A dimethylphosphine oxide-substituted anilinopyrimidine derivative targeting EGFR mutation, preparation method and use thereof, and medicine prepared therefrom

Abstract

The present invention belongs to the field of medical technology and is a dimethylphosphine oxide-substituted anilinopyrimidine derivative targeting EGFR mutation, a preparation method, use and a drug made thereof. The present invention provides a compound shown in formula I. These compounds have an effect on EGFR T790M / C797S / L858R Kinases and EGFR L858R / T790M The kinase has outstanding inhibitory activity (at the nanomolar level) and selectivity, and has a significant inhibitory effect on non-small cell lung cancer cell lines, especially for EGFR mutant cells H1975 cells, with outstanding selectivity and significant inhibitory effect. The compound of the present invention has a significant tumor inhibition effect in a nude mouse xenograft tumor model and has low in vivo toxicity. Therefore, the compound of the present invention can be used as an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of anti-lung cancer drugs, and has good application prospects. #imgabs0# Formula I.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and relates to a dimethylphosphine oxide-substituted anilinopyrimidine derivative targeting EGFR mutation, a preparation method and use thereof, and a medicine prepared therefrom. Background Art

[0002] Cancer is a major social, public health and economic issue in the 21st century, causing three in ten people to die prematurely worldwide. According to the latest statistics from the International Agency for Research on Cancer (IARC) on the global cancer burden in 2022, lung cancer has once again become the world's most common cancer (accounting for 12.4% of total cases) after being surpassed by breast cancer in 2020, followed by female breast cancer (11.6%), colorectal cancer (9.6%), prostate cancer (7.3%) and gastric cancer (4.9%). Lung cancer is also the cancer with the highest cancer mortality rate, accounting for 18.7% of total cancer deaths. This is related to the fact that lung cancer usually has no symptoms in the early stages. Once discovered, it may have developed to the late stage, and the prognosis of lung cancer is poor. The most common type of lung cancer is non-small cell lung cancer (NSCLC), accounting for about 85%.

[0003] In the past 20 years, a lot of progress has been made in the treatment of lung cancer. Currently, the main drug treatments for lung cancer include chemotherapy, immunotherapy, and molecular targeted therapy. In the drug treatment of non-small cell lung cancer, compared with traditional cytotoxic drugs, targeted therapy drugs can act more accurately on tumor tissues, greatly reducing damage to surrounding normal tissues, not only reducing the incidence of adverse drug reactions in patients, but also significantly improving the accuracy of fighting tumors. With the development and advancement of tumor molecular biology technology, targeted drug therapy has become one of the research hotspots in the oncology community in recent years.

[0004] With the development of research on oncogene-driven signaling pathways used in tumor cells, mutations in the proto-oncogene EGFR encoded by the EGFR of the protein tyrosine kinase family and its family-related genes are the main causes of tumor onset in lung cancer patients, especially NSCLC patients. EGFR tyrosine kinase is a transmembrane glycoprotein with cytoplasmic kinase activity, located on the surface of the cell membrane, which can transmit important growth factor signals from the extracellular environment to the cell. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation. EGFR kinase generally exists in monomeric form and requires ligand (EGF) binding activation to have tyrosine kinase activity; after ligand activation, it mainly conducts cell signaling in the form of dimers, thereby triggering a series of physiological activities. In general, abnormal activation of the EGFR signaling pathway may lead to uncontrolled growth of tumor cells, metastatic spread and angiogenesis.

[0005] Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been proven to be effective for patients with non-small cell lung cancer and are currently the most commonly used drugs to prolong patients' lives. There have been three generations of drugs developed for EGFR inhibitors. The first generation of EGFR-TKIs are reversible EGFR-TKIs, which contain 4-aminoquinazoline structures in their molecular structures, such as Gefitinib and Erlotinib. Although patients with NSCLC have shown good therapeutic effects using Gefitinib or Erlotinib, varying degrees of drug resistance usually occur after 9-14 months of continuous medication. T790M mutations have been detected in up to 68% of patients with acquired resistance to EGFR-TKIs. In order to overcome the resistance problem of EGFR T790M mutations generated by the first generation of EGFR-TKIs in the treatment of NSCLC patients, the second generation of EGFR-TKIs was developed. The second-generation EGFR-TKIs introduced an acrylamide structure, which was used to undergo a Michael addition reaction with Cys797 to form a covalent EGFR complex. However, due to dose-limiting rash and gastrointestinal toxicity, which greatly limited its clinical application, the maximum tolerated dose of most second-generation EGFR-TKIs was unacceptably low. The third-generation EGFR-TKIs represented by osimertinib overcame the clinical resistance mutation of EGFR T790M and had good selectivity for the EGFR T790M mutation, thus overcoming the clinical toxicity seen in the second-generation EGFR-TKIs. However, since the mutation of Cys797 to Ser79 interferes with the formation of a covalent bond with the irreversible EGFR inhibitor osimertinib, thereby significantly reducing the activity of the drug, overcoming the resistance of the third-generation mutation is the mission of the fourth-generation EGFR inhibitor. As more and more patients with EGFR-mutated NSCLC receive osimertinib treatment, the importance of developing treatment strategies for this group will increase in the near future.

[0006] Therefore, developing more active and better EGFR inhibitors has positive significance for the treatment of cancers including lung cancer and is an important topic in this field. Summary of the invention

[0007] In view of the problems of the prior art, the present invention provides a dimethylphosphine oxide-substituted anilinopyrimidine derivative targeting EGFR mutation, a preparation method and use thereof, and a drug made therefrom.

[0008] The compound shown in Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a crystalline form thereof, has the structural formula:

[0009]

[0010] Formula I

[0011] in,

[0012] R1 is selected from substituted or unsubstituted 3-10 membered heterocycloalkyl, wherein the substituent is selected from C1-C 10 Alkyl, C1-C 10 Acyl, 3-10 membered heterocycloalkyl, 10 Alkyl-substituted 3-10 membered heterocycloalkyl;

[0013] R2 is selected from nitro, amino, substituted or unsubstituted C1-C 10 Amide, substituted or unsubstituted C1-C 10 Sulfonamide, substituted or unsubstituted C1-C 10 Amine, wherein the substituent is selected from halogen, C6-C 10 Aryl, with at least one R 21 Substituted C6-C 10 Aryl, halosulfonyl;

[0014] R 21 Selected from halogen, C1-C 10 Alkyl, C1-C 10 Alkoxy, halosulfonyl.

[0015] Preferably, the compound has the structural formula:

[0016]

[0017] Formula II

[0018] in,

[0019] R 11 Selected from C1-C 10 Alkyl, C1-C 10 Acyl, 3-10 membered heterocycloalkyl, 10 Alkyl-substituted 3-10 membered heterocycloalkyl.

[0020] Preferably, the compound has the structural formula:

[0021]

[0022] Formula III.

[0023] Preferably, R2 is selected from nitro, amino, substituted or unsubstituted C1-C2 amide, substituted or unsubstituted C1-C2 sulfonamide, substituted or unsubstituted C2 amine, wherein the substituent is selected from F, Cl, phenyl, 21 Substituted phenyl, halosulfonyl;

[0024] R 21 Selected from Cl, methyl, methoxy, halosulfonyl.

[0025] Preferably, R2 is selected from nitro or .

[0026] Preferably, the compound structural formula is selected from:

[0027]

[0028]

[0029] The present invention also provides a method for preparing the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a crystalline form thereof, characterized in that it comprises the following steps:

[0030]

[0031] Step 1, reacting 2-iodoaniline with dimethylphosphine oxide to obtain compound 1;

[0032] Step 2, reacting compound 1 with 2,4,5-trichloropyrimidine to obtain compound 2;

[0033] Step 3, reacting compound 2 with 4-fluoro-2-methoxy-5-nitroaniline to obtain compound 3;

[0034] Step 4, compound 3 and compound Reaction to obtain compound 4;

[0035] Step 5, subjecting compound 4 to a reduction reaction to obtain compound 5;

[0036] Step 6, reacting compound 5 with R2OH, or reacting compound 5 with R2Cl to obtain a compound represented by formula II;

[0037] Wherein, R2OH is a carboxylic acid compound, and R2Cl is an acyl chloride compound;

[0038] In step 1, the reaction is carried out under the action of a catalyst, the catalyst is selected from a combination of potassium phosphate and palladium acetate / Xantphos; the reaction solvent is selected from N,N-dimethylformamide; the reaction temperature is 110-130°C; the reaction time is 2-6 hours;

[0039] In step 2, the reaction is carried out under the action of a base, the base is selected from K2HPO3; the reaction solvent is selected from N,N-dimethylformamide; the reaction temperature is 60-70°C; the reaction time is 3-5 h;

[0040] In step 3, the reaction is carried out under the action of an acid, the acid is selected from p-toluenesulfonic acid, the reaction solvent is selected from at least one of n-butanol and isobutanol; the reaction temperature is 110-130° C.; the reaction time is 1-3 h;

[0041] In step 4, the reaction is carried out under the action of a base, the base is selected from at least one of potassium carbonate and cesium carbonate; the reaction solvent is selected from N,N-dimethylformamide; the reaction temperature is 110-130°C; the reaction time is 3-5 h;

[0042] In step 5, the reducing agent of the reaction is selected from at least one of zinc and iron; the reaction is carried out under the action of a catalyst, and the catalyst is selected from ammonium chloride; the reaction temperature is 60-70°C; the reaction time is 20-40 min;

[0043] In step 6, the reaction is carried out under the action of a condensing agent, a catalyst and a base, the condensing agent is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the catalyst is selected from 1-hydroxybenzotriazole, the base is selected from triethylamine, and the solvent is selected from N,N-dimethylformamide; the reaction temperature is 60-70°C; and the reaction time is 1-4 h.

[0044] The present invention also provides use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a crystal form thereof in the preparation of a tyrosine kinase inhibitor.

[0045] Preferably, the tyrosine kinase inhibitor is a drug that inhibits EGFR phosphorylation.

[0046] Preferably, the tyrosine kinase inhibitor is a drug for treating cancer. Preferably, the cancer is at least one of lung cancer, liver cancer, stomach cancer, kidney cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, uterine cancer, colon cancer, rectal cancer, leukemia, bone cancer, and lymphoma.

[0047] Preferably, the tyrosine kinase inhibitor is a drug for treating lung cancer. Preferably, the lung cancer is non-small cell lung cancer. More preferably, the lung cancer is EGFR mutant non-small cell lung cancer.

[0048] The present invention also provides a pharmaceutical composition, which is a preparation prepared by using the above-mentioned compound, or its pharmaceutically acceptable salt, or its stereoisomer, or its solvate, or its crystal form as an active ingredient, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0049] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0050] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0051] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0052] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a -C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-C6 alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms.

[0053] "Alkyl" refers to a saturated hydrocarbon chain with a specified number of member atoms. For example, C1-C6 alkyl refers to an alkyl group with 1 to 6 member atoms, such as 1 to 4 member atoms. The alkyl group can be straight or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl. The alkyl group may also be a part of other groups, such as C1-C6 alkoxy.

[0054] "Alkoxy" refers to an alkyl group attached to the site of attachment through an oxygen atom, for example, methoxy refers to -OCH3.

[0055] "Aryl" refers to an all-carbon monocyclic or polycyclic ring (including fused ring, spirocyclic or bridged ring) with a conjugated π electron system, such as but not limited to: phenyl, naphthyl, phenanthrenyl, anthracenyl, fluorenyl and indenyl. The aromatic ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as O, N or S. At the same time, the point of connection to the parent must be on a carbon atom on the ring with a conjugated π electron system, such as but not limited to , , , wait.

[0056] "Heterocycle" and "heterocycloalkyl" refer to a saturated or non-aromatic unsaturated ring containing at least one heteroatom and having a single ring; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, or a sulfur atom;

[0057] "Amide" refers to a group attached to a linking site via a carbon chain, wherein the carbon chain contains at least one amide bond (ie, -CONH-).

[0058] "Sulfonamide" refers to a group that is attached to the attachment site through a carbon chain or a nitrogen atom and that contains at least one sulfonamide bond (ie, -SO2NH-) in the carbon chain.

[0059] "Amine group" refers to a group that is attached to a linking site through a carbon chain or a nitrogen atom, and the carbon chain contains at least one nitrogen atom.

[0060] "Acyl" refers to a group attached to the site of attachment through a carbon group (ie, -CO-).

[0061] "Halogen" is fluorine, chlorine, bromine or iodine.

[0062] "Halosulfonyl" refers to -SO2X, where X is selected from halogen.

[0063] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0064] The terms "salt" and "pharmaceutically acceptable salt" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compound. They can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalent amount). These salts may be precipitated in the solution and collected by filtration, or recovered after solvent evaporation, or obtained by freeze drying after reaction in an aqueous medium. The salts described in the present invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluorides, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates or trifluoroacetates of the compound.

[0065] In certain embodiments, one or more compounds of the present invention may be used in combination with each other. It is also possible to select the compounds of the present invention and any other active agent for use in combination to prepare a drug or pharmaceutical composition for regulating cell function or treating a disease. If a group of compounds is used, these compounds may be administered to a subject simultaneously, separately or sequentially.

[0066] The present invention provides a class of dimethylphosphine oxide substituted anilinopyrimidine derivatives shown in formula I, which exhibit good selective inhibition of EGFR T790M / C797S / L858R Kinases and EGFR L858R / T790M Kinase activity, EGFR T790M / C797S / L858RThe kinase inhibitory activity is better than that of osimertinib. The co-crystal structure at the molecular level shows that these compounds can bind to EGFR T790M / C797S / L858R The experimental results show that these compounds have obvious inhibitory effects on non-small cell lung cancer cell lines, especially on EGFR mutant cells NCI-H1975 cells, with good selectivity and significant inhibitory effects, and the compounds of the present invention have low toxicity to normal cells. Therefore, the present invention provides a new drug option for clinical treatment of cancer and has a good application prospect.

[0067] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0068] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The data graphs are of the results of in vivo xenograft experiments of the compound H-21 of the present invention, wherein A is the tumor volume curve of the subcutaneous transplanted tumor in nude mice after administration of H-21; B is the body weight curve of nude mice after administration of H-21; C is the size of the subcutaneous transplanted tumor in nude mice on the last day of administration of H-21; D is the tumor weight of the subcutaneous transplanted tumor in nude mice on the last day of administration of H-21; and E is the pathological section results of mice in different groups. DETAILED DESCRIPTION

[0070] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0071] Example 1

[0072] This embodiment provides: (2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)-5-nitrophenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (compound H-01, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=NO2), the specific preparation method of which is:

[0073] (1) 2-iodoaniline (1.0 equivalent), dimethylphosphine oxide (1.1 equivalent), potassium phosphate (1.1 equivalent) and palladium acetate / Xantphos were added to a 50 mL round-bottom flask and replaced with argon. After adding dry N,N-dimethylformamide, the mixture was heated to 120 °C and reacted for 4 hours. After the reaction was completed as monitored by TLC, N,N-dimethylformamide was extracted with water and ethyl acetate to remove N,N-dimethylformamide, the organic phase was collected and dried, and the product (2-aminophenyl) dimethylphosphine oxide (compound 1) was separated by column chromatography with a yield of 88%.

[0074] (2) (2-aminophenyl) dimethylphosphine oxide (compound 1) (1.0 equivalent), 2,4,5-trichloropyrimidine (1.1 equivalent), and K2HPO3 (1.5 equivalent) were added to a 50 mL round-bottom flask, and dry N,N-dimethylformamide was added, and the mixture was heated to 65 °C for 4 hours. After the reaction was completed as monitored by TLC, N,N-dimethylformamide was extracted with water and ethyl acetate to remove the N,N-dimethylformamide, and the organic phase was collected and dried, and the product (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (compound 2) was separated by column chromatography with a yield of 78%.

[0075] (3) (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphineoxide (Compound 2) (1.0 equivalent), 4-fluoro-2-methoxy-5-nitroaniline (1.1 equivalent), and p-toluenesulfonic acid (1.8 equivalent) were added to a 50 mL round-bottom flask, and dry n-butanol was added, and the mixture was heated to 120 °C for 2 hours. After the reaction was completed as monitored by TLC, the solvent was evaporated, water was added, and the mixture was extracted with dichloromethane. The organic phase was collected and dried, and (2-((5-chloro-2-((4-fluoro-2-methoxy-5-nitrophenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound 3) was obtained by column chromatography with a yield of 80%.

[0076] (4) (2-((5-chloro-2-((4-fluoro-2-methoxy-5-nitrophenyl)amino)pyrimidin-4-yl)amino)phenyl) dimethylphosphine oxide (Compound 3) (1.0 equivalent), 1-methyl-4-(4-piperidinyl)piperazine (1.1 equivalent), and potassium carbonate (1.8 equivalent) were added to a 50 mL round-bottom flask, and dry N,N-dimethylformamide was added, and the mixture was heated to 120 °C for 4 hours. After the reaction was completed as monitored by TLC, N,N-dimethylformamide was washed out by extraction with water and ethyl acetate, and the organic phase was collected and dried by rotation. The product (2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)-5-nitrophenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound H-01) was obtained by column chromatography with a yield of 75%. 1 HNMR (400 MHz, Chloroform- d ) δ 10.86 (s, 1H), 8.83 (s, 1H), 8.45 (m, 1H), 8.12(s, 1H), 7.49 (m, 1H), 7.33 – 7.24 (m, 1H), 7.14 (m, 1H), 6.57 (s, 1H), 3.95(s, 3H),3.32(d, J = 11.0 Hz, 2H), 2.80 (td, J = 11.8, 2.3 Hz, 2H), 2.57 (d, J =64.9 Hz, 8H), 2.39 (td, J = 7.6, 3.8 Hz, 1H), 2.30 (s, 3H), 1.95 – 1.87 (m,2H), 1.76 (td, J = 11.9, 3.7 Hz, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 157.00,156.03, 154.84, 151.99, 143.30 (d, J = 2.6 Hz), 142.75, 136.08, 132.79 (d, J=2.2 Hz), 129.65 (d, J = 10.8 Hz), 123.62, 122.95 (d, J = 12.1 Hz), 122.56 (d, J =7.2 Hz), 120.33 (d, J = 95.6 Hz), 116.31, 107.27, 102.07, 61.57, 56.09, 55.47,52.30, 48.99, 46.02, 28.47, 18.92, 18.21. MS (ESI + ) m / z calcd for C 29 H 39 ClN8O4P + (M+H) + 629.2, found 629.0.

[0077] In the following Examples 2 to 25, the preparation steps not specifically described are all performed according to the steps of Example 1.

[0078] Example 2

[0079] (2-((2-((5-amino-2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl) amino)-5-chloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound H-02, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=NH2).

[0080] The product compound H-01 (1.0 equivalent) in step (4) of Example 1, zinc powder (10 equivalents) and ammonium chloride were added to a 50 mL round-bottom flask, and methanol was added to react at 60-70 °C for 30 min. After the reaction was completed as monitored by TLC, it was filtered through a sand core, the organic phase was collected and dried, and the product (2-((2-((5-amino-2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)-5-chloropyrimidin-4-yl)amino) phenyl)dimethylphosphine oxide (compound H-02) was separated by column chromatography with a yield of 78%. 11H NMR (400 MHz, Chloroform- d ) δ 10.66 (s, 1H), 8.52 (m, 1H), 8.01 (s, 1H), 7.75 (s, 1H), 7.56–7.47 (m, 1H), 7.22 (m, 1H), 7.06 (m, 1H), 6.54 (s, 1H), 3.73 (s, 3H), 3.63 –3.52 (m, 2H), 3.13 –3.06 (m, 2H), 2.66 –2.38 (m, 10H), 2.30 (m, 1H), 2.24 (s, 3H), 1.93 –1.86 (m, 2H), 1.77 (s, 3H), 1.74 (s, 3H), 1.60 (qd, J J = 11.9, 3.9 Hz, 2H). 13 13C NMR (101 MHz, Chloroform- d ) δ 157.38, 155.83, 154.96, 143.83 (d, J J = 2.5 Hz), 141.06, 135.04, 133.12, 132.38 (d, J J = 2.2 Hz), 129.66 (d, J J = 10.8 Hz), 125.84, 123.28 (d, J J = 7.1 Hz), 122.59 (d, J J = 12.2 Hz), 120.76 (d, J J = 95.5 Hz), 106.79, 106.07, 103.92, 61.79, 56.68, 55.31, 51.56, 49.03, 45.91, 29.31, 18.86, 18.15. MS (ESI + ) m / z calcd for C 29 H 41 ClN8O2P + (M + H) + 599.3, found 599.4.

[0081] Example 3

[0082] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-03, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=benzamide).

[0083] The product compound H-02 (1.0 equivalent) in Example 2, benzoic acid (1.1 equivalent), 1-hydroxybenzotriazole (1.2 equivalent), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 equivalent), and triethylamine (1.0 equivalent) were added to a 25 mL round-bottom flask and replaced with argon. Dry N,N-dimethylformamide was added and reacted at 65 °C for 2 hours. After the reaction was completed as monitored by TLC, N,N-dimethylformamide was extracted and washed with water and ethyl acetate, the organic phase was collected and dried, and the product N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (compound H-03) was separated by column chromatography with a yield of 86%. 1 H NMR (400 MHz, Chloroform- d ) δ10.74 (s, 1H), 9.28 (s, 1H), 9.13 (s, 1H), 8.60 (dd, J = 8.6, 4.4 Hz, 1H), 8.17(s, 1H), 7.92 – 7.83(m, 2H), 7.52 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.17 (m,1H), 6.87 (m, 1H), 6.76 (s, 1H), 3.85 (s, 3H), 3.08 (d, J = 11.3 Hz, 2H), 2.79– 2.72 (m, 2H), 2.69 – 2.46 (m, 8H), 2.36 (d, J = 11.1 Hz, 1H), 2.32 (s, 3H), 2.05 (d, J= 12.4 Hz, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.69 (m, 2H). 13 C NMR (101MHz, Chloroform- d ) δ 164.15, 157.54, 155.97, 154.96, 145.31, 143.63 (d, J = 2.6Hz), 136.69, 135.34, 132.38 (d, J = 2.3 Hz), 131.51, 129.40, 129.29, 128.80,126.87, 126.76, 125.90, 122.96 (d, J = 7.1 Hz), 122.36 (d, J = 12.2 Hz), 120.69,119.74, 112.29, 106.80, 103.22, 61.64, 55.99, 55.30, 52.67, 49.35, 45.96,29.78, 18.84,18.13.MS (ESI + ) m / z calcd for C 36 H 45 ClN8O3P + (M+H) + 703.3, found703.0.

[0084] Example 4

[0085] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-4-methoxybenzamide (Compound H-04, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=4-methoxybenzamide).

[0086] The raw material benzoic acid in Example 3 was substituted for 4-methoxybenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-4-methoxybenzamide (Compound H-04) with a yield of 82%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.74(s, 1H), 9.25 (s, 1H), 9.02 (s, 1H), 8.61 (dd, J = 8.5, 4.4 Hz, 1H), 8.17 (s,1H), 7.88 – 7.81(m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.26 (s, 1H), 7.17 (m, 1H),7.03 – 6.97 (m, 2H), 6.88 (m, 1H), 6.75 (s, 1H), 3.90 (s, 3H), 3.85 (s, 3H),3.08 (d, J = 11.3 Hz, 2H), 2.76 (dd, J = 12.1, 2.2 Hz, 2H), 2.73 – 2.43 (m, 8H), 2.37 – 2.33 (m, 1H), 2.32 (s, 3H), 2.06 (d, J = 12.3 Hz, 2H), 1.81 (d, J = 13.1Hz, 6H), 1.69 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 163.80, 162.19,157.57, 155.98, 155.01, 145.17, 143.68 (d, J = 2.8 Hz), 136.59, 132.41 (d, J =2.2 Hz), 129.32 (d, J = 10.9 Hz), 128.69, 127.69, 126.94, 125.87, 122.99 (d,J =7.0 Hz), 122.33 (d, J = 12.1 Hz), 120.68, 119.72, 113.99, 112.34, 106.79,103.18, 61.75, 56.00, 55.49, 55.33, 52.65,49.44, 46.00, 29.86, 18.88,18.17.MS (ESI + ) m / z calcd for C 37 H 47 ClN8O4P + (M+H) + 733.3, found 733.0.

[0087] Example 5

[0088] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amin o)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-4-methylbenzamide (Compound H-05, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=4-methylbenzamide).

[0089] The raw material benzoic acid in Example 3 was substituted for 4-methylbenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-4-methylbenzamide (Compound H-05) with a yield of 82%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.73 (s, 1H), 9.26 (s,1H), 9.08 (s, 1H), 8.60 (m, 1H), 8.17 (s, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.37(t, J= 7.9 Hz, 1H), 7.33 – 7.26 (m, 3H), 7.17 (m, 1H), 6.88 (t, J = 7.3 Hz, 1H),6.75 (s, 1H), 3.85 (s, 3H), 3.08 (d, J = 11.3 Hz, 2H), 2.79 – 2.73 (m, 2H),2.73 – 2.47 (m, 8H), 2.45 (s, 3H), 2.37 (m, 1H), 2.32 (s, 3H),2.04 (d, J = 12.3Hz, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.74 – 1.63 (m, 2H). 13 C NMR (101 MHz,Chloroform- d ) δ 164.16, 157.56, 155.99, 155.00, 145.23,143.65, 141.93,136.64, 132.56, 132.39, 129.46, 129.38, 129.27, 126.89, 125.88, 123.05,122.98, 122.41, 122.29, 120.23 (d, J = 96.0 Hz), 112.33, 106.80, 103.20, 61.69,56.00, 55.36, 52.67, 49.36, 46.01, 29.75, 21.51, 18.86, 18.15.MS(ESI + ) m / zcalcd for C 37 H 47 ClN8O3P + (M+H) + 717.3, found 717.0.

[0090] Example 6

[0091] 4-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-06, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=4-chlorobenzamide).

[0092] The raw material benzoic acid in Example 3 was substituted for 4-chlorobenzoic acid, and the remaining steps were the same as in Example 3 to prepare 4-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-06) with a yield of 79%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.76 (s, 1H), 9.26 (s,1H), 9.10 (s, 1H), 8.60 (m, 1H), 8.17 (s, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.53 –7.46 (m, 2H), 7.36 (t, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.18 (m, 1H), 6.89 (m,1H), 6.76 (s, 1H), 3.86 (s, 3H), 3.10 – 3.03 (m, 2H), 2.76 (m, 2H), 2.64 (m,8H), 2.39 – 2.34 (m, 1H), 2.33 (s, 3H), 2.06 (d, J = 12.4 Hz, 2H), 1.84 (s, 3H), 1.80 (s, 3H), 1.66 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 163.07,157.46, 155.96, 154.93, 145.35, 143.65 (d, J= 2.6 Hz), 137.74, 136.58, 133.73,132.35, 132.33, 129.38 (d, J = 10.9 Hz), 129.06, 128.29, 126.50, 126.00, 122.89(d, J = 7.1 Hz), 122.34 (d, J = 12.2 Hz), 120.69, 119.74, 112.12, 106.90, 103.19,61.57, 55.98, 55.21, 52.65, 49.32, 45.90, 29.85, 18.86,18.15.MS (ESI + ) m / zcalcd for C 36 H 44 Cl2N8O3P + (M+H) + 737.3, found 737.0.

[0093] Example 7

[0094] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-3-methoxybenzamide (Compound H-07, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=3-methoxybenzamide).

[0095] The raw material benzoic acid in Example 3 was substituted for 3-methoxybenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-3-methoxybenzamide (Compound H-07) with a yield of 81%. 1 H NMR (400 MHz, Chloroform- d) δ 10.75(s, 1H), 9.28 (s, 1H), 9.17 (s, 1H), 8.61 (m, 1H), 8.21 – 8.15 (m, 1H), 7.47(s, 1H), 7.39 (d, J = 9.9 Hz, 3H), 7.28 (s, 1H), 7.18 (m, 1H), 7.12 – 7.05(m,1H), 6.90 (t, J = 7.7 Hz, 1H), 6.76 (s, 1H), 3.08 (d, J = 11.4 Hz, 2H), 2.76 (d, J = 11.7 Hz, 2H), 2.73 – 2.49 (m, 9H), 2.39 (d, J = 10.7 Hz, 1H), 2.32 (s, 3H),2.04 (d, J = 12.4 Hz, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.69 (m, 2H). 13 C NMR (101MHz, Chloroform- d ) δ 163.96, 159.98, 157.53, 155.98, 154.96, 145.32, 143.65(d, J = 2.6 Hz), 136.87, 136.65, 132.41 (d, J = 2.2 Hz), 129.78, 129.37 (d, J =10.9 Hz), 126.78, 125.93, 122.93 (d, J = 7.0 Hz), 122.38 (d, J = 12.2 Hz), 120.17(d, J = 96.1 Hz), 118.39, 117.58, 112.65, 112.21, 106.83, 103.28, 61.55, 55.99,55.47, 55.29, 52.68, 49.19,45.91, 29.70, 18.85, 18.14.MS (ESI + ) m / z calcd forC 37 H 47 ClN8O4P + (M+H)+ 733.3, found 733.0.

[0096] Example 8

[0097] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-3-methylbenzamide (Compound H-08, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=3-methylbenzamide).

[0098] The raw material benzoic acid in Example 3 was substituted for 3-methylbenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-3-methylbenzamide (Compound H-08) with a yield of 83%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.74 (s, 1H), 9.27 (s,1H), 9.15 (s, 1H), 8.61 (m, 1H), 8.18 (s, 1H), 7.71 (d, J = 1.9 Hz, 1H), 7.65(m, 1H), 7.46 – 7.31 (m, 4H), 7.18 (m, 1H), 6.90 (m, 1H), 6.75 (s, 1H), 3.85(s, 3H), 3.11 – 3.06 (m, 2H),2.80 – 2.76 (m, 2H), 2.75 – 2.65 (m, 6H), 2.53(s, 2H), 2.44 (s, 3H), 2.41 (d, J = 4.1 Hz, 1H), 2.32 (s, 3H), 2.06 – 2.00(m,2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.69 (m, 2H). 13C NMR (101 MHz, Chloroform- d )δ 164.38, 157.54, 155.97, 154.93, 145.29, 143.61 (d, J = 2.6 Hz), 138.51,136.67, 135.35, 132.42 (d, J = 2.2 Hz), 132.26, 129.36 (d, J = 10.9 Hz), 128.67,127.84, 126.82, 125.87, 123.70, 122.95 (d, J = 7.2 Hz), 122.39 (d, J = 12.3 Hz),120.17 (d, J = 96.0 Hz), 112.24, 106.79, 103.27, 61.49, 55.99, 55.27, 52.66,49.05, 45.89, 29.56, 21.50, 18.82, 18.11.MS (ESI + ) m / z calcd for C 37 H 47 ClN8O3P + (M+H) + 717.3, found 717.0.

[0099] Example 9

[0100] 3-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-09, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=3-chlorobenzamide).

[0101] The raw material benzoic acid in Example 3 was substituted for 3-chlorobenzoic acid, and the remaining steps were the same as in Example 3 to prepare 3-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-09) with a yield of 78%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.76 (s, 1H), 9.28 (s,1H), 9.14 (s, 1H), 8.58 (m, 1H), 8.18 (s, 1H), 7.84 (t, J = 1.9 Hz, 1H), 7.72(m, 1H), 7.52 (m, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.31(s, 1H), 7.19 (m, 1H), 6.88 (m, 1H), 6.76 (s, 1H), 3.86 (s, 3H), 3.07 (d, J =12.4 Hz, 2H), 2.81 – 2.75 (m, 2H), 2.74 – 2.51 (m, 8H), 2.40 (m, 1H), 2.33(s, 3H), 2.04 (d, J = 12.4 Hz, 2H), 1.85 (s, 3H), 1.82 (s, 3H), 1.72 (m, 2H). 13 CNMR (101 MHz, Chloroform- d ) δ 162.59, 157.44, 155.96, 154.97, 145.32, 143.68(d, J = 2.6 Hz), 137.19, 136.51, 134.89, 132.30, 131.47, 130.15, 129.35 (d, J =10.6 Hz), 127.56, 126.47, 126.09, 124.70, 122.93 (d, J= 7.1 Hz), 122.35 (d, J =12.3 Hz), 120.31 (d, J = 95.9 Hz), 111.93, 106.93, 103.24, 61.51, 56.00, 55.29,52.74, 49.08, 45.91, 29.58, 18.86, 18.15.MS (ESI + ) m / z calcd for C 36 H 44 Cl2N8O3P + (M+H) + 737.3, found 736.9.

[0102] Example 10

[0103] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-2-methoxybenzamide (Compound H-10, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=2-methoxybenzamide).

[0104] The raw material benzoic acid in Example 3 was substituted for 2-methoxybenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-2-methoxybenzamide (Compound H-10) with a yield of 75%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.70(s, 1H), 10.17 (s, 1H), 9.16 (s, 1H), 8.59 (m, 1H), 8.20 – 8.10 (m, 2H), 7.55– 7.47 (m, 1H), 7.35 (t, J= 7.9 Hz, 1H), 7.23 (s, 1H), 7.10 (m, 3H), 6.78 (d, J = 7.8 Hz, 1H), 6.72 (s, 1H), 4.09 (s, 3H), 3.85 (s, 3H), 3.15 (d, J = 11.2 Hz, 2H), 2.72 – 2.51 (m, 10H), 2.42 (m, 1H), 2.33 (s, 3H), 1.95 (d, J = 12.0 Hz, 2H), 1.82 (s, 3H), 1.79 (s, 3H), 1.70 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 162.64, 157.69, 157.28, 155.95, 155.00, 145.72, 143.66 (d, J = 2.6 Hz), 138.13, 132.80, 132.61, 132.39 (d, J = 2.2 Hz), 129.26 (d, J = 10.9 Hz), 126.60, 125.05, 123.01 (d, J = 6.8 Hz), 122.76, 122.22 (d, J = 12.3 Hz), 121.39, 120.12 (d, J = 96.1 Hz), 114.55, 111.61, 106.53, 102.67, 61.60, 56.43, 55.98, 55.31, 52.51, 48.82, 45.87, 29.00, 18.84, 18.13. MS (ESI + ) m / z calcd for C 37 H 47 ClN8O4P + (M+H) + 733.3, found 733.0.

[0105] Example 11

[0106] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-2-methylbenzamide (Compound H-11, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=2-methylbenzamide).

[0107] The raw material benzoic acid in Example 3 was substituted for 2-methylbenzoic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)-2-methylbenzamide (Compound H-11) with a yield of 79%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.83 (s, 1H), 9.27 (s,1H), 8.67 (m, 1H), 8.61 (s, 1H), 8.16 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.42(t, J = 7.9 Hz, 1H), 7.36 (m, 1H), 7.28 (d, J = 13.8 Hz, 2H), 7.24 (d, J = 5.6 Hz,1H), 7.22 – 7.17 (m, 1H), 6.98 (m, 1H), 6.76 (s,1H), 3.85 (s, 3H), 3.05 (d, J =11.4 Hz, 2H), 2.78 – 2.71 (m, 2H), 2.71 – 2.48 (m, 8H), 2.47 (s, 3H), 2.34(d, J = 8.1 Hz, 1H), 2.31 (s, 3H), 2.00 (d, J = 12.3 Hz, 2H), 1.82 (s, 3H), 1.79 (s, 3H), 1.57 (m, 2H).13 C NMR (101 MHz, Chloroform- d ) δ 167.11, 157.59,155.95, 154.93, 145.51, 143.80 (d, J = 2.8 Hz), 136.74, 136.62 (d, J = 8.8 Hz),132.45, 132.42, 131.43, 130.09, 129.44 (d, J = 10.9 Hz), 126.96, 126.70,125.93, 125.84, 122.66 (d, J = 7.1 Hz), 122.29 (d, J = 12.2 Hz), 119.94 (d, J =95.8 Hz), 112.50, 106.90, 103.36, 61.50, 55.99, 55.23, 52.70, 49.20, 45.88,29.51, 20.13, 18.90, 18.19.MS (ESI + ) m / z calcd for C 37 H 47 ClN8O3P + (M+H) + 717.3, found 717.0.

[0108] Example 12

[0109] 2-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-12, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=2-chlorobenzamide).

[0110] The raw material benzoic acid in Example 3 was substituted for 2-chlorobenzoic acid, and the remaining steps were the same as in Example 3 to prepare 2-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)benzamide (Compound H-12) with a yield of 74%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.81 (s, 1H), 9.36 (s,1H), 9.14 (s, 1H), 8.67 (m, 1H), 8.18 (s, 1H), 7.72 (m, 1H), 7.48 – 7.34(m,4H), 7.25 (s, 1H), 7.20 (m, 1H), 6.99 (m, 1H), 6.77 (s, 1H), 3.86 (s, 3H), 3.06 (d, J = 11.2 Hz, 2H), 2.78 – 2.71 (m, 2H), 2.70 – 2.43 (m, 8H), 2.35 (t, J =5.7 Hz, 1H), 2.31 (s, 3H), 2.02 – 1.94 (m, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.62 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 163.52, 157.56, 155.98, 154.95,145.53,143.79 (d, J = 2.6 Hz), 136.71, 135.77, 132.50 (d, J = 2.3 Hz), 131.41,130.69, 130.44, 130.29, 129.40 (d, J = 10.9 Hz), 127.22, 126.99, 126.04, 122.79(d, J = 7.2 Hz), 122.29 (d, J = 12.4 Hz), 120.00 (d, J= 95.9 Hz), 112.29, 106.97,103.57, 61.56, 55.97, 55.28, 53.00, 49.23, 45.91, 29.37, 18.91, 18.20.MS (ESI + ) m / z calcd for C 36 H 44 Cl2N8O3P + (M+H) + 737.3, found 736.9.

[0111] Embodiment 13

[0112] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamide (Compound H-13, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=acetamide).

[0113] The raw material benzoic acid in Example 3 was substituted for acetic acid, and the remaining steps were the same as in Example 3 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamide (Compound H-13) with a yield of 76%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.77 (s, 1H), 9.03 (s, 1H), 8.60 (m, 1H), 8.13 (d, J = 6.7 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.25 (d, J = 5.2Hz, 2H), 7.12 – 7.05 (m, 1H), 6.68 (s, 1H), 3.83 (s,3H), 3.03 (d, J= 11.5 Hz,2H), 2.81 – 2.52 (m, 10H), 2.37 (s, 3H), 2.13 (s, 3H), 2.06 (d, J = 11.5 Hz, 2H), 1.84 (s, 3H), 1.81 (s, 3H), 1.70 (m, 2H). 13 C NMR (101 MHz, Chloroform- d )δ 167.49, 157.50, 155.91, 154.88, 145.25,143.77, 136.43, 132.36, 129.45 (d, J =10.7 Hz), 126.45, 125.67, 122.80 (d, J = 6.9 Hz), 122.24 (d, J = 12.4 Hz), 120.15(d, J = 95.7 Hz), 112.68, 106.82, 102.97, 61.40, 55.98, 55.07, 52.39, 49.06,45.68,29.71, 29.68, 24.71, 18.86, 18.15.MS (ESI + ) m / z calcd for C 31 H 43 ClN8O3P + (M+H) + 641.3, found 641.0.

[0114] Embodiment 14

[0115] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acrylamide (Compound H-14, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=acrylamide).

[0116] Acryloyl chloride (1.0 equivalent) was added to a 50 mL round-bottom flask, and dry N,N-dimethylformamide was added. The product compound H-02 (1.0 equivalent) in Example 2 was dissolved in N,N-dimethylformamide and then slowly added dropwise to the 50 mL round-bottom flask under an ice bath for 2 hours. After the reaction was completed as monitored by TLC, N,N-dimethylformamide was washed out by extraction with water and ethyl acetate, the organic phase was collected and dried, and the product N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acrylamide (compound H-14) was obtained by column chromatography with a yield of 60%. 1 HNMR (400 MHz, Chloroform- d ) δ 10.72 (s, 1H), 9.14 (s, 1H), 8.57 (m, 1H), 8.38 (s, 1H), 8.14 (s, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.25 – 7.18 (m, 2H), 7.07 –6.97 (m, 1H), 6.69 (s, 1H), 6.30 (m, 1H), 6.23 (d, J = 9.7 Hz, 1H), 5.70 (m,1H), 3.82 (s, 3H), 3.01 (d, J = 11.5 Hz, 2H), 2.75 – 2.59 (m, 6H), 2.49 (s, 4H), 2.30 (s, 1H), 2.29 (s, 3H), 2.04 (d, J = 12.4 Hz, 2H), 1.82 (s, 3H), 1.78 (s, 3H), 1.64 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 162.63, 157.52,155.98, 154.96, 145.44, 143.68, 136.61, 132.45 (d, J = 2.1 Hz), 131.99, 127.93(d, J = 282.7 Hz), 127.93 (d, J= 305.9 Hz), 125.79, 122.92 (d, J = 7.1 Hz), 122.37(d, J = 12.1 Hz), 120.71, 119.75, 112.49, 106.87, 103.11, 61.40, 55.98, 55.36,52.55, 49.44,45.98, 29.88, 18.85, 18.14.MS (ESI + ) m / z calcd for C 32 H 43 ClN8O3P + (M+H) + 653.3, found 653.0.

[0117] Embodiment 15

[0118] N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)ethenesulfonamide (Compound H-15, R1 = 1-methyl-4-(4-piperidinyl)piperazine, R2 = vinylsulfonamide).

[0119] The raw material acryloyl chloride in Example 14 was substituted for vinylsulfonyl chloride, and the remaining steps were the same as in Example 14 to prepare N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)ethenesulfonamide (Compound H-13) with a yield of 61%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.81(s, 1H), 8.57 (m, 1H), 8.48 (s, 1H), 8.12 (s, 1H), 7.59 (m, 1H), 7.29 (m,1H), 7.18 – 7.11(m, 1H), 6.72 (s, 1H), 6.49 (m, 1H), 6.33 (d,J = 16.6 Hz, 1H),5.90 (d, J = 9.8 Hz, 1H), 3.84 (s, 3H), 2.99 – 2.92 (m, 2H), 2.74 – 2.49 (m,10H), 2.39 (m, 1H), 2.32 (s,3H), 2.02 – 1.94 (m, 2H), 1.84 (s, 3H), 1.81 (s,3H), 1.69 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 157.22, 155.97, 154.56,145.25, 143.54 (d, J = 2.7 Hz), 136.40, 135.49, 132.86 (d, J = 2.2 Hz), 129.59(d, J = 10.9 Hz), 128.00, 126.77, 125.81, 122.71(d, J = 16.2 Hz), 122.68, 120.16(d, J = 95.8 Hz), 110.25, 107.29, 103.96, 61.23, 56.00, 55.31, 53.10, 49.01,45.88, 29.21, 18.86, 18.15.MS (ESI + ) m / z calcd for C 31 H 43 ClN8O4PS + (M+H) + 689.3, found 689.4.

[0120] Example 16

[0121] 1-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)methane sulfonamide (Compound H-16, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=chloromethanesulfonamide).

[0122] The raw material acryloyl chloride in Example 14 was substituted for chloromethanesulfonyl chloride, and the remaining steps were the same as in Example 14 to prepare 1-chloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)methanesulfonamide (Compound H-16) in a yield of 54%. 1 H NMR (400 MHz, Chloroform- d ) δ10.75 (d, J = 6.8 Hz, 1H), 8.51 – 8.41 (m, 2H), 8.05 (d, J = 6.7 Hz, 1H), 7.48(q, J = 7.9, 7.5 Hz, 1H), 7.29 (d, J = 6.6 Hz, 1H), 7.21 (m, 1H), 7.11 – 7.02 (m,1H), 6.70 (d, J = 6.9 Hz, 1H), 4.39 (d, J = 6.6 Hz, 2H), 3.80 (d, J = 6.6 Hz, 3H),2.93 (d, J = 10.9 Hz, 2H), 2.78 – 2.42 (m, 10H), 2.40 – 2.34 (m, 1H), 2.31 (d, J = 6.8 Hz, 3H), 1.92 (d, J = 11.7 Hz, 2H), 1.76 (dd, J = 13.8, 6.3 Hz, 6H), 1.65(m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 157.15, 155.95, 154.68, 145.66,143.53, 136.24, 132.72, 129.62 (d, J = 11.1 Hz), 127.19, 125.29, 122.76 (d, J=12.1 Hz), 122.55 (d, J = 7.0 Hz), 120.32 (d, J = 95.9 Hz), 109.56, 107.43,104.45, 61.18, 56.02, 55.12, 53.20, 53.07, 48.70, 45.65, 29.25, 18.83,18.12.MS (ESI + ) m / z calcd for C 30 H 44 Cl2N8O4PS + (M+3H) + 713.2, found 713.4.

[0123] Embodiment 17

[0124] 2-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)ethane-1-sulfonylfluoride (Compound H-17, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=ethanesulfonyl fluoride ).

[0125] The raw material acryloyl chloride in Example 14 was substituted for ethanesulfonyl fluoride, and the remaining steps were the same as in Example 14 to prepare 2-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)ethane-1-sulfonylfluoride (Compound H-17) in a yield of 52%. 1 H NMR (400 MHz, Chloroform- d) δ 10.79 (s, 1H), 8.53 (m, 1H), 8.04 (s, 1H), 7.69 (s, 1H), 7.45 – 7.39 (m, 1H), 7.34 (s, 1H), 7.27 – 7.21 (m, 1H), 7.08 – 7.01 (m, 1H), 6.63 (s, 1H), 4.88 – 4.79 (m, 1H), 3.75 (s, 3H), 3.44 (s, 2H), 3.38 (t, J = 5.2 Hz, 2H), 3.02 – 2.95 (m, 2H), 2.68 – 2.39 (m, 10H), 2.36 – 2.29 (m, 1H), 2.26 (s, 3H), 1.92 – 1.86 (m, 2H), 1.79 (s, 3H), 1.75 (s, 3H), 1.60 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 156.43, 154.77, 153.87, 142.88 (d, J = 2.8 Hz), 139.85, 133.87, 132.71, 131.55 (d, J = 2.2 Hz), 128.76 (d, J = 10.9 Hz), 125.47, 121.37, 121.27 (d, J = 5.1 Hz), 119.23 (d, J = 95.3 Hz), 105.79, 103.78, 101.97, 60.62, 55.65, 54.23, 51.02, 49.02, 48.89, 47.90, 44.81, 37.50, 28.21, 17.84, 17.13. MS(ESI + ) m / z calcd for C 31 H 44 ClFN8O4PS + (M + H) + 709.3, found 709.0.

[0126] Example 18

[0127] 3-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)carbamoyl)benzenesulfonyl fluoride (Compound H-18, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=3-(fluorosulfonyl)benzamide.

[0128] The raw material benzoic acid in Example 3 was substituted for 3-(fluorosulfonyl)benzoic acid, and the remaining steps were the same as in Example 3 to prepare 3-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)carbamoyl)benzenesulfonyl fluoride (Compound H-18) with a yield of 65%. 1 H NMR (400 MHz, Chloroform- d )δ 10.76 (s, 1H), 9.63 (s, 1H), 9.27 (s, 1H), 8.57 (m, 1H), 8.31 (t, J = 1.8 Hz,1H), 8.13 (s, 1H), 8.06 – 8.01 (m, 2H), 7.52 (t, J = 7.7 Hz, 1H), 7.31 (t, J =7.9 Hz, 1H), 7.24 (s, 1H), 7.18 – 7.12 (m, 1H), 6.91 (m, 1H), 6.59 (s, 1H), 3.76 (s, 3H), 3.02 (t, J = 17.0 Hz, 4H), 2.87 – 2.71 (m, 4H), 2.40 (s, 2H), 2.15 (s, 6H), 1.98 (s, 1H), 1.78 (s, 3H), 1.75 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 160.71, 157.27, 155.91, 154.94, 145.34, 143.71, 137.01,135.99, 134.95, 133.85,133.58 (d, J = 25.4 Hz), 132.13, 130.74, 129.47, 129.37,126.94, 126.29 (d, J = 37.9 Hz), 122.89, 122.31 (d, J = 12.0 Hz), 111.41, 107.06,103.25, 61.55, 56.01, 54.46, 52.68, 48.26, 45.19, 29.32, 18.80,18.09.MS (ESI + ) m / z calcd for C 36 H 44 ClFN8O5PS + (M+H) + 785.3, found 785.3.

[0129] Embodiment 19

[0130] 4-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino) -4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)carbamoyl)benzenesulfonyl fluoride (Compound H-19, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=4-(fluorosulfonyl)benzamide.

[0131] The raw material benzoic acid in Example 3 was substituted for 4-(fluorosulfonyl)benzoic acid, and the remaining steps were the same as in Example 3 to prepare 4-((5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)carbamoyl)benzenesulfonyl fluoride (Compound H-19) with a yield of 64%. 11H NMR (400 MHz, Chloroform- d ) δ 10.77 (s, 1H), 9.29 (s, 1H), 9.22 (s, 1H), 8.55 (m, 1H), 8.13 – 8.07(m,3H), 8.02 (d, J = 8.3 Hz, 2H), 7.32 (t, J = 7.8 Hz, 1H), 7.25 (s, 1H), 7.14 (m,1H), 6.89 – 6.82 (m, 1H),6.71 (s, 1H), 3.81 (s, 3H), 2.99 (d, J = 11.5 Hz, 2H),2.75 – 2.69 (m, 2H), 2.68 – 2.48 (m, 8H), 2.32 (s, 1H), 2.29 (s, 3H), 2.02(d, J = 12.4 Hz, 2H), 1.78 (s, 3H), 1.75 (s, 3H), 1.65 – 1.54 (m, 2H). 13 13C NMR(101 MHz, Chloroform- d ) δ 160.60, 156.33, 154.97, 153.90, 144.57, 142.79,140.93, 135.38, 134.44,134.19,131.27, 128.43 (d, J = 10.9 Hz), 128.05, 127.10,125.36, 125.14, 121.71 (d, J = 7.1 Hz), 121.25 (d, J = 12.4 Hz), 119.68, 118.73,110.69, 106.22, 102.25, 60.48, 54.98, 54.03, 51.70, 48.25, 44.72, 28.96,17.92,17.20.MS (ESI + ) m / z calcd for C 36 H 44 ClFN8O5 P S + (M+H) + 785.3, found 785.4.

[0132] Example 20

[0133] 2,2,2-trichloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrim idin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamide (Compound H-20, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=trichloroacetamide).

[0134] The raw material acryloyl chloride in Example 14 was substituted for trichloroacetyl chloride, and the remaining steps were the same as in Example 14 to prepare 2,2,2-trichloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamide (Compound H-20) with a yield of 40%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.72 (d, J =3.6 Hz, 1H), 9.74 (d, J = 3.1 Hz, 1H), 9.08 (d, J = 5.6 Hz, 1H), 8.48 (m, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.36 (m, 1H), 7.24 – 7.13(m, 2H), 7.00 (m, 1H), 6.69(d, J = 4.2 Hz, 1H), 3.80 (d, J = 5.2 Hz, 3H), 3.01 – 2.92 (m, 2H), 2.73 – 2.38 (m, 10H), 2.33 (s, 1H), 2.27 (d, J = 5.4 Hz, 3H), 1.95 (d, J = 11.5 Hz, 2H), 1.77(d, J = 5.4 Hz, 3H), 1.74 (d, J= 5.6 Hz, 3H), 1.68 – 1.56 (m, 2H). 13 C NMR (101MHz, Chloroform- d ) δ 157.98, 157.28, 155.95, 154.88, 145.96, 143.61 (d, J = 2.6Hz), 136.82, 132.34 (d, J = 2.2 Hz), 129.52 (d, J = 11.0 Hz), 126.42, 125.11,122.73 (d, J = 7.0 Hz), 122.55 (d, J = 12.2 Hz), 120.27 (d, J = 96.0 Hz), 110.96,107.19, 103.35, 93.56, 61.46, 55.97, 55.11, 52.81, 49.02, 45.75, 29.45,18.84, 18.13.MS (ESI + ) m / z calcd for C 31 H 42 Cl4N8O3P + (M+3H) + 745.2, found 745.3.

[0135] Embodiment 21

[0136] 2,2-dichloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidi n-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamide (Compound H-21, R1=1-methyl-4-(4-piperidinyl)piperazine, R2=dichloroacetamide).

[0137] The raw material acryloyl chloride in Example 14 was substituted for dichloroacetyl chloride, and the remaining steps were the same as in Example 14 to prepare 2,2-dichloro-N-(5-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acetamid e (Compound H-21) with a yield of 41%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.70 (s,1H), 9.40 (s, 1H), 8.98 (s, 1H), 8.48 (m, 1H), 8.06 (s, 1H), 7.39 – 7.31 (m,1H),7.18 (m, 1H), 7.02 – 6.94 (m, 1H), 6.66 (s, 1H), 5.92 (s, 1H), 3.78 (s,3H), 3.01 – 2.93(m, 2H), 2.70 – 2.64 (m, 2H), 2.61 (d, J = 12.2 Hz, 4H), 2.53 –2.40 (m, 4H), 2.34 – 2.29 (m, 1H), 2.25 (s, 3H), 1.98 – 1.91 (m, 2H), 1.76(s, 3H), 1.72 (s, 3H), 1.65 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 160.61,157.35, 155.90, 154.80, 145.95, 143.62 (d, J = 2.5 Hz), 137.02, 132.41, 132.39,129.48 (d, J = 10.9 Hz), 125.51 (d, J = 89.9 Hz), 122.73 (d, J = 6.9 Hz), 122.40(d, J= 12.4 Hz), 120.64, 119.69, 111.63, 106.97, 103.22, 67.45, 61.46, 55.95,55.13, 52.66, 48.98, 45.77, 29.29,18.81, 18.10.MS (ESI + ) m / z calculation for C 31 H 41 Cl3N8O3P + (M+H) + 709.2080, found 709.2080.

[0138] Embodiment 22

[0139] (2-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)-5-nitrophenyl)amino) pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound H-22, R1=N-methylpiperazine, R2=NO2).

[0140] The raw material 1-methyl-4-(4-piperidinyl)piperazine in step (4) of Example 1 was replaced by N-methylpiperazine, and the remaining steps were the same as Example 1 to prepare (2-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)-5-nitrophenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound H-22) with a yield of 82%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.83 (s, 1H), 8.85 (s,1H), 8.44 (m, 1H), 8.13 (s, 1H), 7.53 – 7.46 (m, 1H), 7.40 (s, 1H), 7.33 –7.28 (m, 1H), 7.16 (m, MS (ESI) +) m / zcalcd for C 24 H 30 ClN7O4P + (M+H) + 546.2, found 546.0.

[0141] Embodiment 23

[0142] (2-((2-((5-amino-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (Compound H-23, R1=N-methylpiperazine, R2=NH2).

[0143] The raw material compound H-01 in the step of Example 2 was replaced by compound H-22, and the remaining steps were the same as Example 2 to prepare (2-((2-((5-amino-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-chloro pyrimidin-4-yl)amino)phenyl)dimethylphosphineoxide (compound H-23) with a yield of 80%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.76 (s, 1H), 8.60 (m, 1H), 8.09 (s,1H), 7.85 (s, 1H), 7.59 (m, 1H), 7.47 (s, 1H), 7.34 – 7.28(m, 1H), 7.14 (m,1H), 6.67 (s, 1H), 3.81 (s, 3H), 2.93 (t, J = 4.7 Hz, 4H), 2.59 (s, 4H), 2.37(s, 3H), 1.85 (s, 3H), 1.82 (s, 3H).MS (ESI + ) m / z calcd for C 24 H 32 ClN7O2P + (M+H) + 516.2, found 515.9.

[0144] Embodiment 24

[0145] 1-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl) amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)ethan-1-one (compound H-24, R1=1-acetylpiperazine, R2=NO2).

[0146] The raw material 1-methyl-4-(4-piperidinyl)piperazine in step (4) of Example 1 was replaced by 1-acetylpiperazine, and the remaining steps were the same as in Example 1 to prepare 1-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxy-2-nitrophenyl) piperazin-1-yl)ethan-1-one (Compound H-24) with a yield of 84%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.80 (s, 1H), 8.87– 8.79 (m, 1H), 8.36 (m, 1H), 8.05 (m,1H), 7.47 – 7.34 (m, 2H), 7.25 (m, 1H),7.08 (m, 1H), 6.51 (m, 1H), 3.89 (d, J = 1.3 Hz, 3H), 3.75 – 3.66 (m, 2H), 3.56(t, J = 4.8 Hz, 2H), 2.95 (q, J = 4.3 Hz, 4H), 2.06 (d, J = 1.2 Hz, 3H), 1.78 (d, J =1.2 Hz, 3H), 1.74 (d, J = 1.2 Hz, 3H).MS (ESI + ) m / z calcd for C 25 H 30 ClN7O5P + (M+H) + 574.2, found 573.9.-

[0147] Embodiment 25

[0148] 1-(4-(2-amino-4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxyphenyl)piperazin-1-yl)ethan-1-one (compound H-25, R1=1-acetylpiperazine, R2=NH2).

[0149] The raw material compound H-01 in the step of Example 2 was replaced by compound H-24, and the remaining steps were the same as Example 2 to prepare 1-(4-(2-amino-4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-5-methoxyphenyl)piperazin-1-yl)ethan-1-one (compound H-25) with a yield of 80%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.79 (s, 1H), 8.60 (m, 1H), 8.10 (s, 1H), 7.89 (s, 1H), 7.59 (m, 1H), 7.49 (s, 1H), 7.31 (m, 1H), 7.15(m, 1H), 6.59 (s, 1H), 3.81 (s,3H), 3.73 (s, 2H), 3.61 (q, J = 4.9 Hz, 2H),2.88 (dd, J = 10.2, 5.1 Hz, 4H), 2.15 (s, 3H), 1.86 (s, 3H), 1.83 (s, 3H).MS(ESI + ) m / z calcd for C 25 H 32 ClN7O3P + (M+H) + 544.2, found 543.9.

[0150] The technical scheme of the present invention is further illustrated by experiments below. Compounds H-1 to H-21 used in the following experiments were prepared according to the methods of Examples 1 to 21 above.

[0151] Experimental Example 1 Lance Ultra Assay to determine the inhibitory activity of EGFR kinase

[0152] 1. Experimental methods:

[0153] The Lance Ultra Assay method was used to test the EGFR kinase inhibitory activity of the invented compounds, and the compound Staurosporine was used as a standard control. First, the compounds were prepared. The initial test concentration of the compound was 120nM, and it was necessary to prepare it to a 100-fold concentration, that is, 12 μM. When the initial test concentration of the compound was 30nM, it was necessary to prepare it to a 100-fold concentration, that is, 3 μM. Transfer 50μL of 100% DMSO to two empty wells as a control without compound and enzyme. Use Echo 650 to transfer 200 nl of compound to a 384-well test plate. Prepare EGFR T790M / L858R and EGFR T790M / C797S / L858R 1x kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.01% Tween-20, 0.01% BSA, 2 mM DTT) and EGFR 1x kinase buffer (50 mM HEPES, pH 7.5, 10 mM MnCl2, 0.01% Tween-20, 0.01% BSA, 2 mM DTT). Add kinase to 1x kinase buffer to form kinase solution, transfer 10 μL of the above kinase solution to the reaction wells of 384-well plate, add 1x kinase buffer (without enzyme) to the negative control wells, centrifuge at 1000 rpm for 1 minute, and incubate at room temperature for 10 minutes. Add substrate and ATP and other reagents to kinase buffer to form substrate solution. Transfer 10 μL of substrate solution to the reaction wells of 384-well plate and centrifuge at 1000 rpm for 1 minute. Kinase assay reacts at room temperature for 60 minutes. Prepare 2x stop reaction solution containing antibody. Add 20 µL of the above reagents to a 384-well reaction plate and centrifuge at 1000 rpm for 1 minute. Incubate at room temperature for 60 minutes. Read the fluorescence values ​​at 665 mm and 615 mm on an Envision 2104 Multilabel Reader. Convert the above data to inhibition percentage using the following formula: Percent inhibition = (max- SampleLance signal ratio) / (max-min)*100. “min” is the reading of the control well without enzyme; “max” is the reading of the control well with DMSO added. Import the data into MS Excel and use XLFit excel add-inversion 5.4.0.8 for curve fitting. The fitting formula is: Y=Bottom + (Top-Bottom) / (1+(IC 50 / X)^HillSlope).

[0154] 2. Experimental results:

[0155] The results in Table 1 show that the compounds of the present invention have EGFR T790M / C797S / L858R Overall, all the compounds of the invention showed a higher inhibitory activity than osimertinib on EGFR T790M / C797S / L858R Kinases have better inhibitory activity.

[0156] Table 1 Effects of the compounds of the present invention on EGFR WT , EGFR T790M / L858R and EGFR T790M / C797S / L858R Kinase inhibitory activity

[0157]

[0158] -: Not tested.

[0159] Select EGFR T790M / C797S / L858R The compounds H-01, H-09, and H-21 with good kinase inhibitory activity were evaluated for their effects on EGFR WT , EGFR T790M / L858R and EGFR T790M / C797S / L858R IC of kinase inhibition 50 The results in the table show that the compounds have an effect on EGFR T790M / L858R and EGFR T790M / C797S / L858R IC of kinase 50 The values ​​were all in the nanomolar level. The kinase inhibitory activity results confirmed that the compound showed quite good EGFR double and triple mutation inhibitory activity against EGFR WT Be selective.

[0160] Experimental Example 2 Determination of cell growth inhibition rate by MTT method

[0161] 1. Experimental methods:

[0162] BEAS-2B cells are normal lung bronchial epithelial cell lines, cultured in DMEM high-glucose medium (supplemented with 10% fetal bovine serum); BaF3 T790M / C797S / L858R Cells, H1975 T790M / L858R Cells and HCC827 cells are lung cancer cell lines and were cultured in RPMI1640 medium (supplemented with 10% fetal bovine serum); A549 WTCell lung cancer cell line was cultured in DMEM high glucose medium (supplemented with 10% fetal bovine serum). When the cells were in the logarithmic phase, they were seeded in a 96-well cell culture plate at a density of 5000 / well and incubated in a 37°C, 5% CO2 incubator for 12 h. The experimental compounds were added to three replicate wells at different concentrations (50, 16.67, 5.56, 1.85, 0.62, 0.2, 0.068, 0.023 μM), and 0.4% DMSO was used as a control. After incubation in a 37°C, 5% CO2 incubator for 72 h, 10 μL of MTT dye (5 mg / mL) was added to each well and cultured in the incubator for another 4 h. Then, the culture medium was removed, and 100 μL of Formanzan solution (Formanzan was dissolved in 100 μL of DMSO) was added to each well. The cells were shaken until all the crystals were dissolved. The absorbance (OD value) of each well of the 96-well plate in the experiment was measured at a wavelength of 490 nm using a VICTOR® Nivo™ multi-mode plate reader. The cell lethality was calculated as %=[1-(OD490 drug-added well – OD490 background well) / (OD490 control well – OD490 background well)]×100%, and the half-maximal inhibitory concentration (IC) was calculated using SPSS 26.0. 50 Values ​​(mean ± SD, n=3) (the above parallel experiments were repeated three times independently).

[0163] 2. Experimental Results

[0164] The results in Table 2 show that the selected compounds of the present invention are more sensitive to H1975, HCC827, and BaF3 lung cancer cells, especially have outstanding anti-proliferative activity against H1975 cells. In addition, among the various compounds provided by the present invention, compound H-21 has the best anti-proliferative activity against H1975 cells and has significantly better activity than other compounds.

[0165] Table 2 IC of compounds against NSCLC cell proliferation 50 value.

[0166]

[0167] -: Not tested.

[0168] Experimental Example 3 Effects of the compounds of the present invention on the proliferation of lung cancer H1975 cells

[0169] 1. Experimental methods:

[0170] Plate cloning experiment

[0171] Take H1975 cells in good growth state and logarithmic growth phase, digest with routine trypsin, and then blow into cell suspension with RPMI1640 culture medium containing 10% fetal bovine serum. Plant 1000 cells in each well of a six-well plate. Add the compound of the present invention after adherence. Add different concentrations of the compound of the present invention to the experimental group. Add the same amount of culture medium without the compound of the present invention to the control group. Each group has 3 replicates. Culture in an incubator at 37°C, 5% CO2, and saturated humidity for 14 days. Change the solution once every 3 days during this period. Observe frequently, and terminate the culture when visible clones appear in the culture dish. Discard the supernatant and carefully rinse twice with PBS. Add 4% (w / v) paraformaldehyde to fix the cells, and remove the fixative after 15 minutes. Add an appropriate amount of 0.1% crystal violet staining, slowly wash off the staining solution with running water after 30 minutes, air dry, and observe and record the inhibitory effect of the tested compound on the clone formation ability of H1975 cells.

[0172] 2. Experimental Results

[0173] The compound H-21 of the present invention can inhibit the formation of lung cancer H1975 cell clones in a concentration-dependent manner, which is significantly better than the positive drug osimertinib.

[0174] Experimental Example 4 Effect of the compounds of the present invention on apoptosis of lung cancer H1975 cells

[0175] 1. Experimental methods:

[0176] Flow cytometry

[0177] Take cells that are in good growth state and in the logarithmic growth phase, digest with routine trypsin, and then pipette the culture medium into a cell suspension. Seed 3×10 cells per well of a six-well plate. 5 cells. Cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. After the cells adhered, different concentrations of drugs were added for 48 h. After 48 h of culture, the cells were collected and centrifuged again at 2000 rpm for 5 min in a 4 °C precooled centrifuge. After centrifugation, the supernatant was carefully aspirated and 500 μL of 1×Binding Buffer was added to resuspend the cells. 5 μL of Annexin V-FITC (biosharp) was added to the sample and mixed, and then 5 μL of propidiumiodide staining solution was added and mixed. After staining at room temperature in the dark for 15 min, the samples were analyzed using an Agilent NovoCyte flow cytometer.

[0178] 2. Experimental Results

[0179] The compound H-21 of the present invention was determined by flow cytometry to be able to induce apoptosis of lung cancer H1975 cells in a concentration-dependent manner.

[0180] Experimental Example 5 Effects of the compounds of the present invention on migration and invasion of lung cancer H1975 cells

[0181] 1. Experimental methods:

[0182] (1) Scratch test

[0183] H1975 cells were digested and inoculated into six-well plates. When the cells reached a density of 90%, scratches were prepared. Three straight lines were drawn in each well with the tip of a 10 μL sterile pipette. The culture medium was discarded, and the cells were washed three times with PBS to remove the cells that fell due to the scratches. Each well was then replaced with 2 mL of RPMI 1640 culture medium containing 2% fetal bovine serum. Photos were taken under a Fluorescence microscope-research-grade inverted fluorescence microscope to record the state of the scratch area at 0 h. After that, different concentrations of drugs were added for treatment for 24 h, and the same scratch area was photographed and recorded again. The scratch area was calculated using ImageJ software, and the changes in the same area in 24 h were compared.

[0184] (2) Transwell migration assay

[0185] H1975 cells in the logarithmic growth phase were taken, the culture medium was discarded, and after washing once with PBS, serum-free RPMI1640 culture medium was added for starvation treatment for 4 hours. After that, trypsin was used for digestion, and the cells were resuspended in serum-free RPMI 1640 culture medium to adjust the cell concentration to 4×10 5 / mL, take 200 μL and add it to the upper chamber of the transwell chamber, and add 700 μL of RPMI 1640 culture medium containing 20% ​​fetal bovine serum to the lower chamber. Then add different concentrations of drugs to treat the cells. After incubation for 24 hours, discard the culture medium, fix with 4% paraformaldehyde at room temperature for 30 minutes, and then stain with 0.1% crystal violet at room temperature for 15-30 minutes. Gently wash with PBS 3 times, gently wipe off the unmigrated cells in the upper layer with a cotton swab, and then take pictures and observe with a Fluorescence microscope research-grade inverted fluorescence microscope.

[0186] (3) Transwell invasion assay

[0187] Take H1975 cells in the logarithmic growth phase, discard the culture medium, wash once with PBS, and add serum-free RPMI1640 culture medium for starvation treatment for 4 hours. At the same time, Matrigel (BD Biosciences) was quickly diluted with the corresponding serum-free RPMI1640 culture medium on ice at a ratio of 1:7, and 100 μL of diluted Matrigel was pre-coated on the upper chamber of the transwell chamber. After being placed in a 37°C incubator for 30 minutes, 100 μL of warm serum-free culture medium was added to the upper chamber to rehydrate the basement membrane layer for 1 hour. After removing the culture medium in the upper chamber, a 10 μL cell culture medium with a density of 10 was taken. 6 200 μL of H1975 cells / mL was added to the upper chamber, and 700 μL of RPMI 1640 medium containing 20% ​​FBS was added to the lower chamber. At the same time, different concentrations of drugs were added to treat the cells. During this period, some invasive H1975 cells degraded the matrix proteins in Matrigel matrix gel, passed through the pores of the polycarbonate membrane, and finally adhered to the bottom side of the polycarbonate membrane. After incubation for 24 hours, the culture medium was discarded, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then stained with 0.1% crystal violet at room temperature for 15-30 minutes. Gently wash with PBS 3 times, gently wipe off the unmigrated cells on the upper layer with a cotton swab, and then take pictures and observe with a Fluorescence microscope research-grade inverted fluorescence microscope.

[0188] 2. Experimental Results

[0189] The scratch test confirmed that the compound H-21 of the present invention can inhibit the migration of lung cancer H1975 cells in a concentration-dependent manner.

[0190] The compound H-21 of the present invention was confirmed by Transwell migration experiment to be able to inhibit the migration of lung cancer H1975 cells in a concentration-dependent manner.

[0191] The compound H-21 of the present invention was confirmed by the Transwell invasion experiment to be able to inhibit the invasion of lung cancer H1975 cells in a concentration-dependent manner.

[0192] Experimental Example 6 Inhibitory effect of the compound of the present invention on lung cancer tumors in nude mouse subcutaneous transplanted tumor H1975 model

[0193] 1. Experimental methods:

[0194] Five-week-old male nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. They were first allowed to adapt to the SPF animal room for about a week and eat freely to eliminate the impact of the environment on them. Healthy male nude mice were selected and weighed. H1975 cells in good growth condition and in the logarithmic growth phase were obtained. After routine trypsin digestion, the culture medium was blown into a cell suspension. The density of 1×107 NCI-H1975 cells / mL were suspended in sterile PBS, and then 0.2 mL / mouse of cell suspension was injected subcutaneously into the left axillary area of ​​nude mice to establish a subcutaneous transplant tumor model of H1975 non-small cell lung cancer. When the volume of the implanted tumor reached about 100 mm 3 Afterwards, the nude mice were randomly divided into 3 groups (5 mice in each group), namely, a blank control group, a 10 mg / kg compound H-21 administration group, and a 30 mg / kg compound H-21 administration group. Compound H-21 was dissolved in normal saline, and a trace amount of castor oil was used as a cosolvent to obtain a solution of appropriate concentration, which was administered once a day by intraperitoneal injection. The blank control group was intraperitoneally injected with a mixture of normal saline and castor oil in the same volume and proportion for 14 consecutive days. The weight of the mice and the size of the tumor measured with a vernier caliper were recorded every two days. The shorter one is the width of the tumor, represented by W, and the longer one is the length of the tumor, represented by L. The calculation formula for the tumor volume is L×W 2 / 2. On the 14th day after the start of drug administration, the nude mice were killed, the tumors were removed, weighed, and the tumor growth inhibition rate was calculated. The formula for tumor growth inhibition rate (TGI) is [1-(T-T0) / (C-C0)]× 100%, C and C0 represent the tumor volume of the blank control group measured on the last day and the first day, respectively, and T and T0 represent the tumor volume of the drug administration group measured on the last day and the first day, respectively. This study was approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University.

[0195] 2. Experimental results:

[0196] Results Figure 1 , A is the tumor volume curve of the subcutaneous transplanted tumor of nude mice after administration of the selected compound; B is the body weight curve of nude mice after administration of the selected compound; C is the size of the subcutaneous transplanted tumor of nude mice on the last day of administration of the selected compound; D is the tumor weight of the subcutaneous transplanted tumor of nude mice on the last day of administration of the selected compound; E is the pathological section results of mice in different groups. The figure shows that different concentrations of compound H-21 can slow down the growth rate of tumor volume and inhibit tumor growth after administration. The tumor inhibition rate of compound H-21 at high and low concentrations is 72.34%. The results of pathological sections found that compared with the control group, there were no obvious pathological changes in spleen, liver, kidney, heart, and lung tissue pathological studies. This shows that long-term administration of this type of compound has no cumulative toxicity.

[0197] Through the above examples and experimental examples, it can be seen that the compounds of the present invention have an effect on EGFR T790M / C797S / L858R Kinases and EGFR L858R / T790MThe kinase has outstanding inhibitory activity (at the nanomolar level) and selectivity. The compounds of the present invention have low toxicity to normal cells and have obvious inhibitory effects on non-small cell lung cancer cell lines, especially for EGFR mutant cells H1975 cells. They have outstanding selectivity and significant inhibitory effects; the compounds of the present invention can induce apoptosis of EGFR mutant cells H1975, and the cell cycle is blocked in the G0 / G1 phase. At the same time, the compounds of the present invention can effectively inhibit the phosphorylation of EGFR, as well as the phosphorylation of important kinases AKT, STAT3 and ERK1 / 2 in the downstream of the pathway involved in cancer cell proliferation and survival in H1975 cells. The compounds of the present invention have significant tumor inhibition effects in nude mouse xenograft tumor models, and low in vivo toxicity. In particular, the compound H-21 of the present invention has significantly better non-small cell lung cancer cell inhibition effects than compounds of the same structure.

[0198] Therefore, the compound of the present invention can be used as an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of anti-lung cancer drugs, and has good application prospects.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, characterized in that: The compound structural formula is selected from: H-21。 2. Use of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a tyrosine kinase inhibitor.

3. The use according to claim 2, characterized in that: The tyrosine kinase inhibitor is a drug for treating cancer; the cancer is lung cancer.

4. A pharmaceutical composition, characterized in that The preparation is prepared by using the compound described in claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

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