A pyrrolopyrimidine compound, a preparation method and application thereof

Abstract

The application discloses a pyrrolopyrimidine compound and a preparation method and application thereof, and belongs to the technical field of medicines. The pyrrolopyrimidine compound provided by the application has good EGFR kinase inhibiting capacity, and thus can be used as an active ingredient for preparing a therapeutic drug for diseases caused by abnormal activation of EGFR kinase, and the pyrrolopyrimidine compound has a strong effect of inhibiting the proliferation of lung cancer cell strains.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a pyrrolopyrimidine compound and a preparation method and application thereof. BACKGROUND

[0002] Non-small cell lung cancer (NSCLC) accounts for about 80-85% of total lung cancer, mainly including squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Compared with small cell lung cancer, NSCLC cell proliferation and diffusion speed is slower, but it is still one of the main causes of cancer-related deaths.

[0003] With the development of molecular medicine, targeted therapy has become an important treatment strategy for advanced NSCLC. Among them, epidermal growth factor receptor (EGFR) mutation is one of the most common driver gene mutations in NSCLC, accounting for about 40-50% in Asian non-smoking lung adenocarcinoma patients. EGFR mutation can abnormally activate the downstream signaling pathway, promote tumor cell growth and survival.

[0004] Targeted drugs for EGFR mutation, such as first and second generation EGFR tyrosine kinase inhibitors (TKIs) - Gefitinib and Erlotinib have been widely used in clinical. However, patients usually develop drug resistance due to T790M mutation after 9-12 months of treatment, leading to disease progression.

[0005] Osimertinib (AZD9291) is the third generation irreversible EGFR-TKI, which can selectively inhibit EGFR T790M mutations, and also has good inhibitory effect on common activating mutations of EGFR (such as L858R and exon 19 deletion mutation). Osimertinib inhibits mutant EGFR by covalently binding to ATP binding site, while it has weak effect on wild-type EGFR, thereby reducing common adverse reactions such as skin rash and diarrhea. Based on the good efficacy of AURA study, Osimertinib has become the first-line treatment for patients with EGFR mutant NSCLC. However, some patients may still develop disease progression after receiving Osimertinib treatment due to new drug resistance mechanisms such as EGFR C797S mutations, MET or HER2 gene amplification.

[0006] To address the treatment challenges after osimertinib resistance, new generation EGFR-TKIs are being developed. For example, BU-945 is a new, highly selective EGFR inhibitor designed to overcome C797S mutation resistance. Studies have shown that BU-945 has a strong inhibitory effect on C797S mutant EGFR and has shown good safety and tolerability in preclinical studies. In addition, BU-945 has less effect on wild-type EGFR, thereby reducing related adverse reactions, making it a promising treatment option for drug-resistant patients.

[0007] Although EGFR-TKIs have significantly prolonged the survival of patients with EGFR-mutant NSCLC, drug resistance remains a challenge that cannot be ignored. Osimertinib successfully overcomes T790M mutation resistance, and the development of new inhibitors such as BU-945 provides new treatment hope for C797S mutant patients. In the future, through combination therapy or new drug development, it is expected to further optimize the treatment strategy for EGFR-mutant NSCLC and improve the treatment effect of patients. SUMMARY

[0008] The purpose of the present application is to provide a pyrrolopyrimidine compound and its preparation method and application. The provided pyrrolopyrimidine compound has high EGFR single-target antitumor activity.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] One of the technical solutions of the present application: a pyrrolopyrimidine compound is provided, which has a structural formula as shown in formula I or formula II:

[0011]

[0012] In formula I, R1 is

[0013] In formula II, when R3 is , R2 is

[0014] When R3 is , R2 is

[0015] When R3 is , R2 is

[0016] When R3 is , R2 is The second technical solution of the present application: a preparation method of the above-mentioned pyrrolopyrimidine compound is provided,

[0017] The synthesis route of the compound of formula I is as follows:

[0018]

[0019] In the above route, compound 2 is prepared by a nucleophilic substitution reaction; compound 3 is prepared by an aromatic nucleophilic substitution reaction; compound 4 is prepared by an amidation reaction; compound 5 is prepared by an acylation reaction;

[0020] The synthesis route of the compound of formula II is as follows:

[0021]

[0022] In the above route, compound 2 is prepared by a nucleophilic substitution reaction; compounds 3a-3d are prepared by an aromatic nucleophilic substitution reaction; compounds 4a-4d are prepared by an amidation reaction.

[0023] The names of the 42 compounds of formula I and formula II of the present application are as follows:

[0024] [1] N-acryloyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0025] [2] Ethyl 4-oxo-4-(2-(4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazino)butyrate;

[0026] [3] N-(4-methoxybenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0027] [4] N-(cyclopropylcarbonyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0028] [5] N-(2-methoxyacetyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0029] [6] N-valeryl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0030] [7] N-isobutyryl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide;

[0031] [8] N-(cyclohexanecarbonyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0032] [9] 4-((4-phenoxyphenyl)amino)-N-propionyl-7H-pyrrolo[2,3-d]pyrimidine-7- carbohydrazide;

[0033]

[10] N-heptanoyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7- carbohydrazide;

[0034]

[11] N-(3-methoxybenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0035]

[12] N-(4-methylbenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0036]

[13] N-(2-chloropropionyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0037]

[14] N-(4-chlorobutyryl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0038]

[15] N-(cyclobutenecarbonyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0039]

[16] N-(2-methylbenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0040]

[17] N-(4-methylpentanoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide;

[0041]

[18] 2-oxo-2-(2-(4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7- carbonyl)hydrazino)acetic acid ethyl ester;

[0042]

[19] 4-((4-phenoxyphenyl)amino)-N-(2-(thiophen-2-yl)ethyl)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide;

[0043]

[20] N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0044]

[21] N-(thiazol-2-yl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0045]

[22] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0046]

[23] N-(1-methoxybutan-2-yl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0047]

[24] 4-((4-phenoxyphenyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0048]

[25] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0049]

[26] 4-((4-(3-oxomorpholino)phenyl)amino)-N-(pyridin-2-ylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0050]

[27] 4-((4-(3-oxomorpholino)phenyl)amino)-N-(2-(thiazol-5-yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0051]

[28] 4-((4-(3-oxomorpholino)phenyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0052]

[29] N-(1-methoxybutyl)-4-((4-(3-oxomorpholino)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0053]

[30] N-(2-methylthiazol-5-yl)-4-((4-(3-oxomorpholino)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0054]

[31] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((4-(3-oxomorpholinyl)phenyl)amino)-7H- pyrrolo [2, 3-d] pyrimidine-7-carboxamide;

[0055]

[32] N-(1-isopropylpiperidin-4-yl)-4-((4-(3-oxomorpholinyl)phenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide;

[0056]

[33] N-(1-acetylpiperidin-4-yl)-4-((4-(3-oxomorpholinyl)phenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide;

[0057]

[34] N-(1-acetylpiperidin-4-yl)-4-((4-(4-fluorophenoxy)phenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide;

[0058]

[35] N-(5-cyclopropyl-1H-pyrazol-3-yl)-4-((4-(4-fluorophenoxy)phenyl)amino)-7H- pyrrolo [2, 3-d] pyrimidine-7-carboxamide;

[0059]

[36] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-4-((4-(4-fluorophenoxy)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0060]

[37] 4-((4-(4-fluorophenoxy)phenyl)amino)-N-(1-(methylsulfonyl)piperidin-4-yl)-7H- pyrrolo [2, 3-d] pyrimidine-7-carboxamide;

[0061]

[38] 4-((4-(4-fluorophenoxy)phenyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide;

[0062]

[39] 4-[(4-(4-fluorophenoxy)phenyl)amino]-N-[1-(methylsulfonyl)piperidin-3-yl]-7H- pyrrolo [2, 3-d] pyrimidine-7-carboxamide;

[0063]

[40] N-(1-(methylsulfonyl)piperidin-4-yl)-4-((4-(thiophen-2-yl)phenyl)amino)-7H- pyrrolo [2, 3-d] pyrimidine-7-carboxamide;

[0064]

[41] N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-4-((4-(thiophen-2-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide;

[0065]

[42] N-(lH-indol-5-yl)-4-((4-(thiophen-2-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide.

[0066] The second aspect of the present application provides a preparation method of the pyrrolopyrimidine compound,

[0067] The synthesis route of the compound shown in Formula I is as follows:

[0068]

[0069] In the above route, compound 2 is prepared by a nucleophilic substitution reaction; compound 3 is prepared by an aromatic nucleophilic substitution reaction; compound 4 is prepared by an amidation reaction; and compound 5 is prepared by an acylation reaction.

[0070] The synthesis route of the compound shown in Formula II is as follows:

[0071]

[0072] In the above route, compound 2 is prepared by a nucleophilic substitution reaction; compounds 3a-3d are prepared by an aromatic nucleophilic substitution reaction; and compounds 4a-4d are prepared by an amidation reaction.

[0073] The third aspect of the present application provides a pharmaceutically acceptable salt of the pyrrolopyrimidine compound.

[0074] The fourth aspect of the present application provides a pharmaceutical preparation, wherein the active ingredient comprises the pyrrolopyrimidine compound and / or the pharmaceutically acceptable salt of the pyrrolopyrimidine compound.

[0075] Optionally, the dosage form of the pharmaceutical preparation includes, but is not limited to, an injection, a tablet, a capsule, an aerosol, a suppository, a film, a dripping pill, an external preparation, an ointment, or an enteric-coated tablet.

[0076] The fifth aspect of the present application provides a use of the pyrrolopyrimidine compound in the preparation of a medicament for treating and / or preventing cancer.

[0077] It is expected that the cancer to be treated and / or prevented includes breast cancer, lung cancer, liver cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, prostate cancer, bladder cancer, uterine cancer, pancreatic cancer, etc.

[0078] Preferably, the cancer is lung cancer.

[0079] The sixth aspect of the present application provides a use of the above-mentioned pyrrolopyrimidine compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing cancer.

[0080] It is expected that the cancer to be treated and / or prevented includes breast cancer, lung cancer, liver cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, prostate cancer, bladder cancer, uterine cancer, pancreatic cancer, etc.

[0081] Preferably, the cancer is lung cancer.

[0082] The seventh aspect of the present application provides a use of the above-mentioned pharmaceutical preparation in the preparation of a medicament for treating and / or preventing cancer.

[0083] It is expected that the cancer to be treated and / or prevented includes breast cancer, lung cancer, liver cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, prostate cancer, bladder cancer, uterine cancer, pancreatic cancer, etc.

[0084] Preferably, the cancer is lung cancer.

[0085] The beneficial technical effects of the present application are as follows:

[0086] The pyrrolopyrimidine compound provided by the present application has good ability to inhibit EGFR kinase, and thus can be used as an active ingredient for preparing a therapeutic drug for diseases caused by abnormal activation of EGFR kinase. In addition, the pyrrolopyrimidine compound provided by the present application has strong effect of inhibiting the proliferation of lung cancer cell lines. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 The results of the analysis of the structure-activity relationship of the pyrrolopyrimidine compound prepared for Experimental Example 42 and EGFR, wherein A is the results of molecular docking, B is the molecular surface electrostatic potential map, and C is the molecular docking score and RMSD data.

[0088] Figure 2 The effect of the pyrrolopyrimidine compound prepared for Experimental Example 42 on the morphology of cancer cells.

[0089] Figure 3 The effect of the pyrrolopyrimidine compound prepared for Experimental Example 42 on the apoptosis of cancer cells detected by flow cytometry.

[0090] Figure 4 The effect of the pyrrolopyrimidine compound prepared for Experimental Example 42 on the cell cycle of cancer cells detected by flow cytometry.

[0091] Figure 5 The effect of the pyrrolopyrimidine compound prepared for Experimental Example 42 on the content of reactive oxygen species in H1975 cells detected by DCFH-DA probe.

[0092] Figure 6 Figure 1 shows the effect of the pyrrolopyrimidine compounds prepared for Example 42 on the mitochondrial membrane potential of cancer cells. DETAILED DESCRIPTION

[0093] The following detailed description of various example embodiments of the application is not to be considered limiting of the scope or spirit of the application, but rather as a description of its some aspects, features and embodiments. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0094] It should be noted that the present application is not limited in detail to the description thereof as there are numerous variations of the described embodiments within the scope of the present application. Also, various omissions, substitutions and changes in the form of the detail of the methods and materials as set forth are contemplated as being

[0095] Further, for the purposes of the present application, a range of values includes each and every value within the range. Clear of any otherwise explicitly provided range or explicit value, the endpoints are included in the ranges.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. Any and all

[0097] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, and specifically do not exclude additional, unrecited elements or method steps.

[0098] In the embodiments of the present application, "room temperature" and "normal temperature" both refer to a temperature of 20±5℃.

[0099] The synthetic route of the pyrrolopyrimidine compounds (L1-L18) of Examples 1-18 of the present application is shown in Figure Route 1:

[0100] Route 1

[0101]

[0102] The synthetic route of the pyrrolopyrimidine compounds (L19-L42) of Examples 19-42 of the present application is shown in Figure Route 2 (wherein the intermediate 3 of L19-L25 is intermediate 3a, the intermediate 3 of L26-L33 is intermediate 3b, the intermediate 3 of L34-L39 is intermediate 3c, and the intermediate 3 of L40-L42 is intermediate 3d):

[0103] Route 2

[0104]

[0105] The proton nuclear magnetic resonance spectra of each compound in the embodiments of this invention ( 1 H NMR, carbon spectrum 13 ¹³C NMR was performed using a Bruker ARX 400, and mass spectrometry (MS) was performed using an Agilent 1100LC / MSD. All reagents used were of analytical or chemically pure grade.

[0106] Example 1

[0107] Preparation of N-acryloyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxyhydrazide:

[0108] Step 1: Preparation of phenyl 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid ester (intermediate 2)

[0109] Place the prepared ice bath apparatus on a stirrer at room temperature. To reach 0°C, NaCl can be added to the ice water to lower the temperature. Weigh 4 g (25.56 mmol) of 4-chloro-7H-pyrrolopyrimidine (starting material 1) and place it in a round-bottom flask containing 60 mL of tetrahydrofuran. Add 3 mL of triethylamine, and then add 1.6 equivalents of phenyl chloroformate (6.4 g, 40.896 mmol) dropwise. Stir in an ice bath for four hours and monitor the reaction by TLC. After the reaction is complete, add petroleum ether solvent and sonicate. Filter under vacuum, wash the filter cake with the filtrate, and air-dry to obtain a white solid, i.e., intermediate 2, with a yield of 90%.

[0110] Step 2: Preparation of phenyl 4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid ester (intermediate 3)

[0111] Under nitrogen protection, intermediate 2 (2.1 g, 13.5 mmol) was dissolved in 80 mL of ultra-dry 1,4-dioxane, and 20 mL of saturated potassium carbonate aqueous solution was added to make the reaction system weakly alkaline. 0.19 g of 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene and 0.38 mmol of 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) were added. The reaction mixture was heated to 95 °C, and then 4-phenoxyaniline was added. The reaction was carried out for 8 hours, monitored by TLC. After the reaction was complete, most of the organic solvent was removed by rotary evaporation under reduced pressure. Water was added and the mixture was filtered. The filter residue was dried to obtain a yellow crude extract, which was purified by column chromatography to obtain a white solid, intermediate 3, in 65% yield.

[0112] Step 3: Preparation of 4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbamoylhydrazine (intermediate 4)

[0113] Intermediate 3 (2.0 g, 6.45 mmol) and hydrazine hydrate (1.2 g, 7.74 mmol) were dissolved in 60 mL of anhydrous ethanol, and 3 drops of DIPEA were added dropwise to adjust the reaction system to weak alkaline. Stirring at 25 °C for 3 h. The reaction mixture was added with water to obtain a solid-liquid mixture, and the precipitation of solid could be improved by adjusting the ratio of ethanol and water. White solid was obtained by suction filtration, which was intermediate 4, with a yield of 84.2%.

[0114] Step four: preparation of N-acryloyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxyhydrazide

[0115] Intermediate 4 (1 g, 13.6 mmol) was dissolved in 30 mL of super-dry dichloromethane under stirring at room temperature, 1.3 mL of triethylamine was added under stirring, and acrylamide dissolved in super-dry dichloromethane was added dropwise. The reaction solution was observed to change from clear to turbid, and solid precipitated. After the reaction was completed, the solvent was rotary evaporated, NaHCO3 aqueous solution was added, and the mixture was extracted with ethyl acetate and water. The organic layer was concentrated, and the solvent was rotary evaporated under reduced pressure to obtain a solid-liquid mixture. After the solid was dissolved by adding a small amount of ethyl acetate and ultrasonic treatment, an appropriate amount of petroleum ether was added to precipitate the solid. White solid was obtained by suction filtration, which was N-acryloyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxyhydrazide.

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 10.77 (s, 1H), 9.95 (s, 1H), 8.48 (d, J = 2.7 Hz, 1H), 7.82 (dd, J = 9.1, 2.7 Hz, 2H), 7.75 (d, J = 3.8 Hz, 1H), 7.39 (td, J = 7.8, 2.6 Hz, 2H), 7.16 - 7.07 (m, 3H), 7.06 (d, J = 2.5 Hz, 2H), 7.04 - 6.98 (m, 2H), 6.37 (dd, J = 17.1, 10.0 Hz, 1H), 6.31 - 6.21 (m, 1H), 5.81 (dd, J = 9.9, 2.5 Hz, 1H). HRMS (ESI) (m / z): [M+H]+: calcd for C 22 H 18 N6O3: 415.1519, found: 415.1518.

[0117] Example 2

[0118] Preparation of ethyl 4-oxo-4-(2-(4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbonyl)hydrazino)butanoate:

[0119] According to the method of Example 1, the acrylamide in step four was replaced with an equal molar amount of 4-oxobutyryl chloride, to obtain.

[0120] 1 H NMR (400 MHz, DMSO-d6) δ 10.95 (d, J = 2.1 Hz, 1H), 10.47 (d, J = 2.2 Hz, 1H), 9.86 (d, J = 5.4 Hz, 1H), 8.48 (d, J = 5.6 Hz, 1H), 7.85 - 7.79 (m, 2H), 7.74 (t, J = 4.5 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.16 - 6.98 (m, 5H), 4.06 (q, J = 7.2 Hz, 2H), 2.56 (dd, J = 13.5, 8.0 Hz, 4H), 1.18 (q, J = 7.0 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 172.51, 170.72, 157.79, 154.64, 152.47, 152.39, 150.51, 149.21, 135.41, 130.45, 123.52, 123.49, 122.19, 119.77, 118.43, 105.88, 103.18, 60.45, 29.04, 28.29, 14.53. HRMS (ESI) (m / z): [M+H]+: calcd for C25H24N6O5: 489.1886, found: 489.1895.

[0121] Example 3

[0122] Preparation of N-(4-methoxybenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide:

[0123] According to the method of Example 1, the acrylamide in step four was replaced with an equal molar amount of 4-oxobutyryl chloride, to obtain.

[0124] 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.82 (s, 1H), 9.92 (s, 1H), 8.50 (s, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 3.9 Hz, 1H), 7.39 (t, J = 7.7 Hz, 2H), 7.16 - 7.05 (m, 6H), 7.02 (d, J = 7.9 Hz, 2H), 3.84 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 165.39, 162.30, 157.30, 154.05, 152.27, 151.60, 150.61, 148.62, 134.72, 130.04, 129.57, 124.05, 123.37, 123.12, 121.95, 119.37, 118.04, 113.86, 105.51, 103.05, 55.47, 40.06. HRMS (ESI) (m / z): [M+H]+: calcd for C27H22N6O4: 495.1781, found: 495.1781.

[0125] Example 4

[0126] Preparation of N-(cyclopropylcarbonyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0127] According to the method of Example 1, the acrylamide in step four was replaced by equimolar amount of cyclopropylcarbonyl chloride, to give the title compound.

[0128] 1 H NMR (400 MHz, Methanol-d4) δ 10.92 (d, J = 2.0 Hz, 1H), 10.62 (d, J = 2.0 Hz, 1H), 10.04 (s, 1H), 8.47 (d, J = 1.9 Hz, 1H), 7.82 (dd, J = 8.9, 2.0 Hz, 2H), 7.75 (dd, J = 4.0, 1.9 Hz, 1H), 7.39 (ddd, J = 8.7, 7.4, 2.0 Hz, 2H), 7.16 - 7.07 (m, 3H), 7.07 (d, J = 2.0 Hz, 1H), 7.04 - 6.98 (m, 2H), 1.71 (td, J = 7.3, 3.7 Hz, 1H), 0.85 - 0.73 (m, 4H). 13C NMR (151 MHz, DMSO-d6) δ 174.49, 157.22, 153.66, 152.62, 150.82, 150.03, 148.02, 134.26, 130.06, 123.78, 123.18, 122.07, 119.38, 118.10, 105.49, 103.36, 41.84, 40.06, 28.94, 25.38, 25.13.

[0129] Example 5

[0130] Preparation of N-(2-methoxyacetyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0131] According to the method of Example 1, the acrylamide in step four was replaced by equimolar amount of 2-methoxyacetyl chloride, to give the title compound.

[0132] 1 H NMR (400 MHz, Acetonitrile-d3) δ 10.99 (s, 1H), 8.78 (s, 1H), 8.40 (s, 1H), 8.15 (s, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.66 (dd, J = 4.0, 1.1 Hz, 1H), 7.38 (tt, J = 7.4, 1.0 Hz, 2H), 7.13 (td, J = 7.4, 1.1 Hz, 1H), 7.04 (ddt, J = 8.7, 7.9, 1.0 Hz, 4H), 6.73 (d, J = 4.0 Hz, 1H), 4.05 (d, J = 1.1 Hz, 2H), 3.46 (d, J = 1.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 168.65, 157.34, 154.23, 152.05, 151.99, 150.17, 148.79, 134.95, 130.02, 123.10, 123.07, 121.77, 119.34, 118.00, 105.44, 102.78, 70.56, 58.81. HRMS (ESI) (m / z): [M+H]+: calcd for C22H20N6O4: 433.1624, found: 433.1624.

[0133] Example 6

[0134] Preparation of N-pentanoyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carbohydrazide:

[0135] Following the procedure of Example 1, acrylamide in step four was replaced with an equimolar amount of valeroyl chloride to give the title compound.

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 10.89 (d, J = 2.1 Hz, 1H), 10.33 (d, J = 2.2 Hz, 1H), 9.87 (d, J = 2.9 Hz, 1H), 8.47 (d, J = 2.9 Hz, 1H), 7.82 (dd, J = 9.1, 2.8 Hz, 2H), 7.73 (d, J = 3.8 Hz, 1H), 7.39 (ddd, J = 11.1, 6.0, 2.8 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 7.09 - 7.02 (m, 3H), 7.02 - 6.97 (m, 2H), 2.25 - 2.17 (m, 2H), 1.54 (p, J = 7.8, 7.4 Hz, 2H), 1.40 - 1.26 (m, 2H), 0.89 (td, J = 7.3, 2.8 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 171.82, 157.44, 154.29, 152.23, 152.00, 150.34, 148.87, 134.99, 130.17, 123.32, 123.23, 121.95, 119.48, 118.11, 105.55, 102.89, 32.90, 27.22, 21.82, 13.85. HRMS (ESI) (m / z): [M+H]+: calcd for C24H24N6O3: 445.1988, found: 445.1982.

[0137] Example 7

[0138] Preparation of N-isobutyryl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0139] Following the procedure of Example 1, acrylamide in step four was replaced with an equimolar amount of valeroyl chloride to give the title compound.

[0140] 1H NMR (400 MHz, Acetonitrile-d3) δ 10.96 (s, 1H), 8.42 (s, 1H), 8.37 (s, 1H), 8.14 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 3.9 Hz, 1H), 7.38 (t, J = 7.7 Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 7.07 - 6.99 (m, 4H), 6.70 (d, J = 4.1 Hz, 1H), 2.56 - 2.48 (m, 1H), 1.17 (t, J = 5.5 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 175.43, 157.34, 154.21, 152.02, 151.99, 150.79, 150.22, 148.80, 134.96, 130.02, 129.95, 123.09, 123.07, 121.96, 121.78, 119.48, 119.34, 117.99, 117.64, 105.43, 102.71, 32.06, 19.25.

[0141] Example 8

[0142] Preparation of N-(cyclohexanecarboxyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxyhydrazide

[0143] Following the procedure of Example 1, the acrylamide in Step Four was replaced with an equimolar amount of cyclohexanecarboxylic acid chloride to give the title compound.

[0144] 1 H NMR (400 MHz, Methanol-d4) δ 10.85 (s, 1H), 10.30 (s, 1H), 10.12 (s, 1H), 8.47 (d, J = 1.2 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 4.0 Hz, 1H), 7.39 (td, J = 7.3, 1.8 Hz, 2H), 7.16 - 6.98 (m, 6H), 2.28 (dd, J = 13.3, 9.7 Hz, 1H), 1.83 - 1.58 (m, 5H), 1.49 - 1.13 (m, 5H). 13C NMR (151 MHz, DMSO-d6) δ 173.94, 157.56, 154.04, 153.15, 151.20, 150.49, 148.51, 134.51, 130.51 (2C), 124.30, 123.68, 122.57 (2C), 119.80 (2C), 118.55 (2C), 105.85, 103.62, 37.00, 24.90 (2C), 18.42 (2C), 18.17. HRMS (ESI) (m / z): [M+H]+: calcd for C21H18N6O3: 403.1519, found: 403.1511.

[0145] Example 9

[0146] Preparation of 4-((4-phenoxyphenyl)amino)-N-propionyl-7H-pyrrolo[2,3- d]pyrimidine-7-carboxyhydrazide:

[0147] According to the method of Example 1, the acrylamide in step four was replaced by equimolar amount of propionyl chloride, to give the title compound.

[0148] 1 H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.31 (s, 1H), 9.84 (s, 1H), 8.47 (d, J = 2.2 Hz, 1H), 7.85 - 7.79 (m, 2H), 7.74 (t, J = 3.0 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.12 (t, J = 7.5 Hz, 1H), 7.09 - 7.04 (m, 2H), 7.02 (s, 2H), 7.00 (s, 1H), 2.28 - 2.18 (m, 1H), 1.07 (td, J = 7.5, 1.9 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 171.65, 156.72, 153.59, 151.40, 151.36, 149.59, 148.18, 134.35, 129.41 (2C), 122.47, 122.45, 121.17 (2C), 118.73 (2C), 117.37 (2C), 104.82, 102.12, 25.72, 8.91.

[0149] Example 10

[0150] Preparation of N-heptanoyl-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxyhydrazide:

[0151] Following the procedure of Example 1, acrylamide in step four was replaced with an equimolar amount of heptanoyl chloride to give the title compound.

[0152] 1 H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.29 (s, 1H), 9.83 (s, 1H), 8.47 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 3.9 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.16 - 6.98 (m, 6H), 2.22 (t, J = 7.3 Hz, 2H), 1.55 (q, J = 7.3 Hz, 2H), 1.29 (d, J = 6.9 Hz, 6H), 0.88 (t, J = 6.6 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 171.96, 157.78, 154.59, 152.51, 152.30, 150.60, 149.16, 135.35,, 130.46 (2C), 123.57, 123.51, 122.25 (2C), 119.78 (2C), 118.43 (2C), 105.89, 103.22, 33.51, 31.47, 28.68, 25.38, 22.47, 14.41.

[0153] Example 11

[0154] Preparation of N-(3-methoxybenzoyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxyhydrazide:

[0155] Following the procedure of Example 1, acrylamide in step four was replaced with an equimolar amount of 3-methoxybenzoyl chloride to give the title compound.

[0156] 1 H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.29 (s, 1H), 9.83 (s, 1H), 8.47 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 3.9 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.16 - 6.98 (m, 6H), 2.22 (t, J = 7.3 Hz, 2H), 1.55 (q, J = 7.3 Hz, 2H), 1.29 (d, J = 6.9 Hz, 6H), 0.88 (t, J = 6.6 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 166.08, 159.70, 157.78, 154.70, 152.49 (2C), 150.99, 149.34, 135.41, 133.77, 130.47 (2C), 130.26, 123.56, 123.52, 120.26, 119.80, 118.44 (2C), 114.34 (2C), 113.12 (2C), 105.93, 103.30, 55.81. HRMS (ESI) (m / z): [M+H]+: calcd for C27H22N6O4: 495.1781, found: 495.1775.

[0157] Example 12

[0158] Preparation of N-(4-methylbenzoyl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide:

[0159] According to the method of Example 1, the acrylamide in step four was replaced by equimolar amount of p-tolyl chloride, to give the title compound.

[0160] 1 H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 10.87 (s, 1H), 9.90 (s, 1H), 8.50 (s, 1H), 7.83 (d, J = 8.1 Hz, 4H), 7.78 (d, J = 4.1 Hz, 1H), 7.38 (dt, J = 14.5, 7.6 Hz, 4H), 7.13 (d, J = 7.2 Hz, 1H), 7.11 - 7.05 (m, 3H), 7.02 (d, J = 8.0 Hz, 2H), 2.39 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 165.15, 156.66, 153.39, 151.66, 150.92, 149.89, 147.95, 141.65, 134.05, 129.42, 128.51 (2C), 128.51 (2C), 127.02 (2C), 122.77, 122.49, 121.33 (2C), 118.74 (2C), 117.41 (2C), 104.89, 102.50, 20.47. HRMS (ESI) (m / z): [M+H]+: calcd for C27H22N6O3: 479.1832, found: 479.1820

[0161] Example 13

[0162] Preparation of N-(2-chloropropionyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0163] According to the method of Example 1, the acrylamide in step four was replaced by an equimolar amount of 2-chloropropionyl chloride to obtain the title compound.

[0164] 1 H NMR (400 MHz, DMSO-d6) δ 11.07 (d, J = 4.9 Hz, 1H), 10.94 (s, 1H), 10.07 (s, 1H), 8.49 (d, J = 5.0 Hz, 1H), 7.87 - 7.79 (m, 2H), 7.75 (d, J = 4.1 Hz, 1H), 7.45 - 7.35 (m, 2H), 7.12 (dd, J = 9.0, 5.7 Hz, 2H), 7.09 - 7.06 (m, 2H), 7.01 (d, J = 7.7 Hz, 2H), 4.66 (q, J = 6.6 Hz, 1H), 1.61 (d, J = 6.6 Hz, 3H). HRMS (ESI) (m / z): [M+H]+: calcd for C22H19N6O3Cl: 451.1285, found: 451.1285.

[0165] Example 14

[0166] Preparation of N-(4-chlorobutyryl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0167] According to the method of Example 1, the acrylamide in step four was replaced by an equimolar amount of 4-chlorobutyryl chloride to obtain the title compound.

[0168] 1 H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.43 (s, 1H), 9.85 (s, 1H), 8.47 (s, 1H), 7.82 (dd, J = 8.9, 2.5 Hz, 2H), 7.73 (d, J = 3.8 Hz, 1H), 7.39 (t, J = 8.0 Hz, 2H), 7.16 - 6.98 (m, 6H), 3.74 - 3.66 (m, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.01 (p, J = 7.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 171.08, 157.78, 154.65, 152.47, 152.41, 150.64, 149.24, 135.40, 130.47 (2C), 123.55, 123.52, 122.25 (2C), 119.79 (2C), 118.43 (2C), 105.88, 103.23, 45.16, 30.68, 28.43.

[0169] Example 15

[0170] Preparation of N-(2-methylbenzoyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0171] According to the method of Example 1, the acrylamide in step four was replaced by an equimolar amount of 2-methylbenzoyl chloride to give the title compound.

[0172] 1 H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 10.24 (s, 1H), 9.99 (s, 1H), 8.46 (s, 1H), 7.77 (dd, J = 31.4, 6.2 Hz, 3H), 7.39 (t, J = 7.6 Hz, 2H), 7.20 - 6.96 (m, 6H), 3.16 (t, J = 8.4 Hz, 1H), 2.15 (dt, J = 34.7, 9.4 Hz, 4H), 2.01 - 1.76 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 171.08, 157.78, 154.65, 152.47, 152.41, 150.64, 149.24, 135.40, 130.47 (2C), 123.55, 123.52, 122.25 (2C), 119.79 (2C), 118.43 (2C), 105.88, 103.23, 45.16, 30.68, 28.43.

[0173] Example 16

[0174] Preparation of N-(2-methylbenzoyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carbohydrazide:

[0175] According to the method of Example 1, the acrylamide in step four was replaced by an equimolar amount of 2-methylbenzoyl chloride to give the title compound.

[0176] 1H NMR (400 MHz, DMSO-d6) δ 11.03 (d, J = 1.9 Hz, 1H), 10.63 (s, 1H), 9.88 (s, 1H), 8.52 (d, J = 2.8 Hz, 1H), 7.84 (dd, J = 9.2, 2.7 Hz, 2H), 7.78 (d, J = 3.8 Hz, 1H), 7.53 - 7.47 (m, 1H), 7.46 - 7.35 (m, 3H), 7.32 (d, J = 7.8 Hz, 2H), 7.17 - 6.99 (m, 6H), 2.44 (d, J = 2.3 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 168.81, 157.83, 154.70, 152.49, 152.40, 150.90, 149.30, 136.55, 135.51, 134.62, 131.09 (2C), 130.66, 130.47 (2C), 128.02, 126.10, 123.55, 123.49, 122.19, 119.79 (2C), 118.40 (2C), 105.96, 103.56, 19.77.

[0177] Example 17

[0178] Preparation of N-(4-methylpentanoyl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0179] According to the method of Example 1, the acrylamide in step four was replaced with an equimolar amount of 4-methylpentanoyl chloride to give the title compound.

[0180] 1H NMR (400 MHz, DMSO-d6) δ 10.90 (d, J = 2.4 Hz, 1H), 10.35 (t, J = 2.5 Hz, 1H), 9.88 (d, J = 2.9 Hz, 1H), 8.47 (d, J = 3.0 Hz, 1H), 7.83 (dd, J = 9.2, 3.0 Hz, 2H), 7.74 (d, J = 3.7 Hz, 1H), 7.39 (td, J = 8.0, 2.8 Hz, 2H), 7.16 - 7.11 (m, 1H), 7.07 (dq, J = 6.9, 3.9 Hz, 3H), 7.04 - 6.99 (m, 2H), 2.27 - 2.18 (m, 2H), 1.57 (h, J = 6.3 Hz, 1H), 1.47 (dq, J = 9.4, 6.9, 4.8 Hz, 2H), 0.89 (dd, J = 6.6, 2.9 Hz, 6H). HRMS (ESI) (m / z): [M+H]+: calcd for C25H26N6O3: 459.2145, found: 459.2145.

[0181] Example 18

[0182] Preparation of 2-oxo-2-(2-(4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7- carbonyl)hydrazino)acetic acid ethyl ester:

[0183] According to the method of Example 1, the acrylamide in step four was replaced with equimolar amount of 2-oxo-2 acetoacetyl chloride, and the product was obtained.

[0184] 1 H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 2H), 10.66 (s, 2H), 9.89 (d, J = 13.9 Hz, 2H), 8.49 (d, J = 9.6 Hz, 2H), 7.91 - 7.72 (m, 7H), 7.40 (d, J = 7.7 Hz, 5H), 7.13 (d, J = 7.3 Hz, 3H), 7.07 (d, J = 8.6 Hz, 6H), 7.01 (d, J = 8.0 Hz, 5H), 4.65 (s, 3H), 2.11 (d, J = 4.7 Hz, 6H), 2.07 (s, 1H), 1.24 (d, J = 6.7 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 170.42, 166.85, 157.76, 154.68, 152.51, 152.44, 150.50, 149.25, 135.35, 130.48 (2C), 123.59, 123.54, 122.19 (2C), 119.78 (2C), 118.43 (2C), 105.86, 103.32, 61.56, 20.93. HRMS (ESI) (m / z): [M+H]+: calcd for C25H26N6O3: 459.2145, found: 459.2145.

[0185] Example 19

[0186] Preparation of 4-((4-phenoxyphenyl)amino)-N-(2-(thiophen-2-yl)ethyl)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0187] Step one: Preparation of phenyl 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (intermediate 2)

[0188] Put the prepared ice bath device on the room temperature stirrer, and add NaCl in ice water to reduce the temperature to 0°C. Weigh 4 g (25.56 mmol) of 4-chloro-7H-pyrrolopyrimidine (raw material 1) into a round-bottom flask containing 60 mL of tetrahydrofuran, then add 3 mL of triethylamine, and dropwise add 1.6 equivalents of phenyl chloroformate (6.4 g, 40.896 mmol). Stir in the ice bath for four hours, and monitor the reaction by TLC. After the reaction is complete, add petroleum ether solvent to ultrasonic, filter, wash the filter cake with the filtrate, and air dry to obtain a white solid, which is intermediate 2, with a yield of 90%.

[0189] Step two: Preparation of phenyl 4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (intermediate 3a)

[0190] Dissolve intermediate 2 (2.1 g, 13.5 mmol) in 80 mL of super-dry 1,4-dioxane under nitrogen protection, and add 20 mL of saturated potassium carbonate aqueous solution to make the reaction system weakly alkaline. Add 0.19 g of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (0.38 mmol), heat the reaction mixture to 95°C, and then add 4-phenoxyaniline after reaction. Stir for 8 hours, and monitor the reaction by TLC. After the reaction is complete, remove most of the organic solvent by rotary evaporation under reduced pressure, filter with water, dry the filter cake, and purify by column chromatography to obtain a white solid, which is intermediate 3a, with a yield of 65%.

[0191] Step three: Intermediate 3a (1.0 g, 2.5 mmol) was dissolved in dry acetonitrile (20 mL) to form a homogeneous solution. To the above solution, thienyl ethylamine (0.3 g, 2.75 mmol) was added under stirring condition. The reaction was continued under stirring condition. Triethylamine (0.5 mL) was added slowly to the reaction mixture for neutralization of the byproduct formed and to improve the efficiency of the reaction. The reaction mixture was stirred at 60 °C. The progress of the reaction was monitored by thin layer chromatography and completion of the reaction was confirmed. After completion of the reaction, the reaction solution was cooled to room temperature and the solution was washed with water to remove excess base and water soluble byproducts. The organic layer was extracted with ethyl acetate and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using ethyl acetate / pet ether mixture as eluent and the product was collected.

[0192] 1 H NMR (400 MHz, CDC13) δ 9.67 (s, 1H), 8.32 (s, 1H), 7.67 (d, J = 4.0 Hz, 1H), 7.53-7.42 (m, 2H), 7.37-7.28 (m, 2H), 7.17-7.09 (m, 1H), 7.09-6.99 (m, 5H), 6.92 (dd, J = 4.9, 3.1 Hz, 2H), 6.89 (d, J = 3.6 Hz, 1H), 6.04 (d, J = 4.0 Hz, 1H), 3.75 (q, J = 6.6 Hz, 2H), 3.18 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 157.83, 154.53, 152.32, 152.05, 150.65, 149.26, 141.41, 135.56, 130.47 (2C), 127.53, 126.13, 124.84, 123.49, 123.41, 122.45 (2C), 119.80 (2C), 118.39 (2C), 105.88, 102.08, 41.99, 29.82. HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 21 N5O2S: 456.1494, found: 456.1494.

[0193] Example 20

[0194] Preparation of N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborolan-5-yl)-4-((4- phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0195] Following the procedure of Example 19, the thienylethylamine in step three was replaced with an equimolar amount of 5-amino-benzo[c][l,2]oxaborolan-l(3H)-ol to give the title compound.

[0196] 1 H NMR (400 MHz, CD3CN) δ 11.76 (s, 1H), 9.88 (s, 1H), 9.31 (s, 1H), 8.58 (s, 1H), 8.06 (s, 1H), 7.84 (dd, J = 14.6, 8.5 Hz, 4H), 7.50 (d, J = 8.3 Hz, 1H), 7.42 (t, J = 7.8 Hz, 2H), 7.16 (d, J = 7.1 Hz, 1H), 7.11 (d, J = 8.9 Hz, 3H), 7.04 (d, J = 8.0 Hz, 2H), 5.02 (s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 157.82, 154.66, 152.42, 152.16, 150.22, 149.23, 148.23, 136.64, 135.48, 131.99, 130.48 (2C), 130.40, 123.50 (2C), 123.23, 122.63, 122.01, 119.81 (2C), 118.40 (2C), 118.08, 106.12, 102.90, 70.22. HRMS (ESI) (m / z): [M+H] + : calcd for C 26 H 20 N5O4B: 478.1691, found: 478.1689.

[0197] Example 21

[0198] Preparation of N-(thiazol-2-yl)-4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3- d]pyrimidine-7-carboxamide:

[0199] Following the procedure of Example 19, the thienylethylamine in step three was replaced with an equimolar amount of 5-methylthiazol-2-amine to give the title compound.

[0200] 1 H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.56 (s, 1H), 7.85 - 7.78 (m, 3H), 7.39 (t, J = 7.8 Hz, 2H), 7.23 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 7.9 Hz, 3H), 7.02 (d, J = 7.9 Hz, 2H), 2.40 (s, 3H).13 C NMR (151 MHz, DMSO-d6) δ 157.73, 154.68, 152.61, 152.42, 151.24, 149.39, 147.92, 135.80, 135.23, 130.49 (2C), 123.63 (2C), 123.56, 122.07, 119.75 (2C), 118.49 (2C), 106.10, 103.93, 11.68. HRMS (ESI) (m / z): [M+H] + : calcd for C 23 H 18 N6O2S: 443.1290, found: 443.1284.

[0201] Example 22

[0202] Preparation of N-(l-(methylsulfonyl)piperidin-4-yl)-4-((4- phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0203] Following the procedure of Example 19, the thienylethylamine in Step three was replaced with an equimolar amount of l-(methylsulfonyl)-piperidin-4-amine to give the title compound.

[0204] 1 H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.27 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.61 - 7.53 (m, 2H), 7.37 - 7.28 (m, 2H), 7.08 (tt, J = 7.5, 1.5 Hz, 1H), 7.00 - 6.92 (m, 2H), 6.90 - 6.83 (m, 2H), 6.79 (d, J = 8.5 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 3.81 (dp, J = 8.5, 7.0 Hz, 1H), 3.29 (qt, J = 12.3, 7.1 Hz, 4H), 2.84 (s, 3H), 1.87 (qd, J = 7.1, 1.2 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 157.82, 154.53, 152.34, 152.25, 149.89, 149.29, 135.53, 130.47 (2C), 128.51, 125.96, 123.49 (2C), 119.82 (2C), 118.39 (2C), 105.95, 102.15, 46.88, 44.48 (2C), 34.85, 31.24 (2C). HRMS (ESI) (m / z): [M+H] +: calcd for C 25 H 26 N6O4S: 507.1815, found: 507.1810.

[0205] Example 23

[0206] Preparation of N-(l-methoxybutan-2-yl)-4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0207] Following the procedure of Example 19, the thienylethylamine in Step three was replaced with an equimolar amount of l-methoxy-2-butylamine to give the product.

[0208] 1 H NMR (400 MHz, CD3CN) δ 9.47 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 4.5 Hz, 1H), 8.03 (s, 1H), 7.77 - 7.72 (m, 2H), 7.71 (d, J = 4.0 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 7.04 (t, J = 7.8 Hz, 4H), 6.66 (d, J = 4.0 Hz, 1H), 4.03 (d, J = 5.3 Hz, 1H), 3.52 (qd, J = 9.8, 4.7 Hz, 2H), 3.35 (s, 2H), 1.66 (dddt, J = 30.8, 23.6, 15.5, 7.1 Hz, 2H), 0.97 (dt, J = 16.2, 7.5 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 157.84, 154.53, 152.30, 152.23, 150.42, 149.30, 135.57, 130.46 (2C), 134.47, 123.39, 122.49 (2C), 119.83 (2C), 118.37 (2C), 102.02, 73.82, 58.97, 51.76, 24.80, 10.81. HRMS (ESI) (m / z): [M+H] + : calcd for C 24 H 25 N5O3: 432.2036, found: 432.2028.

[0209] Example 24

[0210] Preparation of 4-((4-phenoxyphenyl)amino)-N-(2-(pyrrolidin-l- yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0211] Following the procedure of Example 19, step three, the thienylethylamine was replaced with an equimolar amount of 2-(pyrrolidin-l-yl)ethanamine to give the title compound.

[0212] 1 H NMR (400 MHz, CDC13) δ 9.70 (d, J = 5.9 Hz, 1H), 8.42 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 3.9 Hz, 1H), 7.53 - 7.46 (m, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 7.08 - 7.01 (m, 5H), 6.07 (d, J = 4.0 Hz, 1H), 3.65 (q, J = 6.3 Hz, 2H), 2.78 (t, J = 6.7 Hz, 2H), 2.63 (d, J = 6.1 Hz, 4H), 1.81 (q, J = 3.5 Hz, 4H). HRMS (ESI) (m / z): [M + H] + : calcd for C 25 H 26 N6O2: 443.2195, found: 443.2199.

[0213] Example 25

[0214] Preparation of N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-4-((4- phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0215] Following the procedure of Example 19, step three, the thienylethylamine was replaced with an equimolar amount of 3-(aminomethyl)-4,6-dimethyl-2(lH)-pyridinone to give the title compound.

[0216] 1 H NMR (400 MHz, CDC13) δ 9.70 (d, J = 5.9 Hz, 1H), 8.42 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 3.9 Hz, 1H), 7.53 - 7.46 (m, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 7.08 - 7.01 (m, 5H), 6.07 (d, J = 4.0 Hz, 1H), 3.65 (q, J = 6.3 Hz, 2H), 2.78 (t, J = 6.7 Hz, 2H), 2.63 (d, J = 6.1 Hz, 4H), 1.81 (q, J = 3.5 Hz, 4H). HRMS (ESI) (m / z): [M + H] 13C NMR (151 MHz, DMSO-d6) δ 163.46, 157.85, 154.45, 152.24, 152.15, 150.47, 149.43, 149.19, 143.80, 135.60, 130.46 (2C), 123.45 (2C), 123.36, 122.54, 122.05, 119.82 (2C), 118.35 (2C), 118.09, 107.86, 105.87, 101.96, 36.25, 19.29, 18.71. HRMS (ESI) (m / z): [M+H] + : calcd for C 27 H 24 N6O3: 481.1988, found: 481.1986.

[0217] Example 26

[0218] Preparation of 4-((4-(3-oxymorpholinophenyl)amino)-N-(pyridin-2- ylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0219] Step one: Preparation of phenyl 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7- carboxylate (intermediate 2)

[0220] Put the prepared ice bath device on the room temperature stirrer, to achieve 0°C, add NaCl in ice water to reduce the temperature. Weigh 4 g (25.56 mmol) of 4-chloro-7H-pyrrolopyrimidine (raw material 1) into a round-bottom flask containing 60 mL of tetrahydrofuran, add 3 mL of triethylamine, and dropwise add 1.6 equivalents of phenyl chloroformate (6.4 g, 40.896 mmol), stir in the ice bath for four hours, and monitor the reaction by TLC. After the reaction is complete, add petroleum ether solvent to ultrasonic, filter, wash the filter cake with the filtrate, and air dry to obtain white solid, intermediate 2, with a yield of 90%.

[0221] Step two: Preparation of phenyl 4-((4-phenoxyphenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxylate (intermediate 3b)

[0222] Intermediate 2 (2.1 g, 13.5 mmol) was dissolved in 80 mL of super dry 1,4-dioxane under nitrogen protection, 20 mL saturated potassium carbonate aqueous solution was added to make the reaction system weakly alkaline. 0.19 g 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (0.38 mmol) were added, and the reaction mixture was heated to 95°C. After reaction, 4-morpholinone aniline was added, and the reaction was carried out for 8 hours and monitored by TLC. After the reaction was completed, most of the organic solvent was removed by rotary evaporation under reduced pressure, water was added and filtered, and the dry residue was obtained as a yellow crude product, which was purified by column chromatography to obtain a white solid, intermediate 3b, with a yield of 65%.

[0223] Step three: Intermediate 3b (1.0 g, 2.5 mmol) was dissolved in anhydrous acetonitrile (20 mL) to form a uniform solution. Under stirring conditions, 3-(aminomethyl)-4,6-dimethyl-2(1H)-pyridinone (0.3 g, 2.75 mmol) was added to the above solution, and stirring was continued. Triethylamine (0.5 mL) was slowly added to the reaction system to neutralize the generated by-products and improve the reaction efficiency. The reaction system was stirred at 60°C. The progress of the reaction was monitored by thin layer chromatography to confirm the completion of the reaction. After the reaction was completed, the reaction solution was cooled to room temperature, and the solution was washed with water to remove excess base and water-soluble by-products. The organic layer was extracted with ethyl acetate, and the organic phase was combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of ethyl acetate / petroleum ether as the eluent, and the product was collected.

[0224] 1 H NMR (400 MHz, DMSO-d6) δ 9.82 (d, J = 7.4 Hz, 2H), 8.63 (d, J = 3.0 Hz, 1H), 8.51-8.45 (m, 2H), 7.85 (dd, J = 9.3, 3.0 Hz, 3H), 7.75 (d, J = 3.8 Hz, 1H), 7.42-7.35 (m, 3H), 7.01 (t, J = 3.5 Hz, 1H), 4.67 (t, J = 4.6 Hz, 2H), 4.20 (d, J = 3.2 Hz, 2H), 3.98 (t, J = 4.9 Hz, 2H), 3.77-3.70 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 164.30, 152.31, 150.06, 148.77, 147.29, 147.25 (2C), 146.78, 133.59, 132.71, 124.07 (2C), 121.98 (2C), 120.53, 119.61 (2C), 103.99, 100.11, 66.09, 61.85, 47.45, 39.43. HRMS (ESI) (m / z): [M+H] + : calcd for C 23 H 21 N7O3: 444.1784, found: 444.1784.

[0225] Example 27

[0226] Preparation of 4-((4-(3-oxomorpholinyl)phenyl)amino)-N-(2-(thiazol-5-yl)ethyl)- 7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0227] Following the procedure of Example 26, 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone in Step three was replaced with equimolar amount of 2-(thiophen-2- yl)ethylamine to give the product.

[0228] 1 H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.66 (d, J = 6.0 Hz, 1H), 8.41 (s, 1H), 7.94 - 7.80 (m, 3H), 7.74 (d, J = 3.9 Hz, 1H), 7.45 - 7.30 (m, 4H), 6.98 (d, J = 3.9 Hz, 1H), 5.89 (s, 1H), 4.40 (d, J = 5.6 Hz, 2H), 4.20 (d, J = 2.1 Hz, 3H), 3.98 (t, J = 5.0 Hz, 3H), 3.73 (t, J = 5.1 Hz, 3H), 2.27 (s, 3H), 2.11 (s, 3H). HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 25 N7O4: 488.2046, found: 488.2040.

[0229] Example 28

[0230] Preparation of 4-((4-(3-oxomorpholinyl)phenyl)amino)-N-(2-(pyrrolidin-1- yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0231] Following the procedure of Example 26, 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone in Step three was replaced with an equimolar amount of 2-(pyrrolidin-l- yl)ethanamine to give the title compound.

[0232] 1 H NMR (400 MHz, DMSO-d6) δ 9.78 (d, J = 2.8 Hz, 1H), 9.50 (d, J = 6.2 Hz, 1H), 8.46 (d, J = 2.9 Hz, 1H), 7.85 (dd, J = 9.2, 2.6 Hz, 2H), 7.75 (t, J = 3.4 Hz, 1H), 7.42 - 7.30 (m, 2H), 7.00 (t, J = 3.5 Hz, 1H), 4.21 (d, J = 2.7 Hz, 2H), 3.98 (dd, J = 6.0, 3.8 Hz, 2H), 3.77 - 3.70 (m, 2H), 3.51 (q, J = 5.9 Hz, 2H), 2.69 - 2.63 (m, 2H), 1.74 - 1.66 (m, 4H). HRMS (ESI) (m / z): [M+H] + : calcd for C 23 H 27 N7O3: 450.2254, found: 450.2255.

[0233] Example 29

[0234] Preparation of N-(l-methoxybutyl)-4-((4-(3-oxomorpholino)phenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0235] Following the procedure of Example 26, 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone in Step three was replaced with an equimolar amount of 1-methoxy-2- butanamine to give the title compound.

[0236] 1 H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.40 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 2.1 Hz, 1H), 7.95 - 7.82 (m, 2H), 7.75 (d, J = 3.9 Hz, 1H), 7.43 - 7.36 (m, 2H), 7.01 (d, J = 3.9 Hz, 1H), 4.21 (d, J = 2.3 Hz, 2H), 3.99 (t, J = 5.0 Hz, 3H), 3.74 (t, J = 4.9 Hz, 2H), 3.57 - 3.43 (m, 2H), 3.35 (s, 3H), 1.66 (dq, J = 31.9, 7.1 Hz, 2H), 0.95 (t, J = 7.4 Hz, 3H).13 C NMR(151MHz,DMSO-d6)δ154.45,152.17,150.39,149.36,138.00,137.12,126.21,122.65,121.76 ,106.18,102.00,73.81,68.23,63.99,58.97,51.77,49.58,24.80,10.80.HRMS(ESI)(m / z):[M+H] + :calcd for C 22 H 26 N6O4:439.2094, found:439.2090.

[0237] Example 30

[0238] Preparation of N-(2-methylthiazolyl-5-yl)-4-((4-(3-oxomorpholino)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0239] Following the method of Example 26, in step three, 3-(aminomethyl)-4,6-dimethyl-2(1H)-pyridone was replaced with an equimolar amount of 5-methylthiazol-2-amine to obtain the final product.

[0240] 1 H NMR (400MHz, DMSO-d6) δ8.56(d,J=4.8Hz,1H),8.34(s,1H),7.86-7.78(m,1H),7.49(d,J=7.9Hz,1H),7.37-7.25(m ,4H),6.97(dd,J=5.0,3.5Hz,1H),6.90(d,J=8.0Hz,3H),6.10(t,J=5.8Hz,1H),5.10(s,2H),2.95(t,J=7.0Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ166.44,154.58(2C),152.37(2C),149.47,137.72,137.37,135.86,126.2 2(2C),122.24,121.94(2C),106.31,103.90,68.23,63.99,49.55,11.69.HRMS(ESI)(m / z):[M+H] + :calcd for C 21 H 19 N7O3S:450.1348,found:450.1349.

[0241] Example 31

[0242] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((4-(3-oxomorpholinyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0243] Following the procedure of Example 26, replace 3-(aminomethyl)-4,6-dimethyl-2(1H)- pyridinone in Step Three with an equimolar amount of 1-(methylsulfonyl)-piperidin-4-amine to afford the title compound.

[0244] 1 H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.49 (d, J = 7.1 Hz, 1H), 8.49 (s, 1H), 7.87 - 7.82 (m, 2H), 7.75 (d, J = 4.0 Hz, 1H), 7.42 - 7.37 (m, 2H), 7.01 (d, J = 4.0 Hz, 1H), 4.21 (s, 2H), 3.98 (t, J = 5.1 Hz, 3H), 3.74 (t, J = 5.0 Hz, 2H), 3.52 (d, J = 11.7 Hz, 2H), 3.02 (t, J = 11.2 Hz, 2H), 2.92 (d, J = 3.1 Hz, 3H), 2.08 (d, J = 12.5 Hz, 2H), 1.70 (s, 1H), 1.66 (d, J = 10.1 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 166.43, 154.44, 152.18, 149.86, 149.35, 137.97, 137.14, 130.22, (2C) 126.21, 121.77 (2C), 106.14, 102.13, 68.22, 63.99, 49.58, 46.88, 44.47 (2C), 34.83, 31.23 (2C). HRMS (ESI) (m / z): [M+H] + : calcd for C 23 H 27 O5N7S: 514.1873, found: 514.1874.

[0245] Example 32

[0246] Preparation of N-(1-(methylsulfonyl)piperidin-4-yl)-4-((4-(3-oxomorpholinyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0247] Following the procedure of Example 26, replace 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone in Step Three with an equimolar amount of 1 -isopropylpiperidin-4-amine to give the title compound.

[0248] 1 H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.46 (d, J = 7.4 Hz, 1H), 8.47 (s, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 3.9 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 3.8 Hz, 1H), 4.21 (s, 2H), 3.98 (t, J = 4.9 Hz, 2H), 3.74 (t, J = 5.2 Hz, 3H), 2.72 (q, J = 7.5, 6.8 Hz, 3H), 2.31 (t, J = 10.0 Hz, 2H), 1.96 (d, J = 11.9 Hz, 2H), 1.56 (q, J = 10.5 Hz, 2H), 0.98 (d, J = 6.5 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 166.43, 154.44, 152.07, 149.70, 149.31, 137.99, 137.12, 126.20 (2C), 122.53, 121.76 (2C), 106.17, 101.97, 68.23, 63.99, 54.19, 49.57 (2C), 47.83, 46.66, 32.43 (2C), 18.44 (2C). HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 31 N7O3: 478.2567, found: 478.2567.

[0249] Example 33

[0250] Preparation of N-(l-acetylpiperidin-4-yl)-4-((4-(3-oxomorpholino)phenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0251] Following the procedure of Example 26, replace 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone in Step Three with an equimolar amount of 1 -acetylpiperidin-4-amine to give the title compound.

[0252] 1H NMR (400 MHz, DMSO-d6) δ 9.81 (s, 1H), 9.48 (d, J = 7.6 Hz, 1H), 8.47 (s, 1H), 7.86 (s, 2H), 7.84 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 4.0 Hz, 1H), 7.39 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 4.1 Hz, 1H), 4.20 (s, 3H), 3.99 (d, J = 6.5 Hz, 4H), 3.74 (d, J = 5.5 Hz, 4H), 2.03 (s, 7H). 13 CNMR (151 MHz, DMSO-d6) δ 168.59, 166.43, 154.43, 152.11, 149.79, 149.32, 137.97, 137.13, 126.20 (2C), 122.52, 121.77 (2C), 106.15, 102.08, 68.22, 63.99, 49.57 (2C), 47.49, 44.67, 32.36, 31.72, 21.75. HRMS (ESI) (m / z): [M+H] + : calcd for C 24 H27N7O4: 478.2203, found: 478.2206.

[0253] Example 34

[0254] Preparation of N-(l-acetylpiperidin-4-yl)-4-((4-(4-fluorophenoxy)phenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0255] Step one: Preparation of phenyl 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (Intermediate 2)

[0256] Put the prepared ice bath device on the room temperature stirrer, to achieve 0°C, add NaCl in ice water to reduce the temperature. Weigh 4 g (25.56 mmol) of 4-chloro-7H-pyrrolopyrimidine (raw material 1) into a round-bottom flask containing 60 mL of tetrahydrofuran, then add 3 mL of triethylamine, dropwise add 1.6 equivalents of phenyl chloroformate (6.4 g, 40.896 mmol), stir in the ice bath for four hours, monitor the reaction by TLC. After the reaction is completed, add petroleum ether solvent to ultrasonic, filter, wash the filter cake with the filtrate, and air dry to obtain a white solid, which is intermediate 2, with a yield of 90%.

[0257] Step two: Preparation of phenyl 4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7- carboxylate (Intermediate 3c)

[0258] Under nitrogen protection, intermediate 2 (2.1 g, 13.5 mmol) was dissolved in 80 mL of super dry 1,4-dioxane, 20 mL of saturated potassium carbonate aqueous solution was added to make the reaction system weakly alkaline. 0.19 g of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (0.38 mmol) were added, and the reaction mixture was heated to 95°C. After reaction, (4-(4-fluorophenoxy)) aniline was added, and the reaction was carried out for 8 hours, which was monitored by TLC. After the reaction was completed, most of the organic solvent was removed by rotary evaporation under reduced pressure, water was added and filtered, and the dry residue was obtained as a yellow crude product, which was purified by column chromatography to obtain a white solid, intermediate 3c, with a yield of 65%.

[0259] Step three: Intermediate 3c (1.0 g, 2.5 mmol) was dissolved in anhydrous acetonitrile (20 mL) to form a uniform solution. Under stirring conditions, 1-acetylpiperidin-4-amine (0.3 g, 2.75 mmol) was added to the above solution, and stirring was continued. Triethylamine (0.5 mL) was slowly added to the reaction system to neutralize the generated byproducts and improve the reaction efficiency. The reaction system was stirred at 60°C. The progress of the reaction was monitored by thin layer chromatography to confirm the completion of the reaction. After the reaction was completed, the reaction solution was cooled to room temperature, and the solution was washed with water to remove excess base and water-soluble byproducts. The organic layer was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of ethyl acetate / petroleum ether as the eluent, and the product was collected.

[0260] 1 H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.49 (d, J = 7.5 Hz, 1H), 8.44 (s, 1H), 7.84 - 7.76 (m, 2H), 7.73 (d, J = 3.9 Hz, 1H), 7.23 (t, J = 8.6 Hz, 3H), 7.10 - 6.99 (m, 5H), 6.96 (d, J = 4.0 Hz, 1H), 4.15 (d, J = 13.5 Hz, 1H), 4.02 (s, 1H), 3.77 (d, J = 13.7 Hz, 1H), 3.27 (s, 1H), 2.94 (t, J = 11.9 Hz, 1H), 2.06 - 1.92 (m, 5H), 1.58 (d, J = 11.5 Hz, 1H), 1.42 (d, J = 11.7 Hz, 1H). HRMS (ESI) (m / z): [M+H] + : calcd for C 26 H 25 N6O3F: 489.2050, found: 489.2049.

[0261] Example 35

[0262] Preparation of N-(5-cyclopropyl-lH-pyrazol-3-yl)-4-((4-(4-fluorophenoxy)phenyl)amino)- 7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0263] Following the procedure of Example 34, replace 1-acetylpiperidin-4-amine in Step Three with an equimolar amount of 5-cyclopropyl-lH-pyrazol-3-amine to give the title compound.

[0264] 1 H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 11.73 (s, 1H), 9.85 (s, 1H), 8.53 (s, 1H), 7.82 (d, J = 8.7 Hz, 3H), 7.23 (t, J = 8.7 Hz, 2H), 7.07 (dd, J = 10.4, 6.3 Hz, 5H), 6.24 (s, 1H), 1.92 (s, 1H), 0.95 (d, J = 8.2 Hz, 2H), 0.72 (d, J = 5.2 Hz, 2H). HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 20 N7O2F: 470.1741, found: 470.1715.

[0265] Example 36

[0266] Preparation of N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-4-((4-(4- fluorophenoxy)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0267] Following the procedure of Example 34, replace 1-acetylpiperidin-4-amine in Step Three with an equimolar amount of 3-(aminomethyl)-4,6-dimethyl-2(lH)-pyridinone to give the title compound.

[0268] 1H NMR (400 MHz, Chloroform-d) δ 9.59 (d, J = 7.5 Hz, 1H), 8.54 - 8.48 (m, 1H), 8.33 (d, J = 1.7 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.59 (q, J = 8.4, 6.5 Hz, 3H), 7.28 (s, 1H), 7.17 (d, J = 1.6 Hz, 4H), 6.93 (d, J = 8.8 Hz, 1H), 6.88 (s, 1H), 6.02 (d, J = 3.7 Hz, 1H), 5.22 (s, 2H), 3.97 (s, 1H), 3.67 (d, J = 12.5 Hz, 2H), 2.91 (t, J = 11.3 Hz, 2H), 2.74 (d, J = 1.7 Hz, 3H), 2.11 (d, J = 12.9 Hz, 2H), 1.75 (d, J = 11.5 Hz, 2H).

[0269] Example 37

[0270] Preparation of 4-((4-(4-fluorophenoxy)phenyl)amino)-N-(1-(methylsulfonyl)piperidin-4- yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0271] Following the procedure of Example 34, Step three, 1-acetylpiperidin-4-amine was replaced with an equimolar amount of 1-(methylsulfonyl)-piperidin-4-amine to give the title compound.

[0272] 1 H NMR (400 MHz, Chloroform-d) δ 9.70 (d, J = 7.6 Hz, 1H), 8.42 (s, 1H), 7.69 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.08 - 6.99 (m, 7H), 6.93 (s, 1H), 6.10 (d, J = 4.0 Hz, 1H), 4.06 (s, 1H), 3.77 (d, J = 12.5 Hz, 2H), 3.00 (t, J = 11.4 Hz, 2H), 2.83 (s, 3H), 2.20 (d, J = 13.0 Hz, 2H), 1.84 (d, J = 11.6 Hz, 2H).

[0273] Example 38

[0274] Preparation of 4-((4-(4-fluorophenoxy)phenyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0275] Following the procedure of Example 34, the 1-acetylpiperidin-4-amine in Step three was replaced with an equimolar amount of 2-(pyrrolidin-l-yl)ethanamine to give the title compound.

[0276] 1 H NMR (400 MHz, Chloroform-d) δ 9.70 (s, 1H), 8.41 (s, 1H), 7.71 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.09 - 6.98 (m, 7H), 6.08 (d, J = 3.9 Hz, 1H), 3.65 (q, J = 6.3 Hz, 2H), 2.80 (t, J = 6.7 Hz, 2H), 2.63 (d, J = 5.9 Hz, 4H), 1.81 (d, J = 5.7 Hz, 4H). HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 25 FN6O2: 461.2101, found: 41.2101.

[0277] Example 39

[0278] Preparation of 4-[(4-(4-fluorophenoxy)phenyl)amino]-N-[l-(methylsulfonyl)piperidin-3-yl]-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0279] Following the procedure of Example 34, the 1-acetylpiperidin-4-amine in Step three was replaced with an equimolar amount of l-(methylsulfonyl)-piperidin-3-amine to give the title compound.

[0280] 1 H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.61 (d, J = 6.8 Hz, 1H), 8.59 (d, J = 4.8 Hz, 1H), 8.47 (s, 1H), 8.04 (s, 1H), 7.88 (t, J = 7.7 Hz, 1H), 7.74 (d, J = 3.9 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.26 (d, J = 9.1 Hz, 1H), 6.95 (d, J = 3.9 Hz, 1H), 5.27 (s, 2H), 4.51 (q, J = 5.8 Hz, 1H), 3.62 (dd, J = 10.5, 6.1 Hz, 1H), 3.43 (d, J = 7.5 Hz, 2H), 2.30 (dd, J = 13.5, 6.9 Hz, 1H), 2.03 (m, 1H). HRMS (ESI) (m / z): [M+H] +: calcd for C 24 H 24 N7O4SCl: 542.1377, found: 542.1374.

[0281] Example 40

[0282] Preparation of N-(l-(methylsulfonyl)piperidin-4-yl)-4-((4-(thiophen-2-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide.

[0283] Step one: Preparation of phenyl 4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (Intermediate 2)

[0284] Put the prepared ice bath device on the room temperature stirrer, and add NaCl to the ice water to lower the temperature to 0°C. Weigh 4 g (25.56 mmol) of 4-chloro-7H-pyrrolopyrimidine (raw material 1) into a round-bottom flask containing 60 mL of tetrahydrofuran, and then add 3 mL of triethylamine. Dropwise add 1.6 equivalents of phenyl chloroformate (6.4 g, 40.896 mmol), and stir in the ice bath for four hours. Monitor the reaction by TLC. After the reaction is complete, add petroleum ether solvent and ultrasonic, filter, wash the filter cake with the filtrate, and air dry to obtain a white solid, which is intermediate 2, with a yield of 90%.

[0285] Step two: Preparation of phenyl 4-((4-phenoxyphenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (Intermediate 3d)

[0286] Dissolve intermediate 2 (2.1 g, 13.5 mmol) in 80 mL of super-dry 1,4-dioxane under nitrogen protection, and add 20 mL of saturated potassium carbonate aqueous solution to make the reaction system weakly alkaline. Add 0.19 g of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (0.38 mmol), heat the reaction mixture to 95°C, and then add thiophene aniline after reaction. Monitor the reaction by TLC for 8 hours. After the reaction is complete, remove most of the organic solvent under reduced pressure, filter with water, dry the filter cake, and purify by column chromatography to obtain a white solid, which is intermediate 3d, with a yield of 65%.

[0287] Step three: Intermediate 3d (1.0 g, 2.5 mmol) was dissolved in dry acetonitrile (20 mL) to form a homogeneous solution. To the above solution, 1-(methylsulfonyl)-piperidin-4-amine (0.3 g, 2.75 mmol) was added under stirring condition. The reaction was continued under stirring. To the reaction, triethylamine (0.5 mL) was added slowly to neutralize the side product formed and to improve the efficiency of the reaction. The reaction was stirred at 60 °C. The progress of the reaction was monitored by thin layer chromatography to confirm the completion of the reaction. After completion of the reaction, the reaction solution was cooled to room temperature and the solution was washed with water to remove excess base and water soluble side products. The organic layer was extracted with ethyl acetate and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using ethyl acetate / pet. ether mixture as eluent and the product was collected.

[0288] 1 H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.43 (d, J = 7.3 Hz, 1H), 8.46 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 4.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.43 (dd, J = 16.1, 4.4 Hz, 2H), 7.07 (t, J = 4.4 Hz, 1H), 6.98 (d, J = 4.0 Hz, 1H), 3.86 (s, 1H), 3.46 (d, J = 11.7 Hz, 2H), 2.95 (t, J = 11.2 Hz, 2H), 2.86 (s, 3H), 2.02 (d, J = 12.0 Hz, 2H), 1.63 (d, J = 11.2 Hz, 2H).

[0289] Example 41

[0290] Preparation of N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-4-((4- (thiophen-2-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0291] Following the procedure of Example 40, 1-(methylsulfonyl)-piperidin-4-amine in step three was replaced with equimolar quantity of 3-(aminomethyl)-4,6-dimethyl-2(lH)- pyridinone to obtain the title compound.

[0292] 1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.81 (s, 1H), 8.44 (s, 1H), 7.94 (dd, J = 29.1, 8.4 Hz, 3H), 7.75 (d, J = 3.9 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.51 - 7.44 (m, 3H), 7.13 (dd, J = 5.0, 3.6 Hz, 2H), 7.01 (d, J = 4.0 Hz, 1H), 5.89 (s, 1H), 4.40 (d, J = 5.6 Hz, 2H), 2.27 (s, 3H), 2.11 (s, 3H).

[0293] Example 42

[0294] Preparation of N-(lH-indol-5-yl)-4-((4-(thiophen-2-yl)phenyl)amino)-7H- pyrrolo[2,3-d]pyrimidine-7-carboxamide:

[0295] Following the procedure of Example 40, Step three, 1-(methylsulfonyl)-piperidin-4-amine was replaced with an equimolar amount of 5-aminoindole to give the title compound.

[0296] 1 H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 11.16 (s, 1H), 9.94 (s, 1H), 8.62 (d, J = 2.6 Hz, 1H), 7.98 - 7.85 (m, 5H), 7.69 (d, J = 8.6 Hz, 2H), 7.54 - 7.41 (m, 4H), 7.39 (t, J = 2.8 Hz, 1H), 7.34 - 7.27 (m, 1H), 7.17 - 7.08 (m, 2H), 6.46 (s, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 153.92 (2C), 151.55, 148.80 (2C), 147.80, 143.31, 138.82, 133.33, 128.90, 128.61, 128.39 (2C), 127.74, 126.40, 125.69, 124.86, 122.85, 122.18, 121.30 (2C), 115.26, 111.71, 111.63, 105.93, 101.86, 101.50, 101.18. HRMS (ESI) (m / z): [M+H] + : calcd for C 25 H 18 N6OS: 451.1341, found: 451.1348.

[0297] The structural formula of the pyrrolopyrimidine compounds prepared in Examples 1-42 is shown in Table 1.

[0298] Table 1 Structural formula of pyrrolopyrimidine prepared in Examples 1-42

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] The in vitro anti-tumor activity of the pyrrolopyrimidine compounds prepared in Examples 1-42 was determined:

[0305] The selected cancer cells were lung cancer cells H1975, lung cancer cells A549 and lung cancer cells PC-9, and the control was Osimertinib.

[0306] (1) After the cells were recovered and subcultured for 3 times, they were digested from the bottom of the culture bottle with trypsin solution (0.25%). The cell digestion solution was aspirated into a centrifuge tube, and then culture medium was added to terminate digestion. After centrifuging the centrifuge tube at 1000 r / min for 3 min, the supernatant was discarded, 3 mL of culture medium was added, the cells were mixed by blowing, 10 μL of cell suspension was aspirated and added to a cell counting plate for counting, and the cell concentration was adjusted to 10 4 cells / well. Except for the blank wells at the top, bottom and left side of the 96-well plate, 180 μL of cell suspension was added to the rest of the wells. The 96-well plate was placed in an incubator for 24 h.

[0307] (2) The test sample was dissolved in 20 μL of dimethyl sulfoxide, then 980 μL of culture medium was added to dissolve the sample to 1 mg / mL of the example liquid, and then the sample was diluted to 1, 0.333, 0.111, 0.037, 0.012 μg / mL in an EP tube. Each concentration of the example was added to 3 wells, 20 μL per well, and the cell growth in the surrounding two rows and two columns was greatly affected by the environment, so only blank cell wells were used. The 96-well plate was placed in an incubator for 72 h.

[0308] (3) Discard the drug-containing culture solution in the 96-well plate, rinse the cells twice with phosphate buffer solution (PBS), add 100 μL of MTT (tetrazolium, 0.5 mg / mL) to each well, and place in the incubator for 4 h. Discard the MTT solution, and add 100 μL of dimethyl sulfoxide. Shake on a magnetic shaker to dissolve the MTT reaction product formazan in the surviving cells, and place in an enzyme marker instrument to measure the results at 492 nm.

[0309] The results of the activity of the pyrrolopyrimidine compounds prepared in Examples 1-42 in inhibiting lung cancer cells H1975, lung cancer cells A549, and lung cancer cells PC-9 are shown in Table 2, in which NA indicates that the IC 50 >100.00 μM.

[0310] Table 2 Anti-cell proliferation activity of the pyrrolopyrimidine compounds prepared in Examples 1-42

[0311]

[0312]

[0313] As can be seen from Table 2, the pyrrolopyrimidine compound prepared in Example 42 has the best inhibitory activity on cancer cells.

[0314] The pyrrolopyrimidine compound prepared in Example 42 was subjected to molecular docking with EGFR to study the structure-activity relationship thereof with EGFR, and the results are shown in Figure 1 , in which A is the molecular docking result, B is the molecular surface electrostatic potential map, and C is the molecular docking score and RMSD data.

[0315] As can be seen from Figure 1 , the pyrrolopyrimidine compound is stably combined in the active pocket of EGFR and forms important interactions with key residues. The indole nitrogen atom of the ligand forms a hydrogen bond with Met793 in the hinge region This action helps to stabilize the molecule in the binding pocket. The nitrogen heterocyclic structure located in the affinity pocket enhances the affinity with the protein. In addition, the aromatic ring structure penetrates into the hydrophobic pocket and forms hydrophobic interactions with residues such as Leu718 and Val726, thereby further stabilizing the binding mode. As shown in Figure 1 , the docking calculation result shows that the best binding mode (Mode 1) of the molecule has the lowest binding free energy (-10.96 kcal / mol).

[0316] The effect of the pyrrolopyrimidine compound prepared in Experimental Example 42 on the morphology of cancer cells was observed by AO staining experiment:

[0317] Acridine Orange (AO) fluorescence staining is a technique for observing cell morphology and evaluating survival status. This method takes advantage of the binding properties of AO to nucleic acids, which exhibit different fluorescent colors in cells of different states, thereby distinguishing between living cells, apoptotic cells, and necrotic cells.

[0318] The specific operation steps are as follows:

[0319] (1) The cell density of H1975 cell suspension was 4 x 10 4 cells / mL, and 1 mL was added to each well of a 24-well plate, which was then incubated in a cell incubator for 24 h.

[0320] (2) The original culture solution was aspirated, and PBS was washed twice. 1 mg of the pyrrolopyrimidine compound prepared in Example 42 was dissolved in 20 μL of DMSO, and then serum-free medium was added to prepare a solution with a concentration of 1 μM and 2 μM. The drug solution was added at 1 mL per well, gently shaken to mix, and then incubated in a cell incubator for 12 h. A blank control was prepared without adding the drug solution, and AZD-9291 at a concentration of 1 μM was used as a positive control.

[0321] (3) The culture medium was aspirated and washed twice with 1 mL of PBS. After being washed once with 500 μL of 1 x buffer, 200 μL of AO staining solution (acridine orange: 1 x buffer = 1:20) was added, and the plate was incubated in a cell incubator for 15 min.

[0322] (4) The buffer was aspirated, and each well was washed 3 times with PBS. 500 μL of PBS was added, and the plate was observed under a microscope. The filter was rotated to 4 / 5, the laser was adjusted to blue light, and the plate was photographed and saved.

[0323] The staining results are shown in Figure 2 .

[0324] Figure 2 As shown, under a fluorescence microscope, the H1975 cells in the control group had an intact morphology, the nucleus and cytoplasm exhibited uniform green fluorescence, the cells were closely distributed, and no obvious apoptotic characteristics were observed. After being treated with the pyrrolopyrimidine compound (1 μmol / L) for 24 h, some of the cells showed morphological changes, the nucleus and cytoplasm were slightly reduced in size, and a small number of cells exhibited yellow fluorescence, indicating the occurrence of early apoptosis. As the concentration of the pyrrolopyrimidine compound increased to 2 μmol / L, more cells were observed to have reduced volume, fuzzy edges, and orange-red fluorescent cells, indicating an increase in late apoptosis. In addition, the space between the cells became larger, indicating that some of the cells had died. The changes in the pyrrolopyrimidine compound (2 μmol / L) group were similar to those in the AZD-9291 (1 μmol / L) group, and showed a certain degree of apoptotic characteristics. Overall, the pyrrolopyrimidine compound prepared in Example 42 can dose-dependently induce H1975 cell apoptosis, and the effect is more obvious at a high concentration.

[0325] The effect of the pyrrolopyrimidine compound prepared in Experimental Example 42 on apoptosis of cancer cells was observed by FITC / PI double staining method combined with flow cytometry:

[0326] The apoptosis was detected by FITC / PI (fluorescein isothiocyanate / propidium iodide) double staining method. FITC-labeled Annexin V can specifically recognize and bind to the phosphatidylserine (PS) on the early apoptotic cell membrane. And PI can only enter the late apoptotic or necrotic cells with damaged cell membrane, making the nucleus stained red fluorescence. By this method, normal cells, early apoptotic cells and late apoptotic / necrotic cells can be distinguished.

[0327] The specific operation steps are as follows:

[0328] (1) After the H1975 cells were digested and centrifuged by the subculture method, the number of cells was counted, and 2.0×10 5 cells per well were inoculated into a 6-well plate. Then, the cells were incubated in a 37℃, 5% CO2 incubator for 24 h to adhere to the wall.

[0329] (2) After 24 h, 3 mL of serum-free medium was added to each well, and 300 μL of the prepared pyrrolopyrimidine compound (1 μM, 4 μM) and positive drug (AZD-9291, 1 μM) of Experimental Example 42 were added, respectively. The blank control was not added with drug solution. Then, the plate was placed in a 37℃, 5% CO2 incubator for 24 h.

[0330] (3) The 6-well plate was placed in a clean bench, and the original culture medium was aspirated and washed twice with PBS. 1 mL of trypsin without EDTA was added to each well, and after the cells were completely detached, 1 mL of culture medium was added to terminate the digestion, and then the cells were washed once and transferred to a 5 mL centrifuge tube and centrifuged. The supernatant was discarded, 1 mL of PBS was added to the centrifuge tube, and the cells were gently blown to make them uniform. This step was repeated twice.

[0331] (4) The centrifuge tube was wrapped with tin paper to avoid light. 500 μL of buffer was added to each centrifuge tube, and the cells were gently blown for 8-10 times. 5 μL of Aximnex V and 5 μL of PI dye were added to the blank control group, and the prepared pyrrolopyrimidine compound of Experimental Example 42 and the positive group were repeated with the blank control group. The mixture was incubated in the dark for 30 min.

[0332] (5) The cell staining mixture was counted by flow cytometry at 1.0×10 4 cells, and the data was recorded.

[0333] The detection results are shown in Figure 3 .

[0334] Figure 3 The total apoptosis rate of the blank control group was only 1.38%, while the apoptosis rates of the 1 μM and 4 μM treatment groups were increased to 4.18% and 8.17%, respectively. The effect of the 4 μM treatment group was comparable to that of the positive control drug AZD9291 (1 μM), and the proportion of late apoptosis was increased by 4%. These results show that the pyrrolopyrimidine compound prepared in Example 42 can significantly induce H1975 cell apoptosis and mainly promote the cells to enter the late apoptosis stage, exhibiting good antitumor activity.

[0335] The effect of the pyrrolopyrimidine compound prepared in Example 42 on the cell cycle of cancer cells was detected by flow cytometry:

[0336] The specific operation steps are as follows:

[0337] (1) After the H1975 cells were digested and centrifuged by the subculture method, the number of cells was counted, and 2.0 x 10 5 cells per well were inoculated into a 6-well plate. Then, the cells were incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere.

[0338] (2) After 24 h, 3 mL of serum-free medium was added to each well, and 300 μL of the pyrrolopyrimidine compound prepared in Example 42 (1 μM, 4 μM) and the positive drug (AZD-9291, 1 μM) were added, respectively. The blank control was not added with the drug. Then, the cells were incubated in a 37°C, 5% CO2 incubator for 24 h.

[0339] (3) The H1975 cells were digested with trypsin without EDTA, centrifuged in a 5 mL centrifuge tube, washed with 1 mL of PBS twice (centrifuged), and 500 μL of PBS was added to blow the cells evenly. 500 μL of 70% cold ethanol (ethanol + PBS) was slowly added, and the cells were fixed in a 4°C refrigerator.

[0340] (4) After 2 h, the cells were taken out from the refrigerator and centrifuged, washed with PBS twice. 500 μL of PBS was added and blown evenly. 200 μL of dye (Rnase A: PI = 1:9) was added to each tube, and the mixture was placed in the dark for 30 min.

[0341] (5) The cell dye mixture was counted by flow cytometry at 1.0 x 10 4 cells, and the data was recorded.

[0342] The detection results are shown in Figure 4 .

[0343] From Figure 4As can be seen, after treatment with the pyrrolopyrimidine compound prepared in Experimental Example 42, the proportion of H1975 cells in the G2 phase showed a certain concentration-dependent upward trend. Compared with the control group (20.08% of cells in the G2 phase), the proportion of cells in the G2 phase in the 1 μM treatment group was 19.22%, which was not significantly changed, while the proportion of cells in the G2 phase in the 4 μM treatment group rose to 23.023%. In contrast, the proportion of cells in the G2 phase in the positive control AZD-9291 (1 μM) treatment group was 25.25%, indicating that the G2 phase blocking effect was more significant.

[0344] Effect of the pyrrolopyrimidine compound prepared in Experimental Example 42 on the content of reactive oxygen species in H1975 cells detected by the DCFH-DA probe:

[0345] The specific operation steps are as follows:

[0346] (1) Cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of 2.0 x 10 5 cells per well and cultured in an incubator for 24 h.

[0347] (2) 3 mL of serum-free medium was added to each well, and then 300 μL of the pyrrolopyrimidine compound prepared in Experimental Example 42 (1, 5 μM) and the positive drug (AZD9291, 1 μM) of the specified concentration were added, respectively, with no addition of the drug as the blank control group. After culture for the specified time, the old culture solution was discarded, and two PBS washes were performed before the staining solution was added.

[0348] (3) After three PBS washes, 1 mL of culture solution containing DCFH-DA was added to each well.

[0349] (4) Excess formaldehyde was removed, and two PBS washes were performed.

[0350] (5) Finally, 1 mL / well of crystal violet was added for staining, and the cells were allowed to stand for 15 minutes.

[0351] (6) After the last two PBS washes, the fluorescence microscope was used for observation and photography.

[0352] The detection results are shown in Figure 5 .

[0353] Figure 5 As can be seen, the green fluorescence signal of the H1975 cells in the blank control group was weak, and the ROS production level was extremely low. After treatment with the pyrrolopyrimidine compound prepared in Experimental Example 42 at a concentration of 1 μM, the green fluorescence in the cells was slightly enhanced, indicating that the ROS level was increased. When the concentration was increased to 5 μM, the intensity of the green fluorescence in the cells was increased, indicating that the ROS level showed a significant upward trend.

[0354] The influence of the pyrrolopyrimidine compound prepared in Experimental Example 42 on the mitochondrial membrane potential of cancer cells was detected by using the JC-1 method.

[0355] The specific operation steps are as follows:

[0356] (1) The cell density of H1975 cell suspension was 4x10 4 cells / mL, and 1 mL of the cell suspension was added to each well of a 24-well plate, and the cells were cultured in a cell incubator for 24 h.

[0357] (2) The original culture solution was removed, and the cells were washed twice with PBS. 1 mg of the pyrrolopyrimidine compound prepared in Experimental Example 42 was dissolved in 20 μL of DMSO, and then serum-free medium was added to prepare a solution with a concentration of 1 μM and 2 μM. 1 mL of the drug solution was added to each well, and the plate was gently shaken to mix the solution. The plate was then placed in a cell incubator and cultured for 12 h. A blank control group without the drug solution was used, and AZD-9291 with a concentration of 1 μM was used as a positive control group.

[0358] (3) The culture solution was removed, and the cells were washed twice with 1 mL of PBS. Then, 1 mL of JC-1 working solution (50 uL of JC-1 (200x): 8 mL of ultrapure water) was added, and the plate was mixed thoroughly and placed in a cell incubator for 20 min.

[0359] (4) The working solution was removed, and the cells were washed twice with 1x JC-1 staining buffer.

[0360] (5) 2 mL of 200x JC-1 staining buffer was added, and the plate was placed under a fluorescence microscope for observation and photography.

[0361] Detection results Figure 6 .

[0362] The stability of the mitochondrial membrane potential (MPP) is crucial for the normal physiological function of cells, and a decrease in the mitochondrial membrane potential often indicates that the cells are moving towards apoptosis. The JC-1 fluorescent probe can well reflect this change: when the mitochondrial membrane potential is normal, the cells exhibit red fluorescence, and when the potential decreases, the fluorescence changes to green, so it can be used to directly determine whether the cells are damaged or undergoing apoptosis. Figure 6 As shown, the cells in the control group remained in a healthy state, and the mitochondrial membrane potential was stable, so the fluorescence signal was mainly red. When the cells were exposed to 1 μM of AZD-9291, part of the fluorescence changed from red to green, indicating that the mitochondrial membrane potential decreased, and some cells may have begun to undergo apoptosis. In contrast, the pyrrolopyrimidine compound prepared in Experimental Example 42 caused more obvious green fluorescence in the treatment groups at concentrations of 1 μM and 2 μM, especially at a concentration of 2 μM, where the green fluorescence was more obvious, indicating that the mitochondrial membrane potential decreased more sharply, and the degree of cell damage or apoptosis was deeper. This indicates that the pyrrolopyrimidine compound prepared in Experimental Example 42 can induce cell death by affecting mitochondrial function, and to some extent, it exhibits strong biological activity.

[0363] The above experimental results show that the pyrrolopyrimidine compound prepared in Experimental Example 42 of the present application is expected to be a potential EGFR inhibitor.

[0364] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art, without departing from the design spirit of the present application, shall fall within the scope of the present application as defined by the claims.

Claims

1. A pyrrolopyrimidine compound, characterized in that, The structural formulas of the pyrrolopyrimidine compounds are shown in Formula I or Formula II: In Equation I, R1 is , , , , , , , , , , , , , , , , or ; In Equation II, when R2 is When R3 is , , , , or ; When R2 is When R3 is , , , or ; When R2 is When R3 is , , , , or ; When R2 is When R3 is , or .

2. A pyrrolopyrimidine compound, characterized in that, The structural formula of the pyrrolopyrimidine compound is shown below: , or .

3. A method for preparing the pyrrolopyrimidine compound of claim 1, characterized in that, The synthetic route for compound 5, represented by Formula I, is as follows: ； In the synthetic route of the compounds shown in Formula I, compound 2 is prepared by nucleophilic substitution reaction; compound 3 is prepared by aromatic nucleophilic substitution reaction; compound 4 is prepared by amidation reaction; and compound 5 is prepared by acylation reaction. The synthetic routes for compounds 4a-4d, as shown in Formula II, are as follows: ； In the synthetic route of the compounds shown in Formula II, compound 2 is prepared by nucleophilic substitution reaction; compounds 3a-3d are prepared by aromatic nucleophilic substitution reaction; and compounds 4a-4d are prepared by amidation reaction.

4. A pharmaceutically acceptable salt of the pyrrolopyrimidine compound of claim 1 or 2.

5. A pharmaceutical preparation, characterized in that, The active ingredient comprises at least one of the following: the pyrrolopyrimidine compound of claim 1, the pyrrolopyrimidine compound of claim 2, and a pharmaceutically acceptable salt of the pyrrolopyrimidine compound of claim 4.

6. The use of the pyrrolopyrimidine compound of claim 1 in the preparation of medicaments for the treatment and / or prevention of lung cancer.

7. Use of a pharmaceutically acceptable salt of the pyrrolopyrimidine compound of claim 4 in the preparation of a medicament for the treatment and / or prevention of lung cancer.

8. The use of the pharmaceutical preparation of claim 5 in the preparation of a drug for treating and / or preventing lung cancer.

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