05(s,1H),9.08-9.04(m,1H),8.36-8.35(d,J＝4.0Hz,1H),8.06-8(m,1H),7.75(s,1H),7.39-7.36(m,1H),7.29-7.25(m,3H),7.21-7.18(m,1H),6.72(s,1H),6.2-6.56(m,1H),6.47-6.42(m,1H),6.13(m,1H),5.75-5.72(m,1H),4.82(s,1H),3.98(s,3H),3.87(s,3H),3.34-3.22(m,2H),2.94-2.87(m,2H),2.57(s,3H),1.67-1.58(m,5H),1.4(m,9H).

Synthesis of tert-butyl (S) - (1- ((3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) propyl) amino) -1-oxopropan-2-yl) carbamate (8H)

Alanine (0.16g,0.84mmol) and 10mL of DMF were added to a single vial, and stirred to dissolve, HATU (0.34g,0.89mmol) and triethylamine (0.17mL) were added, stirred at room temperature for 20min, and slowly added (0.5g,1.1mmol) (7c) to react at 25 ℃ for 24h, and TLC was used to monitor the reaction, after completion of the reaction, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and dried under vacuum to give a beige solid, which was chromatographed on a silica gel column (dichloromethane: methanol (100:1)) to give 0.09g of a final beige solid product in 13.4% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.8(s,1H),9.06-9.04(m,2H),8.36-8.35(d,J＝4.0Hz,1H),8.06-8.04(m,1H),7.75(s,1H),7.4-7.37(m,1H),7.29-7.25(m,2H),7.2-7.18(m,1H),6.72(s,1H),6.59-6.55(m,1H),6.47-6.42(m,1H),6.16(s,1H),5.75-5.72(m,1H),4.92(s,1H),4.1-4.05(m,1H),3.98(s,3H),3.87(s,3H),3.32-3.24(m,2H),2.92-2.89(m,2H),2.58(s,3H),1.61-1.57(m,2H),1.45-1.41(m,12H).

Synthesis of tert-butyl (2- ((3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl ] [ methyl) amino) propyl) amino) -2-oxoethyl) carbamate (8i)

Glycine (0.19g,1.08mmol) and DMF10mL were added to a single-neck flask, stirred to dissolve, and HATU (0.43g, 1) was added13mmol), triethylamine (0.17mL) was stirred at room temperature for activation for 20min, slowly added (0.5g,1.1mmol) (7c), reacted at 25 ℃ for 24h, TLC monitored the reaction, after the reaction was completed, the reaction solution was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, dried under vacuum to give a beige solid, and separated by silica gel column chromatography (dichloromethane: methanol (80:1)) gave 0.1g of the final beige solid product in 16.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.79(s,1H),9.01-8.98(m,2H),8.36-8.35(d,J＝4.0Hz,1H),8.09-8.04(m,1H),7.74(s,1H),7.39-7.37(m,1H),7.29-7.25(m,2H),7.2-7.18(m,1H),6.72(s,1H),6.58-6.51(m,1H),6.47-6.42(m,1H),6.12(s,1H),5.76-5.73(s,1H),5.12(s,1H),3.97(s,3H),3.87(s,3H),3.75-3.73(m,2H),3.31-3.26(m,2H),2.92-2.88(m,2H),2.59(s,3H),1.61-1.58(m,2H),1.41(m,9H).

Synthesis of RX-1

After (8d) (0.5g,0.73mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain a product of 0.35g, wherein the yield is 83.3%, and the Mp is 140.3-142.5 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.78(s,1H),9.04(s,2H),8.31(d,J＝4.0Hz,1H),8.07-8.05(d,J＝4.0Hz,1H),7.70(s,1H),7.56(s,1H),7.31(s,1H),7.28-7.25(m,2H),7.2-7.19(d,J＝4.0Hz,1H),6.73(s,1H),6.48-6.46(q,J＝8.0Hz 2H),5.75-5.72(m,1H),3.93(s,3H),3.88(s,3H),3.38-3.37(d,J＝4.0Hz m,3H),3.07-3.06(s,2H),2.66(s,3H),1.70-1.66(s,1H),1.28(s,2H),0.94-0.93(d,J＝4.0Hz,,3H),0.89-0.87(d,J＝8.0Hz,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ175.84,162.56,162.09,159.47,157.86,144.39,138.18,134.96,134.16,132.55,127.87,127.50,126.23,125.91,121.79,120.92,120.25,113.60,110.04,109.78,108.01,103.88,56.27,56.09,53.34,44.18,43.82,37.35,33.05,24.82,23.42,21.11.MS(m/z):HRMS(ESI)Calcd for C ₃₂ H ₄₀ N ₈ O ₃ ([M+H] ⁺ ):585.3302,found:585.3302.

Synthesis of RX-2

After (8g) (0.5g,0.71mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain 0.34g of product, wherein the yield is 80.9%, and the Mp is 143.3-145.6 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.74(s,1H),9.07-8.97(m,1H),8.35(d,J＝4.0Hz,1H),8.06-7.96(m,1H),7.68(s,1H),7.38-7.25(s,4H),7.18-7.16(d,J＝8.0Hz 1H),6.75s,1H),6.6-6.53(m,1H),6.45-6.41(m,1H),5.75-5.72(m,1H),3.95(s,3H),3.86(s,3H),3.35-3.2(m,3H),2.92-2.86(m,2H),2.57(s,3H),1.97(s,2H),1.65-1.64(d,J＝4.0Hz 2H),1.31-1.25(m,2H),0.91-0.90(d,J＝8.0Hz,3H),0.88-0.87(d,J＝4.0Hz,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ175.29,162.95,162.07,159.43,157.76,144.38,138.13,134.92,134.25,132.55,128.02,127.12,126.13,125.85,121.76,120.89,120.22,113.50,110.23,110.02,107.89,103.62,56.05,53.33,53.04,44.05,43.89,36.20,33.02,27.55,24.78,23.32,21.20.MS(m/z):HRMS(ESI)Calcd for C ₃₃ H ₄₂ N ₈ O ₃ ([M+H] ⁺ ):599.3458,found:599.3455.

Synthesis of RX-3

After (8e) (0.5g,0.77mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain a product of 0.34g, wherein the yield is 80.9%, and the Mp is 140.3-142.5 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.82-9.81(m,1H),8.92(s,2H),8.78(s,1H),8.38-8.32(m,1H),8.09-8.04(m,1H),7.7-7.64(m,1H),7.24-7.21(d,J＝12.0Hz,2H),7.16-7.15(s,1H),6.76-6.67(m,1H),6.58-6.52(m,2H),5.73-5.71(d,J＝8.0Hz,1H),3.95(s,3H),3.87-3.84(s,3H),3.44-3.43(s,2H),3.29-3.27(m,1H),3.02(s,2H),2.6(s,2H),2.59(s,1H),1.64(m,3H),1.29-1.23(m,3H).

¹³ C NMR(100MHz,CDCl ₃ )δ175.98,162.79,162.26,159.61,157.98,144.59,138.33,135.03,134.37,132.62,127.92,127.62,126.45,126.04,121.97,121.09,120.42,113.72,110.19,110.06,108.16,104.02,56.28,56.25,50.77,44.31,37.50,33.20,21.72.MS(m/z):HRMS(ESI)Calcd for C ₂₉ H ₃₄ N ₈ O ₃₅ ([M+H] ⁺ ):543.2832,found:543.2832.

Synthesis of RX-4

After (8h) (0.5g,0.76mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 h. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain a product of 0.31g, wherein the yield is 73.8%, and the Mp is 144.3-145.6 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.8(s,1H),9.06-9.02(s,1H),8.37-8.36(s,1H),8.06-8.04(s,1H),7.7(s,1H),7.39-7.23(m,4H),7.19-7.18(d,J＝4.0Hz,2H),6.72(s,1H),6.66-6.54(m,1H),6.47-6.42(m,1H),5.86-5.66(m,1H),3.97(s,3H),3.8(s,3H),3.5-3.46(m,2H),3.32(s,1H),2.91-2.88(m,2H),2.58(S,3H),1.63-1.6(m,2H),1.31-1.29(m,3H). ¹³ C NMR(100MHz,d ₆ -DMSO)δ175.57,162.87,162.06,159.46,157.84,144.30,138.16,135.03,133.94,132.61,128.22,127.29,126.10,125.89,121.73,120.87,120.20,113.57,110.08,110.03,107.90,103.59,56.08,53.08,50.67,44.19,36.18,33.04,27.63,21.79.MS(m/z):HRMS(ESI)Calcd for C ₃₀ H ₃₆ N ₈ O ₃ ([M+H] ⁺ ):557.2989,found:557.2986.

Synthesis of RX-5

After (8f) (0.5g,0.79mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain a product of 0.36g, wherein the yield is 85.7%, and the Mp is 144.3-146.2 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.82-9.78(d,J＝8.0Hz,1H),8.97(s,1H),8.78(s,1H),8.36-8.35(d,J＝4.0Hz,1H),8.08-8.05(m,1H),7.69(s,1H),7.51-7.49(d,J＝8.0Hz,1H),7.39-7.36(m,1H),7.29-7.25(m,2H),7.24(s,1H),7.19-7.18(d,J＝4.0Hz,1H),6.73(s,1H),6.47-6.44(m,2H),5.75-5.72(m,1H),3.96(s,3H),3.87(s,3H),3.37-3.36(d,J＝4.0Hz,2H),3.29(s,2H),3.08-3.05(m,2H),2.65(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.92,162.80,162.26,159.65,158.00,144.61,138.35,135.02,134.35,132.61,127.99,127.68,126.44,126.07,121.98,121.10,120.44,113.76,110.19,110.06,108.19,104.08,56.32,56.27,44.66,44.26,37.43,33.21.MS(m/z):HRMS(ESI)Calcd for C ₂₉ H ₃₄ N ₈ O ₃ ([M+H] ⁺ ):529.2676,found:529.2675.

Synthesis of RX-6

After (8i) (0.5g,0.77mmol) was dissolved in 10mL of dioxane, 3mL of 4M HCl-dioxane solution (V ═ 1:2) was added dropwise to the reaction solution, and the reaction was allowed to react at room temperature for 3 hours. Evaporating the reaction solution to dryness, adding saturated potassium carbonate solution to adjust pH to alkalinity, adding DCM for extraction, evaporating to dryness to obtain a product of 0.33g, wherein the yield is 78.5%, and the Mp is 146.3-147.8 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.8(s,2H),9.15-8.96(m,2H),8.4-8.33(m,2H),8.11-8.04(m,1H),7.76-7.72(s,2H),7.39-7.35(m,1H),7.27-7.25(m,1H),7.19-7.18(s,1H),6.73(s,1H),6.53-6.41(m,2H),5.76-5.72(m,1H),3.96(s,3H),3.87(s,3H),3.49-3.42(m,2H),3.32-3.29(m,3H),2.11-2.07(m,2H),2.64-2.58(s,3H),1.65-1.52(m,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.64,162.81,162.07,159.43,157.80,144.31,138.15,134.99,133.94,132.53,128.19,127.30,126.17,125.87,121.75,120.88,120.19,113.54,110.03,109.97,107.90,103.63,56.07,53.34,44.53,44.10,36.25,33.03,27.66.MS(m/z):HRMS(ESI)Calcd for C ₂₉ H ₃₄ N ₈ O ₃ ([M+H] ⁺ ):543.2832,found:543.2831.

Synthesis of RX-7

ACS81(0.16g,0.89mmol) and 10mL of DMF were added to a single-neck flask, stirred to dissolve, HATU (0.35g,0.92mmol) and triethylamine (0.14mL) were added, stirred at room temperature and activated for 20min, slowly added (0.5g,0.85mmol) (9d) and reacted at 25 ℃ for 24h, TLC was used to monitor the reaction, after the reaction was completed, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and dried under vacuum to give a beige solid, which was chromatographed over a silica gel column (dichloromethane: methanol (120:1)) to give 0.09g of a final beige solid product at a yield of 14.2% and Mp:132.44-135.6 ℃. ¹ H NMR(400MHz,d ₆ -DMSO)δ9.15(s,1H),8.95(s,1H),8.59(s,1H),8.28(d,J＝4.0Hz,1H),8.21(d,J＝8.0Hz,1H),8.05-7.99(m,2H),7.89(s,1H),7.25(d,J＝8.0Hz,1H),7.19(t,J＝8.0Hz,2H),7.13(t,J＝12.0Hz,1H),6.92(s,1H),6.39(q,1H),6.75(q,1H),6.23(t,J＝16.0Hz,2H),5.74-5.64(m,2H),5.10-4.99(m,2H),4.31-4.25(m,1H),3.87(s,3H),3.83(s,3H),3.15(q,J＝8.0Hz,2H),3.09(d,J＝8.0Hz,2H),3.0-2.98(m,2H),2.60(s,3H),2.57-2.53(m,2H),2.37-2.31(m,2H),1.41-1.28(m,2H),1.19(s,1H),0.79(t,J＝8.0Hz,3H),0.77(d,J＝4.0Hz,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.41,171.60,163.21,162.19,159.44,157.67,144.79,138.18,135.35,134.73,133.18,132.47,127.01,126.95,126.61,125.90,121.95,121.06,120.43,118.74,113.54,111.49,110.11,108.00,103.53,56.10,55.24,51.96,43.90,41.87,40.13,37.67,35.50,33.72,33.12,24.77,22.98,21.93.MS(m/z):HRMS(ESI)Calcd for C ₃₈ H ₄₈ N8O ₄ S ₂ ([M+H] ⁺ ):745.3318,found:745.3315.

Synthesis of RX-8

ACS81(0.16g,0.89mmol) and 10mL of DMF were added to a single-neck flask, stirred to dissolve, HATU (0.34g,0.89mmol) and triethylamine (0.14mL) were added, stirred and activated at room temperature for 20min, slowly added (0.5g,0.83mmol) (9g) and reacted at 25 ℃ for 24h, TLC was used to monitor the reaction, after the reaction was completed, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and dried under vacuum to give a beige solid, which was chromatographed over a silica gel column (dichloromethane: methanol (120:1)) to give 0.092g of a final beige solid product at a yield of 14.6% and Mp:144.3-145.1 ℃. ¹ H NMR(400MHz,d ₆ -DMSO)δ9.38(s,1H),8.76(s,1H),8.59(s,1H),8.28(d,J＝8.0Hz,1H),8.05(d,J＝8.0Hz,1H),7.12(t,J＝12.0Hz,1H),6.88(s,1H),6.76(q,J＝16.0Hz,1H),6.23(q,J＝12.0Hz,1H),5.82-5.68(m,2H),5.17-5.06(m,2H),4.21(d,J＝4.0Hz,1H),3.87(s,3H),3.82(s,3H),3.33(t,J＝4.0Hz,2H),3.30(s,1H),3.27(s,1H),3.05(d,J＝4.0Hz,2H),2.89-2.81(m,4H),2.52(s,3H),1.52(t,J＝16.0Hz,2H),1.38(t,J＝16.0Hz,2H),1.19(s,1H),0.82(d,J＝8.0Hz,3H),0.78(d,J＝4.0Hz,3H). ¹³ C NMR(100MHz,d ₆ -DMSO)δ171.87,171.18,163.01,162.12,159.34,144.48,138.18,134.97,134.42,133.14,132.55,127.56,127.06,126.26,125.89,121.83,120.97,120.28,118.74,113.58,110.56,110.06,107.87,103.53,77.32,77.00,76.68,56.08,52.68,51.83,43.65,41.81,40.51,36.60,35.41,33.53,33.07,27.06,24.76,22.91,21.90.MS(m/z):HRMS(ESI)Calcd for C ₃₉ H ₅₀ N8O ₄ S ₂ ([M+H] ⁺ ):759.3475,found:759.3475.

Synthesis of RX-9

To a single-neck flask, ACS81(0.18g,1mmol) and 10mL of DMF were added and stirred to dissolveHATU (0.38g,1mmol) and triethylamine (0.15mL) are added, stirred and activated for 20min at room temperature, 4- (3-chloro-4-fluoroanilino) -6-aminoquinazoline (0.317g,1.1mmol) (9e) is slowly added, the mixture reacts for 24h at 25 ℃, TLC is used for monitoring the reaction, after the reaction is finished, the reaction solution is poured into 100mL of ice water, the mixture is extracted by ethyl acetate after stirring, washed by brine, dried by anhydrous sodium sulfate, the solvent is evaporated under reduced pressure and dried in vacuum to obtain beige solid, and the beige solid product is separated by silica gel column chromatography (dichloromethane: methanol (100:1)) to obtain 0.1g of final beige solid product with the yield of 15.6 percent Mp:78.4-80.3 ℃. ¹ H NMR(400MHz,d ₆ -DMSO)δ9.21(s,1H),8.85(s,1H),8.72(s,1H),8.49(s,2H),7.16-7.14(d,J＝8.0Hz,1H),6.99(s,1H),6.76-6.69(m,1H),6.22-6.18(m,1H),5.91-5.69(m,2H),5.17-5.07(m,2H),4.27-4.23(m,1H),3.88(s,3H),3.8(s,3H),3.(s,2H),2.84-2.81(m,2H),2.67(s,3H),2.50-2.46(m,2H),1.13-1.11(d,J＝8Hz,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.44,171.38,163.23,162.24,159.56,157.78,144.88,138.25,135.47,134.74,133.22,132.44,127.05,126.68,125.97,122.02,121.12,120.50,118.83,113.64,111.50,110.16,108.09,103.63,56.17,55.35,49.03,43.89,41.94,37.74,35.58,33.71,33.20,17.53.MS(m/z):HRMS(ESI)Calcd for C ₃₅ H ₄₂ N ₈ O ₄ S ₂ ([M+H] ⁺ ):703.2849,found:703.2845.

Synthesis of RX-10

Adding ACS81(0.17g,0.95mmol) and 10mL of DMF into a single-neck flask, stirring to dissolve, adding HATU (0.37g,0.97mmol) and triethylamine (0.15mL), stirring and activating at room temperature for 20min, slowly adding HATU (0.5g,0.89mmol) (9h), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction solution into 100mL of ice water after the reaction is finished, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum to obtain beige solid, and separating by silica gel column chromatography (dichloromethane: methanol (100:1)) to obtain a final beige solid product of 0.094g, yield of 14.6% and Mp:74.2-76.3 ℃. ¹ HNMR(400MHz,CDCl ₃ )δ9.73(s,1H),8.95(s,1H),8.35-8.34(m,2H),8.05(s,1H),7.74(s,1H),7.36(s,1H),7.28-7.25(m,2H),7.18-7.12(s,1H),6.70(s,1H),6.59-6.52(m,1H),6.46-6.35(m,3H),5.8-5.73(m,2H),5.16-5.08(m,2H),4.4(m,1H),3.96(s,3H),3.88(s,3H),3.30-3.19(m,4H),2.93-2.81(m,4H),2.64-2.49(m,5H),1.62-1.56(m,2H),1.33-1.3(m,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ172.08,170.91,162.98,162.13,159.33,157.58,144.49,138.15,134.90,134.37,133.13,132.49,127.64,127.05,126.30,125.86,121.83,120.96,120.27,118.74,113.53,110.51,110.05,107.87,103.55,56.07,52.77,48.95,43.75,41.83,36.65,35.41,33.57,33.06,27.13,17.96.MS(m/z):HRMS(ESI)Calcd for C ₃₆ H ₄₄ N ₈ O ₄ S ₂ ([M+H] ⁺ ):717.3005,found:717.3002.

Synthesis of RX-11

Adding ACS81(0.18g,1mmol) and 10mL of DMF into a single-neck flask, stirring to dissolve, adding HATU (0.39g,1.02mmol) and triethylamine (0.16mL), stirring and activating at room temperature for 20min, slowly adding (0.5g,0.94mmol) (9f), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction solution into 100mL of ice water after the reaction is finished, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum to obtain a beige solid, and separating by silica gel column chromatography (dichloromethane: methanol (80:1)) to obtain a final beige solid product of 0.11g, yield of 16.9% and Mp:76.2-77.3 ℃. ¹ H NMR(400MHz,d ₆ -DMSO)δ9.21(s,1H),8.71(s,1H),8.5(s,1H),8.21-8.18(m,3H),7.96(s,1H),6.99(s,1H),6.76-6.70(m,1H),6.22-6.18(m,1H),5.80-5.70(m,2H),3.88(s,3H),3.80(s,3H),3.66-3.51(m,2H),3.35-3.33(m,2H),2.99(s,2H),2.86-2.82(m,2H),2.68(s,3H),2.49-2.46(d,J＝12.0Hz,2H). ¹³ C NMR(100MHz CDCl ₃ ,)δ171.96,169.41,163.28,162.23,159.60,157.85,144.95,138.24,135.56,134.58,133.21,132.31,126.94,126.82,125.94,122.10,121.17,120.52,118.85,113.62,111.48,110.19,108.17,103.73,56.16,55.31,43.79,43.66,41.91,37.76,35.34,33.69,33.22.MS(m/z):HRMS(ESI)Calcd for C ₃₄ H ₄₀ N ₈ O ₄ S ₂ ([M+H] ⁺ ):689.2692,found:689.2686.

Synthesis of RX-12

Adding ACS81(0.18g,1mmol) and 10mL of DMF into a single-neck flask, stirring to dissolve, adding HATU (0.38g,1mmol) and triethylamine (0.15mL), stirring and activating at room temperature for 20min, slowly adding (0.5g,0.92mmol) (9i), reacting at 25 ℃ for 24h, monitoring the reaction by TLC, pouring the reaction solution into 100mL of ice water after the reaction is finished, stirring, extracting with ethyl acetate, washing with saline, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure, drying under vacuum to obtain beige solid, and separating by silica gel column chromatography (dichloromethane: methanol (80:1) to obtain the final beige solid product 0.09g with the yield of 14% Mp:79.2-80.3 ℃. ¹ H NMR(400MHz,CDCl ₃ )δ9.7(s,1H),8.9-8,82(s,2H),8.36-8.34(d,J＝8Hz,2H),8.07-8.06(m,1H),7.69(s,1H),7.39-7.36(m,1H),7.27-7.25(m,2H),7.18-7.17(d,J＝4.0Hz,1H),6.73-6.67(m,2H),6.55-6.45(m,2H),6.36-6.33(m,1H),5.78-5.72(m,2H),5.15-5.07(m,2H),3.95(s,3H),3.86(s,3H),3.82(m,2H),3.25-3.15(m,4H),2.88(m,4H),2.59(s,3H),2.55-2.52(m,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ171.54,168.86,163.05,162.05,159.43,157.79,144.53,138.13,134.67,134.39,133.08,132.35,127.34,126.95,126.46,125.83,121.88,120.98,120.28,118.78,113.54,110.53,110.07,107.96,103.56,56.05,52.85,43.49,43.38,41.76,36.69,35.10,33.51,33.08,26.90.MS(m/z):HRMS(ESI)Calcd for C ₃₅ H ₄₂ N ₈ O ₄ S ₂ ([M+H] ⁺ ):703.2849,found:703.2845.

Synthesis of 2P, 2EL, 2S, 2X, 2B, 2L, 2G and 2BA

Synthesis of tert-butyl (S) -2- ((2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) carbamoyl) pyrrolidine-1-carboxylate (2P)

Proline (0.269g,1.16mmol) and DMF10mL were added to a single vial, stirred to dissolve, HATU (0.44g,1.16mmol) and triethylamine (0.17mL) were added, stirred at room temperature for 20min, and (7b) (0.5g,1.06mmol) was slowly added to react at 25 ℃ for 24h, TLC was used to monitor the reaction, after completion of the reaction, the reaction mixture was poured into 100mL of ice water, stirred, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and dried under vacuum to give a beige solid, which was chromatographed on a silica gel column (ethyl acetate: petroleum ether (2:1)) to give 0.14mg of a final beige solid product with a yield of 19.7%. ¹ H NMR(400MHz,Chloroform-d)δ9.73(s,1H),8.99(s,1H),8.69(s,1H),8.36(d,J＝5.3Hz,1H),8.07(d,J＝7.4Hz,1H),7.40(dd,J＝6.6,2.4Hz,1H),7.32–7.26(m,2H),7.20(d,J＝5.4Hz,1H),6.73(s,1H),6.59(d,J＝26.6Hz,2H),6.50(d,J＝1.9Hz,1H),5.77(dd,J＝9.9,1.9Hz,1H),5.08(s,1H),4.19(q,J＝7.4Hz,1H),3.98(s,3H),3.90(s,3H),3.50–3.40(m,1H),3.29(dd,J＝14.0,6.1Hz,1H),3.06(t,J＝6.1Hz,2H),2.65(s,3H),2.57–2.43(m,2H),2.07(s,3H),1.83–1.83(m,1H),1.40(s,9H). ¹³ C NMR(101MHz,Chloroform-d)δ170.41,161.23,159.92,157.83,153.71,143.41,136.16,135.46,131.04,124.78,124.26,123.75,120.29,119.35,111.04,108.69,105.71,102.45,76.61,54.37,53.42,51.96,41.26,35.50,31.33,30.41,28.25,26.57,22.11,13.21.

After reacting compound 7b (0.5g,1.06mmol) with isoleucine (268mg,1.16mmol), 184mg of the objective compound 2EL was purified as a white solid in 25.4% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.75(s,1H),9.00(s,1H),8.74(s,1H),8.36(d,J＝5.3Hz,1H),8.07(d,J＝8.7Hz,1H),7.76(s,1H),7.41-7.37(m,1H),7.31-7.26(m,2H),7.20(d,J＝5.4Hz,1H),6.73(s,1H),6.55(d,J＝9.8Hz,1H),6.49(d,J＝2.0Hz,1H),6.28(s,1H),5.77(dd,J＝9.8,2.0Hz,1H),4.97(s,1H),3.98(s,3H),3.89(s,3H),3.82(t,J＝7.8z Hz,1H),3.39-3.48(m,1H),3.32-3.19(m,1H),3.05(t,J＝6.2Hz,2H),2.65(s,3H),1.92-1.83(m,1H),1.41(s,11H),1.14–1.05(m,1H),0.91-0.85(m,6H). ¹³ C NMR(101MHz,Chloroform-d)δ163.15,162.44,159.32,157.41,144.65,138.35,135.40,132.81,128.39,126.16,121.94,121.11,120.38,113.69,110.20,107.96,103.82,56.28,47.27,44.47,38.72,36.77,36.61,33.19,28.48,24.80,20.96.

Compound 7b (0.5g,1.06mmol) and methionine (289mg,1.16mmol) were reacted and purified to give 181mg of the title compound 2S as a white solid in 24.3% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.20(d,J＝9.0Hz,1H),8.89(s,1H),8.59(d,J＝4.1Hz,1H),8.27(d,J＝5.3Hz,1H),8.22(d,J＝8.0Hz,1H),8.03-7.90(m,2H),7.49(d,J＝8.2Hz,1H),7.20(dd,J＝8.1,6.3Hz,2H),7.12(t,J＝7.5Hz,1H),6.92(s,1H),6.73(dd,J＝17.0,10.1Hz,1H),6.22(dd,J＝16.9,2.0Hz,1H),5.71(dd,J＝12.24,5.48Hz,1H),4.07-3.95(m,2H),3.87(s,3H),3.82(s,3H),3.29-3.16(m,4H),3.01-2.94(m,2H),2.66(s,3H),2.12-2.12(s,1H),1.99(s,1H),1.79-1.66(m,3H),1.34(s,3H),1.25(s,6H). ¹³ C NMR(101MHz,CHLOROFORM-D)δ163.08,162.23,159.64,157.97,144.47,138.34,135.21,132.84,128.47,127.47,126.07,121.88,121.02,120.35,113.74,110.16,108.04,103.77,80.59,77.48,77.36,77.16,76.84,60.50,56.25,52.76,47.25,44.46,36.61,33.16,28.47,21.14,14.29,1.12,0.09.

Compound 7b (0.5g,1.06mmol) and valine (251mg,1.16mmol) were reacted and purified to give 204mg of the title compound, 2X, as a white solid in 28.7% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.76(s,1H),8.98(s,1H),8.72(s,1H),8.38(d,J＝5.3Hz,1H),8.08(d,J＝7.5Hz,1H),7.71(s,1H),7.42-7.37(m,1H),7.32-7.27(m,2H),7.20(d,J＝5.3Hz,1H),6.73(s,1H),6.60-6.44(m,2H),6.26(s,1H),5.77(dd,J＝9.6,2.2Hz,1H),4.98(s,1H),3.98(s,3H),3.89(s,3H),3.78(t,J＝7.7Hz,1H),3.44(dd,J＝13.7,6.6Hz,1H),3.28(s,1H),3.05(t,J＝6.1Hz,2H),2.65(s,3H),2.15-2.04(m,1H),1.41(s,9H),0.91(dd,J-＝12.6,6.8Hz,6H). ¹³ C NMR(101MHz,Chloroform-d)δ172.25,162.94,162.24,159.53,157.88,156.14,144.63,138.30,135.07,134.35,132.55,127.76,127.51,126.64,126.03,121.95,121.08,120.42,113.69,110.59,110.19,108.10,103.66,80.16,77.48,77.36,77.16,76.84,60.52,56.19,55.88,44.28,37.79,33.19,30.45,28.37,19.54,18.05,1.14,0.12.

From compound 7b (0.5g,1.06mmol) and phenylalanine(307mg,1.16mmol) and purified to give 172mg of the title compound 2B as a white solid in 22.6% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.71(s,1H),8.98(s,1H),8.66(s,1H),8.32(d,J＝5.7Hz,1H),8.06(d,J＝7.1Hz,1H),7.80(s,1H),7.38(dd,J＝7.5,1.6Hz,1H),7.31-7.27(m,2H),7.25-7.15(m,7H),6.70(s,1H),6.48-6.44(m,2H),6.24(s,1H),5.76(dd,J＝7.1,4.9Hz,1H),5.03(s,1H),3.96(s,3H),3.88(s,3H),3.35(dd,J＝13.6,6.5Hz,1H),3.26–3.16(m,1H),3.29-3.05(m,4H),2.62(s,3H),1.37(s,9H). ¹³ C NMR(101MHz,Chloroform-d)δ171.80,162.89,162.41,144.66,138.32,136.82,135.28,134.48,132.49,129.36,128.75,127.70,127.34,127.09,126.71,126.01,122.00,121.16,120.41,113.64,110.58,110.23,108.00,103.65,80.47,77.48,77.16,76.84,56.20,55.73,44.17,38.73,38.24,37.80,33.21,28.32.

After reacting compound 7b (0.5g,1.06mmol) with leucine (268mg,1.16mmol), it was purified to obtain 151mg of the objective compound 2L as a white solid in 24.4% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.71(s,1H),9.00(s,1H),8.74(s,1H),8.34(d,J＝5.3Hz,1H),8.07(dd,J＝9.5,3.1Hz,1H),7.84(s,1H),7.40(dd,J＝6.6,2.4Hz,1H),7.31-7.27(m,2H),7.20(d,J＝5.4Hz,1H),6.72(s,1H),6.67-6.54(m,2H),6.47(dd,J＝16.9,1.8Hz,1H),5.76(dd,J＝10.0,1.8Hz,1H),4.93(s,1H),4.16-4.06(m,1H),3.98(s,3H),3.89(s,3H),3.47-3.40(m,1H),3.32-3.23(m,1H),3.10–3.00(m,3H),2.65(s,3H),1.40(s,12H),1.32(d,J＝7.0Hz,3H),1.30-1.20(m,2H).

Compound 7b (0.5G,1.06mmol) and glycine (203mg,1.16mmol) were reacted and purified to give 122mg of the objective compound 2G as a white solid in 21.8% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.64(s,1H),8.98(s,1H),8.69(s,1H),8.32(d,J＝5.5Hz,1H),8.06(d,J＝7.4Hz,1H),7.90(s,1H),7.41-7.37(m,1H),7.32-7.27(m,2H),7.19(d,J＝5.5Hz,1H),6.73(s,1H),6.55(dd,J＝16.8,9.8Hz,1H),6.46(dd,J＝16.8,2.1Hz,2H),5.94-5.64(m,1H),5.17(s,1H),3.97(s,3H),3.89(s,3H),3.72(d,J＝6.0Hz,2H),3.39(q,J＝6.0Hz,2H),3.08(q,J＝7.1,6.0Hz,2H),2.66(s,3H),1.40(s,9H).

Reaction of compound 7b (0.5g,1.06mmol) with alanine (219mg,1.16mmol) purified 129mg of the title compound 2BA as a white solid in 22.5% yield。 ¹ H NMR(400MHz,Chloroform-d)δ9.71(s,1H),8.98(s,1H),8.66(s,1H),8.38-8.27(m,1H),8.06(d,J＝7.5Hz,1H),7.80(s,1H),7.40-7.36(m,1H),7.32-7.27(m,2H),7.16(s,1H),6.70(d,J＝1.4Hz,1H),6.50-6.42(m,2H),6.24(s,1H),5.76(dd,J＝7.1,4.9Hz,1H),5.03(s,1H),4.26(d,J＝7.5Hz,1H),3.96(s,3H),3.88(s,3H),3.05-2.92(m,4H),2.62(s,3H),1.36(s,9H),1.29-1.22(m,3H).

Synthesis of 3P, 3EL, 3S, 3X and 3B

Synthesis of tert-butyl (S) -2- ((3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) propyl) carbamoyl) pyrrolidine-1-carboxylate (3P)

Proline (0.22g,1.16mmol) and 10mL of DMF are added into a single-neck flask, stirred to be dissolved, HATU (0.44g,1.16mmol) and triethylamine (0.17mL) are added, stirred and activated for 20min at room temperature, 0.5g (1.06 mmol) (7c) is slowly added to react for 24h at 25 ℃, TLC is used for monitoring the reaction, after the reaction is finished, the reaction solution is poured into 100mL of ice water, stirred, extracted by ethyl acetate, washed by saline, dried by anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, dried in vacuum to obtain beige solid, and separated by silica gel column chromatography (ethyl acetate: petroleum ether (2:1)) to obtain the final beige solid product with the yield of 0.15g and 21.2%. ¹ H NMR(400MHz,Chloroform-d)δ9.77(s,1H),9.13(d,J＝15.8Hz,2H),8.32(s,1H),8.07-8.01(m,1H),7.43–7.39(m,1H),7.32-7.27(m,3H),7.21(d,J＝5.5Hz,1H),6.74(s,1H),6.63(s,1H),6.45(dd,J＝16.9,1.9Hz,1H),5.75(dd,J＝10.2,1.8Hz,1H),4.31(t,J＝6.7Hz,1H),4.26(s,1H),4.00(s,3H),3.90(s,3H),3.44-3.22(m,4H),2.98-2.86(m,2H),2.59(s,3H),2.34(s,1H),2.06(d,J＝21.4Hz,1H),1.95-1.78(m,4H),1.43(s,9H). ¹³ C NMR(101MHz,Chloroform-d)δ162.34,159.41,157.62,144.70,138.36,135.28,132.77,127.68,126.31,126.06,121.96,121.12,120.42,113.73,110.20,108.03,103.70,80.72,56.24,47.33,37.72,33.20,28.49.

From compound 7c (0.5g,1.06mmol) and isoleucine (268mg,1.16mmol)) After reaction and purification, 192mg of the objective compound 3EL was obtained as a white solid in 26.5% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.80(s,1H),9.09(d,J＝31.1Hz,2H),8.38–8.32(m,1H),8.09–8.00(m,1H),7.74(s,1H),7.42-7.36(m,1H),7.31-7.26(m,2H),7.24(d,J＝5.6Hz,2H),7.22-7.18(m,1H),6.74(s,1H),6.48–6.42(m,1H),5.77-5.71(m,1H),4.34-4.18(m,1H),3.99(d,J＝1.1Hz,3H),3.88(d,J＝1.0Hz,3H),2.98-2.86(m,4H),3.00-2.85(m,3H),2.80(d,J＝1.0Hz,1H),2.58(s,3H),2.09(d,J＝1.1Hz,1H),1.87(s,4H),1.75-1.69(m,1H),1.43(s,10H). ¹³ C NMR(101MHz,Chloroform-d)δ172.31,162.98,162.30,159.48,157.73,156.09,144.69,138.33,135.11,134.49,132.61,127.76,127.50,126.55,126.05,121.96,121.10,120.43,113.70,110.73,110.18,108.07,103.73,80.18,59.75,56.22,55.90,44.22,37.81,36.93,36.68,33.16,28.38,24.83,15.78,11.31.

From the reaction of compound 7c (0.5g,1.06mmol) and methionine (289mg,1.16mmol), 211mg of the title compound 3S was purified as a white solid in 27.8% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.80(s,1H),9.04(s,2H),8.36(d,J＝5.3Hz,1H),8.12-8.00(m,1H),7.81(s,1H),7.42-7.38(m,1H),7.31-7.27(m,2H),7.23(d,J＝7.7Hz,1H),7.20(d,J＝5.4Hz,1H),6.74(s,1H),6.58(dd,J＝16.8,9.9Hz,1H),6.46(dd,J＝16.8,1.9Hz,1H),6.23(s,1H),5.75(dd,J＝9.9,2.0Hz,1H),5.13(s,1H),4.25-4.07(m,1H),3.99(s,3H),3.89(s,3H),3.38-3.23(m,2H),2.95-2.88(m,2H),2.60(s,3H),2.58–2.48(m,2H),2.13-2.03(m,5H),1.95-1.84(m,2H),1.42(s,9H).

From the reaction of compound 7c (0.5g,1.06mmol) and valine (251mg,1.16mmol), 184mg of the objective compound 3X was purified as a white solid in 25.4% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.80(s,1H),9.09(s,2H),8.34(s,1H),8.06(s,2H),7.90(s,1H),7.40(s,1H),7.29(s,1H),7.21(s,1H),6.74(s,1H),6.67–6.52(m,1H),6.46(d,J＝16.2Hz,1H),6.00(s,1H),5.76(d,J＝8.3Hz,1H),4.98(s,1H),4.00(d,J＝4.0Hz,3H),3.90(s,3H),3.83(d,J＝7.3Hz,1H),3.31(s,2H),2.92(s,2H),2.60(s,3H),2.21-2.10(m,1H),1.43(s,11H),0.94(dd,J＝15.8,9.3Hz,6H). ¹³ C NMR(101MHz,CHLOROFORM-D)δ171.80,163.04,162.38,156.08,144.53,138.35,135.41,133.95,132.77,128.46,127.38,126.26,126.05,121.94,121.11,120.36,113.69,110.33,110.23,107.98,103.75,80.17,77.48,77.36,77.16,76.84,60.48,56.24,52.88,44.59,36.75,33.22,30.56,28.40,27.78,19.52,17.91.HR-MS(m/z)calculated for C ₃₇ H ₄₈ N ₈ O ₅ [M+H]+:685.3748,found 685.4

Compound 7c (0.5g,1.06mmol) and phenylalanine (307mg,1.16mmol) were reacted and purified to give 182mg of the title compound 3B as a white solid in 23.4% yield. ¹ H NMR(400MHz,Chloroform-d)δ9.78(s,1H),9.09(d,J＝12.3Hz,2H),8.31(s,1H),8.10(d,J＝6.8Hz,1H),8.05(d,J＝7.1Hz,1H),7.43-7.39(m,1H),7.33-7.27(m,3H),7.25-7.16(m,6H),6.72(s,1H),6.60(dd,J＝16.8,10.0Hz,1H),6.47(dd,J＝16.8,1.9Hz,1H),5.82(s,1H),5.77(dd,J＝9.9,1.9Hz,1H),5.00(s,1H),4.28(dd,J＝16.4,8.8Hz,1H),4.00(s,3H),3.90(s,3H),3.72(q,J＝7.0Hz,1H),3.30-3.14(m,2H),3.05(d,J＝7.0Hz,2H),2.83(t,J＝6.8Hz,2H),1.49(t,J＝6.8Hz,3H),1.39(s,9H),1.29-1.22(m,3H). ¹³ C NMR(101MHz,Chloroform-d)δ171.33,162.99,162.34,144.49,138.35,135.35,133.87,132.76,129.37,128.82,128.47,127.12,126.34,126.06,121.94,121.10,120.37,113.72,110.22,108.04,103.75,77.48,77.36,77.16,76.84,56.24,53.00,44.51,38.54,36.88,33.23,28.36,27.66.

Synthesis of 2PT, 2ET, 2XT and 2BT

Synthesis of (S) -N- (2- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) ethyl) pyrrolidine-2-carboxamide (2PT)

2P (0.133g,0.2mmol) and 2mL of DCM are added into a single-neck bottle, stirring is carried out to dissolve the mixture, 1mL of TFA is added into a cold well, stirring is carried out for 3h at room temperature, TLC is used for monitoring the reaction, after the reaction is finished, the pH value of a saturated sodium bicarbonate solution is adjusted to 9, suction filtration is carried out, a filter cake is dried in vacuum, and 0.105g of final beige solid is obtained, wherein the yield is 93%. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.93(s,1H),9.67(s,1H),9.15(d,J＝6.0Hz,1H),8.84(s,1H),8.50(s,1H),8.25(s,2H),7.59(d,J＝8.2Hz,1H),7.43(d,J＝6.7Hz,1H),7.31(t,J＝7.6Hz,1H),7.20(s,1H),7.06(s,1H),6.95(dd,J＝16.8,10.2Hz,1H),6.21(d,J＝16.9Hz,1H),5.73(d,J＝10.6Hz,1H),4.26(t,J＝7.2Hz,1H),3.93(s,3H),3.83(s,4H),3.38-3.31(m,2H),3.22-3.14(m,3H),2.77(s,3H),2.35-2.25(m,1H),1.90-1.64(m,3H),1.39-1.20(m,1H).

Boc removal of compound 2EL (137mg,0.2mmol) gave 101mg of the title compound 2ET as a yellow-green solid in 87.1% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.19(s,1H),8.96(s,1H),8.62(d,J＝11.8Hz,1H),8.32(d,J＝5.3Hz,1H),8.26(d,J＝8.0Hz,1H),8.06(s,1H),7.90(d,J＝6.1Hz,1H),7.52(d,J＝8.1Hz,1H),7.26-7.13(m,4H),6.96(s,1H),6.76(dd,J＝16.8,10.2Hz,1H),6.27(dd,J＝16.84,1.92Hz,1H),5.75(dd,J＝10.2,1.5,Hz,1H),3.91(s,4H),3.87(s,3H),3.86(s,1H),3.07(d,J＝5.4Hz,1H),2.99(t,J＝6.4Hz,2H),2.66(s,3H),1.07-0.99(m,1H),0.87-0.83(m,2H),0.82-0.74(m,6H).HR-MS(m/z)calculated for C ₃₂ H ₄₀ N ₈ O ₃ [M+H]+:585.3257,found 585.6.

Boc removal from compound 2X (134mg,0.2mmol) gave 99mg of the title compound 2XT as a white solid in 87.4% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.19(s,1H),8.96(s,1H),8.61(s,1H),8.32(d,J＝5.4Hz,1H),8.26(d,J＝8.1Hz,1H),7.97(t,J＝5.6Hz,1H),7.90(s,1H),7.52(d,J＝8.2Hz,1H),7.26-7.21(m,2H),7.16(t,J＝7.5Hz,1H),6.96(s,1H),6.76(dd,J＝16.9,10.3Hz,1H),6.26(dd,J＝17.0,2.0Hz,1H),5.75(dd,J＝10.1,2.0Hz,1H),3.91(s,3H),3.87(s,3H),3.28-3.20(m,2H),2.98(t,J＝6.4Hz,2H),2.93(d,J＝5.2Hz,1H),2.65(s,3H),1.90–1.81(m,2H),1.26-1.15(m,1H),0.83(d,J＝6.8Hz,3H),0.74(d,J＝6.8Hz,3H).HR-MS(m/z)calculated for C ₃₁ H ₃₈ N ₈ O ₃ [M+H]+:571.3100,found 571.4.

Boc removal of compound 2B (144mg,0.2mmol) gave 113mg of the title compound 2BT as a white solid in 92.0% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.26(s,1H),8.96(s,1H),8.61(s,1H),8.31(d,J＝5.3Hz,1H),8.26(d,J＝7.8Hz,1H),7.88(s,1H),7.52(d,J＝8.2Hz,1H),7.25-7.14(m,9H),6.94(s,1H),6.83(dd,J＝16.8,10.1Hz,1H),6.25(dd,J＝16.6,1.9Hz,1H),5.72(dd,J＝10.4,1.9Hz,1H),3.90(s,3H),3.87(s,3H),3.51(d,J＝6.8Hz,1H),3.22(s,2H),2.97-2.89(m,3H),2.63(s,3H),1.23(s,2H),0.94-0.81(m,1H).HR-MS(m/z)calculated for C ₃₅ H ₃₈ N ₈ O ₃ [M+H]+:619.3100,found 619.3.

Synthesis of 3PT, 3ET, 3XT and 3BT

Synthesis of (S) -N- (3- ((2-acrylamido-5-methoxy-4- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) (methyl) amino) propyl) pyrrolidine-2-carboxamide (3PT)

3P (0.136g,0.2mmol) and 2mL of DCM are added into a single-neck flask, stirring is carried out to dissolve, 1mL of TFA is added into a cold well, stirring is carried out for 3h at room temperature, the reaction is monitored by TLC, after the reaction is finished, the pH value is adjusted to 9 by saturated sodium bicarbonate solution, suction filtration is carried out, and a filter cake is dried in vacuum, so that 0.116g of final beige solid is obtained, and the yield is 88%. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.86(s,1H),9.62(s,1H),9.12(s,1H),8.84(s,1H),8.56–8.45(m,1H),8.35-8.15(m,2H),7.60(d,J＝8.3Hz,1H),7.42(d,J＝6.8Hz,1H),7.31(t,J＝7.6Hz,1H),7.20(s,1H),7.04(s,1H),6.94(dd,J＝16.9,10.2Hz,1H),6.20(d,J＝16.9Hz,1H),5.73(d,J＝10.7Hz,1H),4.30-4.21(m,1H),3.93(s,3H),3.83(s,4H),3.35(dd,J＝11.5,5.6Hz,2H),3.24-3.10(m,4H),2.76(s,3H),2.34-2.24(m,1H),2.00(q,J＝7.4Hz,1H),1.87-1.79(m,2H),1.77-1.67(m,1H),1.23(s,2H).

Boc removal of compound 3EL (139mg,0.2mmol) gave 120mg of the title compound 3ET as a yellow solid in 86.5% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.40(s,1H),8.97(s,1H),8.62(s,1H),8.32(d,J＝5.3Hz,1H),8.26(d,J＝8.0Hz,1H),7.89(t,J＝5.4Hz,2H),7.52(d,J＝8.1Hz,1H),7.26-7.21(m,2H),7.19-7.14(m,1H),6.92(s,1H),6.80(dd,J＝16.9,10.2Hz,1H),6.26(dd,J＝16.9,2.1Hz,1H),5.73(dd,J＝10.1,2.0Hz,1H),3.91(s,3H),3.86(s,3H),3.16-3.09(m,2H),3.00(d,J＝5.4Hz,1H),2.90(t,J＝6.9Hz,2H),2.58(s,3H),1.64–1.56(m,3H),1.44-1.37(m,1H),1.26-1.21(m,1H),1.10–0.99(m,1H),0.86-0.76(m,7H).

From compounds3X (134mg,0.2mmol) was Boc removed to give 108mg of the title compound, 3XT, as a yellow solid in 92.4% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.39(s,1H),8.97(s,1H),8.62(s,1H),8.32(d,J＝5.3Hz,1H),8.26(d,J＝8.0Hz,1H),7.89(s,1H),7.85(t,J＝5.9Hz,1H),7.52(d,J＝8.1Hz,1H),7.26-7.21(m,2H),7.19-7.14(m,1H),6.92(s,1H),6.80(dd,J＝16.9,10.2Hz,1H),6.25(dd,J＝17.0,2.0Hz,1H),5.73(dd,J＝10.1,2.0Hz,1H),3.91(s,3H),3.86(s,3H),3.15-3.08(m,1H),2.90(dd,J＝9.0,6.0Hz,3H),2.58(s,3H),1.90-1.82(m,1H),1.78(s,1H),1.63-1.54(m,1H),1.24-1.15(m,1H),0.86(d,J＝6.8Hz,3H),0.77(d,J＝6.8Hz,3H).HR-MS(m/z)calculated for C ₃₂ H ₄₀ N ₈ O ₃ [M+H]+:585.3257,found 585.4.

Boc removal of compound 3B (146mg,0.2mmol) gave 107mg of the title compound 3BT as a yellow solid in 84.7% yield. ¹ H NMR(400MHz,DMSO-d ₆ )δ9.38(s,1H),9.00(s,1H),8.62(s,1H),8.32(d,J＝5.3Hz,1H),8.26(d,J＝8.0Hz,1H),7.89-7.85(m,2H),7.73-7.65(m,1H),7.52(d,J＝8.1Hz,1H),7.27-7.14(m,9H),6.92(s,1H),6.80(dd,J＝16.9,10.2Hz,1H),6.26(dd,J＝16.9,2.0Hz,1H),5.74(dd,J＝10.5,2.0Hz,1H),4.14(dd,J＝5.8,3.4Hz,1H),3.90(s,3H),3.86(s,3H),3.13-3.06(m,2H),2.95-2.88(m,1H),2.84(t,J＝7.0Hz,2H),2.66-2.59(m,1H),2.58(s,3H).HR-MS(m/z)calculated for C ₃₆ H ₄₀ N ₈ O ₃ [M+H]+:633.3257,found 633.4.

The following are some of the pharmacological tests and results of representative compounds of the invention:

1. CCK-8 method for determining single-concentration inhibition rate of compound

In the experiment for detecting the influence of the compound on the in-vitro proliferation of the human tumor cells, cancer cells growing in the logarithmic phase are connected to a 96-well cell culture plate, 100 mu L of cell suspension is added into each well, the cell suspension is cultured for 24h and then is administrated, the administration concentration is set to be 10 mu M, CCK-8 solution is added after the drug acts for 72h, the OD value is measured at 450nm, and the inhibition rate is calculated. The results of the experiment are shown in table 1.

Table 1 compound inhibition data

ZWQ-5 is positive control AZD 9291.

As can be seen from Table 1: the compound concentration shows certain difference on the proliferation inhibition effect of H1975 tumor cells and PC9 tumor cells under the condition of 10 mu M because of different substituents. Among them, the inhibitory activities of the compounds ZL-1 and ZL-2 on two adenocarcinoma cells are mostly not ideal and are greatly different from positive control drugs. And RX-3, RX-4 and RX-5 in the compound have higher inhibition rate on two cells. In addition, RX-10, RX-11 and RX-12 in the compound relatively well inhibit tumor cells. The inhibition of 2EL and 3EL in the compound on tumor cells was slightly better than that of the positive control.

2. CCK-8 method for determining Compound IC ₅₀

According to the results of the preliminary screening, compounds RX-2, RX-3, RX-4, RX-5, RX-8, RX-9, RX-10, RX-11 and RX-12 with high single-concentration inhibition rate on H1975 cells and PC9 cells are selected to determine the half Inhibition Concentration (IC) of the compounds on the growth of tumor cells ₅₀ ) Selecting several compounds with better inhibition rate at the same time, and testing the IC of the compounds on wild type cell strain A549 ₅₀ Compound was plated in 3 duplicate wells per drug concentration and the results are shown in table 2.

IC of the Compounds of Table 2 on H1975 cells, PC9 cells and cell line A549 ₅₀ Data of

ZWQ-5 as positive control AZD9291

As can be seen from Table 2: the pair of compounds tested was H1975,PC9 cell has good cell proliferation inhibiting activity, and its IC ₅₀ The value was in the range of 3.448-12.799. mu.M. Among them, compound RX-5 has the best inhibitory effect on H1975, IC ₅₀ The best inhibition activity of RX-2 on PC-9 cells was obtained at 3.448. mu.M, IC ₅₀ The best inhibition activity of RX-2 on PC-9 cells, IC, was found at 3.448. mu.M ₅₀ It was worth further investigation at 4.075 μ M. By comparing the concentrations of the compounds over the wild type and mutant cells, it was found that the tested compounds were not highly selective for the mutant.

3. Study of inhibitory Activity of Compounds against kinases

In the experiment, the inhibition rate of the compound on EGFR (L858R/T790M) is measured by using a Kinase-Glo Plus luminescence Kinase assay kit when the concentration of the compound is 1 mu M, and the result is shown in Table 3.

TABLE 3 inhibition Rate data of EGFR by Compounds

ZWQ-5 as positive control AZD9291

As can be seen from Table 3, the tested compounds have certain inhibitory effect on EGFR (L858R/T790M) at a concentration of 1 μ M, wherein the inhibition rate of RX-5 is equivalent to that of the control and reaches 100%. We selected compounds RX-2, RX-3, RX-4, RX-5, RX-10 of which the inhibition rates were higher and further tested their IC's in EGFR (WT), EGFR (T790M) and EGFR (T790M/L858R) ₅₀ The value is obtained. The results are shown in tables 4, 5 and 6.

IC of Table 4 Compounds for EGFR (WT) ₅₀ Data of

ZWQ-5 as positive control AZD9291

IC of Table 5 Compounds for EGFR (T790M) ₅₀ Data of

ZWQ-5 as positive control AZD9291

IC of Table 6 Compounds on EGFR (T790M/L858R) ₅₀ Data of

ZWQ-5 as positive control drug AZD9291

As can be seen from tables 4, 5 and 6, the tested compounds showed the best effect on EGFR (WT), and the EGFR (T790M) was inferior and the EGFR (T790M/L858R) was inferior. Among them, compounds RX-5 showed the best inhibitory activity against EGFR (WT), EGFR (T790M), and EGFR (L858R/T790M), and IC ₅₀ Are 12.7 + -2.4, 13.2 + -3.2 and 46 + -9 nM, respectively. The inhibitory effect of compound RX-10 was less than ideal for the control.

4. Detection of phosphorylation levels of EGFR and related proteins by Western blot method

We selected compound RX-5, and after it acted on H1975 cells 24, we examined the expression of p-EGFR protein in H1975 cells, and the results are shown in FIG. 1.

As is clear from FIG. 1, compound RX-5 has an inhibitory effect on the expression of p-EGFR protein in H1975 cells, and the inhibition of the expression of p-EGFR protein in H1975 cells is more significant as the concentration of compound RX-5 is increased.

5PI single staining method for detecting cell cycle

In order to examine the blocking of the compound RX-5 on the H1975 cell cycle, the cell cycle tissue condition of the compound RX-5 after acting on the H1975 cells for 72 hours is tested by a flow cytometer. The results are shown in FIG. 2.

As can be seen from FIG. 2, as the concentration of compound RX-5 increased, the cycle of H1975 cells was arrested in the G2/M phase, and H1975 cells exhibited concentration dependence, as compared to the blank control, indicating that the compound could inhibit lung cancer by arresting the cell cycle of lung cancer cells.

6 detection of hydrogen sulfide release by hydrogen sulfide fluorescent probe

This experiment was carried out by H ₂ S fluorescent Probe WSP-1, test Compound RX-10 for H at a concentration of 50. mu.M ₂ S release levels, the results are shown in figure 3.

As can be seen from fig. 3, panel a is a blank of the control group, panel b is the control group, panel c is a blank of the experimental group, and panel d is the experimental group. By comparing the experimental group with the control group, we found that the fluorescence of the experimental group is enhanced, indicating that RX-10 can release H ₂ S。

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

x represents hydrogen, amino, -NH-R ¹ 、-NH-CH ₂ -R ¹ 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ¹ 、-NH-CO-CH ₂ -CH ₂ -R ¹ 、-NH-R ² 、-NH-CH ₂ -R ² 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ² 、-NH-CO-CH ₂ -CH ₂ -R ² 、-NH-Boc、C ₁ -C ₄ Alkyl or nitro;

y represents oxygen, a covalent bond, -CH ₂ -、-CH ₂ -CH ₂ -、-NH-、-CO-NH-、-NH-CO-、-NH-CH ₂ -、-NH-CH ₂ -CH ₂ -or-NH-CO-CH ₂ -CH ₂ -；

Z represents hydrogen、C ₁ -C ₄ Alkyl, -NH-Boc, phenyl, -S-CH ₃ 、-S-CH ₂ -CH ₃ 、C ₁ -C ₄ Alkoxy radical, R ¹ Or R ² ；

n represents 1,2, 3 or 4;

the R is ¹ Or R ² The structural formula of (A) is as follows:

2. a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X represents hydrogen, amino, -NH-R ¹ 、-NH-Boc、-NH-CH ₂ -R ¹ 、-NH-CO-CH ₂ -R ¹ 、-NH-CH ₂ -CH ₂ -R ¹ or-NH-CO-CH ₂ -CH ₂ -R ¹ (ii) a Preferably, X represents hydrogen, amino, -NH-Boc or-NH-CO-CH ₂ -CH ₂ -R ¹ 。

3. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Y represents oxygen, a covalent bond, -CH ₂ -、-NH-、-NH-CH ₂ -or-NH-CH ₂ -CH ₂ -; preferably, Y represents oxygen, a covalent bond, -CH ₂ -or-NH-.

4. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Z represents hydrogen, methyl, ethyl, isopropyl, sec-butyl, isobutyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² (ii) a Preferably, Z represents hydrogen, isopropyl, sec-butyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² 。

5. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n represents 2, 3 or 4; preferably, n represents 2 or 3.

6. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X represents hydrogen, amino, -NH-Boc or-NH-CO-CH ₂ -CH ₂ -R ¹ (ii) a Y represents oxygen, a covalent bond, -CH ₂ -or-NH-; z represents hydrogen, isopropyl, sec-butyl, -NH-Boc, phenyl, -S-CH ₃ 、R ¹ Or R ² (ii) a n represents 2 or 3.

7. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from:

8. a pharmaceutical composition comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof as an active ingredient or a major active ingredient, together with a pharmaceutically acceptable carrier.

9. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with a tumour.

10. The use according to claim 9, of a compound or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for an EGFR inhibitor.

Images