WO2017101862A1

WO2017101862A1 - 5,8-dihydropteridine-6,7-diketone derivative as egfr inhibitor and use thereof

Info

Publication number: WO2017101862A1
Application number: PCT/CN2016/110437
Authority: WO
Inventors: 李洪林; 徐玉芳; 丁健; 郝永佳; 孙德恒; 王霞; 童依; 张臣; 谢华
Original assignee: 华东理工大学
Priority date: 2015-12-18
Filing date: 2016-12-16
Publication date: 2017-06-22
Also published as: CN106892922B; CN106892922A

Abstract

The present invention relates to a pyrimidopyrimidine dione derivative as an EGFR inhibitor and a use thereof. In particular, the present invention relates to a compound as shown in formula I, the pharmaceutical composition containing the compound of formula I and the use of the compound in preparing drugs for treating EGFR-related diseases or inhibiting EGFR.

Description

5,8-dihydropteridin-6,7-dione derivative as EGFR inhibitor and application thereof

Technical field

The present invention relates to the field of medicinal chemistry; in particular, the present invention relates to novel 5,8-dihydropteridin-6,7-dione derivatives, a synthetic method thereof and a medicament for preparing a tumor-related disease as an EGFR inhibitor Application in .

Background technique

Cancer, also known as malignant tumor, is a large class of diseases characterized by abnormal cell proliferation and metastasis. It has the characteristics of high morbidity and high mortality, and is one of the malignant diseases that threaten human health and cause death. Research data shows that there were 12.7 million cancer patients worldwide in 2008, including more than 7 million deaths. In the world, 20% of new cancer patients are in China, and 24% of cancer patients are in China. If no effective measures are taken to prevent it or a better treatment plan, it is estimated that by 2030, there will be 26 million new cancer cases every year worldwide, and the number of cancer deaths will reach 17 million. Among the existing cancers, lung cancer is the most common malignant tumor with morbidity and mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer patients. According to the World Health Organization (WHO), by 2025, the number of new lung cancer cases in China will exceed 1 million per year. Once diagnosed with lung cancer, the patient has only a survival prospect, and the 5-year survival rate is less than 15%.

Since the 1980s, with the deepening of molecular biology research, the molecular mechanism of tumorigenesis and development has become increasingly clear. Among the many factors inducing cancer, certain protein kinases in cancer cells that are highly expressed by gene mutations are one of the main factors leading to abnormal signal transduction pathways. Protein tyrosine kinase is an important factor in the signal transduction process and participates in a series of cellular activities, which are closely related to cell growth, differentiation and proliferation. It catalyzes the transfer of the γ-phosphate group of ATP to the tyrosine residues of many important proteins, phosphorylating the phenolic hydroxyl group, and transmitting signals. Therefore, the development of selective protein kinase inhibitors to block or regulate diseases caused by abnormalities in these signaling pathways has been recognized as an effective research strategy for anti-tumor drug development. Among many tyrosine kinases, epidermal growth factor receptor tyrosine kinase (EGFR) is an indispensable and important component. EGFR consists of 1186 amino acids and encodes a transmembrane glycoprotein with a molecular weight of 170-kDa. EGFR can mediate multiple signal transduction pathways and transmit extracellular signals to the intracellular cells, which play an important role in regulating the proliferation, differentiation and apoptosis of normal cells and tumor cells (Cell, 2000, 100, 113-127). EGFR is a constitutive expression component of many normal epithelial tissues (such as skin and hair follicles), and in most solid tumors, EGFR is overexpressed or highly expressed. For example, in lung cancer, the expression rate of EGFR reaches 40-80%. Therefore, selectively inhibiting EGFR and interfering with its signal transduction pathway can achieve the purpose of treating lung cancer, and opens up a feasible way for targeted treatment of lung cancer.

Clinical treatment, combined with traditional radiotherapy, chemotherapy, EGFR targeted drugs such as gefitinib (Iressa), erlotinib (Tarceva) and other first-line drugs have been proved to be very effective in the treatment of lung cancer. However, clinical practice has shown that most patients with non-small cell lung cancer develop acquired resistance within 6-12 months after treatment with gefitinib or erlotinib. The resistance of approximately 50% of cases is related to a mutation in one amino acid residue in the EGFR kinase domain (mutation of the 790 threonine residue to methionine, T790M) (The New England Journal of Medicine, 2005, 352, 786) -792). The T790M mutation results in steric hindrance when the inhibitor binds to EGFR or increases the affinity of EGFR to ATP, making the anticancer effect of such reversible binding competitive inhibitors greatly diminished. The emergence of drug resistance not only reduces the patient's medication The sensitivity of the object also greatly reduces the quality of life of patients with cancer. In order to overcome the drug resistance caused by T790M mutation, a series of irreversible ATP competitive inhibitors (such as CI-1033, HKI-272, PF00299804, etc.) have entered the clinical research stage. The irreversible inhibitor contains a Michael acceptor fragment that forms a covalent bond with a conserved amino acid residue (Cys797) of the ATP binding site of EGFR, thereby obtaining a stronger EGFR binding affinity than the reversible inhibitor. Despite this, due to the poor selectivity of these drugs for wild-type and mutant EGFR, their maximum tolerated dose (MTD) is low, and the clinical trial results are not obvious.

Therefore, research and development of third-generation EGFR targeted drugs that selectively inhibit T790M mutations and overcome clinical drug resistance have great clinical significance and application prospects.

Summary of the invention

It is an object of the present invention to provide compounds which are capable of selectively inhibiting the T790M mutation as EGFR inhibitors.

Another object of the present invention is to provide a pharmaceutical composition comprising the above compound.

Still another object of the present invention is to provide use of the above compounds for the preparation of a medicament for treating an EGFR-related disease or inhibiting EGFR.

In a first aspect, the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

Wherein A is a benzene ring, a five- or six-membered heterocyclic ring, or a C ₃ -C ₈ cycloalkyl group;

R ^{1 is} each independently selected from the group consisting of hydrogen, halogen, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkyl, C ₁ -C ₄ alkylamido, substituted piperazinyl, substituted homopiperazinyl, substituted? Lolinyl, substituted thiomorpholinyl, 4-N-methylpiperazinyl, 4-N-acetylpiperazinyl, 4-N,N-dimethylpiperidinyl, substituted piperidinyl, N, N-dimethylaminoethylamino, N,N-dimethylaminoethoxy, 4-(4-N-methylpiperazinyl)piperidinyl, -NR _a R _b , wherein R _a And R _b may be selected from the group consisting of alkyl and nitrogen-containing alkyl groups;

B is selected from the following group:

R ^{2 is} each independently selected from the group consisting of:

R ^{3 is} selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl, substituted C ₁ -C ₁₀ alkyl, optionally substituted C ₃ -C ₈ cycloalkyl, optionally substituted benzyl, optionally substituted Heterocyclic group;

m is any integer from 0 to 7, preferably from 1 to 7.

In a specific embodiment, the compound is as shown in Formula II:

Wherein B, R ¹ , R ² and R ^{3 are} as defined in claim 1;

m is an integer from 0 to 5, preferably from 1 to 5.

In a specific embodiment, the compound is as shown in Formula III:

In the formula,

R ^{2 is} selected from

R ^{3 is} selected from H, or C ₁ -C ₆ alkyl, preferably methyl or isopropyl;

R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ^{8 are} independently selected from the group consisting of

In a specific embodiment, R ^{3 is} selected from C ₁ -C ₆ alkyl, preferably methyl or isopropyl; R ⁵ , R ⁷ and R ⁸ are H; and R ⁴ and R ^{6 are} independently selected from the group consisting of

In a second aspect, the invention provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof:

In a third aspect, the present invention provides a pharmaceutical composition comprising the compound of the first or second aspect of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In a preferred embodiment, the pharmaceutical composition is a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, powders.

In a fourth aspect, the invention provides the use of a compound of the first or second aspect of the invention for the manufacture of a medicament for the treatment or prevention of an EGFR mediated disease, or inhibition of EGFR.

In a specific embodiment, the EGFR mediated disease is cancer.

In a specific embodiment, the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck Squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumors.

In a fifth aspect, the present invention provides a method of treating or preventing an EGFR-mediated disease comprising administering a compound of the first or second aspect of the invention or the pharmaceutical composition of the third aspect of the invention to a need thereof Object.

In a preferred embodiment, the EGFR-mediated disease is cancer; preferably, the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer , glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumor .

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

detailed description

After extensive and intensive research, the inventors unexpectedly discovered a number of structurally novel 5,8-dihydropteridin-6,7-dione derivatives that selectively inhibit EGFR T790M mutations, EGFR ^{T790M / L858R} kinase inhibition activity IC ₅₀ value reaches the level of sub nM; IC ₅₀ values of inhibitory activity against cancer cells (EGFR ^{L858R / T790M} mutation) proliferation levels reached nM. The present invention has been completed on this basis.

The present inventors synthesized a candidate compound having EGFR inhibitory activity. Structural optimization of the candidate compounds was carried out, and a series of 5,8-dihydropteridin-6,7-dione compounds which were not reported in the literature were designed and synthesized and characterized. A series of compounds were tested for activity at the molecular and cellular levels, resulting in a batch of compounds that selectively inhibit the EGFR T790M mutation. Wherein the compound 005 pairs of EGFR ^{T790M / L858} kinase inhibitory activity IC ₅₀ of 0.3nM, H1975 (non-small cell lung cancer cells, EGFR ^{L858R / T790M)} cell proliferation inhibitory activity IC ₅₀ of 18nM. Art-recognized, and inhibiting cell extremely excellent inhibition IC ₅₀ ratio of the difference toxic mutant of EGFR comprising greater than 100 means that the compound may have in vivo comprising the wild-type EGFR. On the other hand, the compound of the present invention has an IC ₅₀ inhibitory activity against H1975 (non-small cell lung cancer cells, EGFR ^L858R/T790M ) cell proliferation, and the compound of the present invention also has excellent IC ₅₀ ratio, wherein the above IC ₅₀ ratio of compound 005 More than 400.

Definition of Terms

Some of the groups mentioned in this article are defined as follows:

As used herein, "alkyl" refers to a saturated branched or straight-chain alkyl group having a carbon chain length of from 1 to 10 carbon atoms, and preferred alkyl groups include 2-8, 1-6, 1-4, 3-8 alkyl groups of 1-3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, and the like. The alkyl group may be substituted by one or more substituents, for example by halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.

As used herein, "alkoxy" refers to an oxy group substituted with an alkyl group. Preferred alkoxy groups are alkoxy groups having from 1 to 6 carbon atoms, more preferably alkoxy groups having from 1 to 4 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and the like.

As used herein, "halogen" refers to fluoro, chloro, bromo and iodo.

As used herein, "heterocyclyl" includes, but is not limited to, a 5- or 6-membered heterocyclic group containing from 1 to 3 heteroatoms selected from O, S or N, including but not limited to furyl, thienyl, pyrrolyl, Pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl and the like.

As used herein, "amido" refers to a group of the formula "-R'-NH-C(O)-R", wherein R' may be selected from hydrogen or alkyl, and R may be selected from alkyl, alkenyl. , alkynyl, NR _c R _d is substituted alkyl, NR _c R _d is substituted alkenyl and NR _c R _d group substituted alkynyl, halogen substituted alkyl, alkenyl substituted with cyano group, Wherein R _c and R _d may be selected from the group consisting of alkyl and alkenyl.

As used herein, "optionally substituted" means that the substituent to which it is modified may be optionally substituted with from 1 to 5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C. _1-4 aldehyde group, C _1-6 linear or branched alkyl group, cyano group, nitro group, amino group, hydroxyl group, hydroxymethyl group, halogen-substituted alkyl group (for example, trifluoromethyl group), halogen-substituted alkoxy group A group (e.g., trifluoromethoxy), a carboxyl group, a C _1-4 alkoxy group, an ethoxycarbonyl group, an N(CH ₃ ) group, and a C _1-4 acyl group.

Compound of the invention

The compound of the present invention is a compound of the following formula I or a pharmaceutically acceptable salt thereof:

R ^{1 is} each independently selected from the group consisting of hydrogen, halogen, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkyl, C ₁ -C ₄ alkylamido, substituted piperazinyl, substituted homopiperazinyl, substituted? Lolinyl, substituted thiomorpholinyl, 4-N-methylpiperazinyl, 4-N-acetylpiperazinyl, 4-N,N-dimethylpiperidinyl, substituted piperidinyl, N, N,N'-trimethylethylenediamine, N,N-dimethylethanolamine, 1-methyl-4-(piperidinyl)piperazinyl, -NR _a R _b , wherein R _a and R _b may be selected from an alkyl group and a nitrogen-containing alkyl group;

B is selected from the following group:

R ^{2 is} each independently selected from the group consisting of:

m is any integer from 0 to 7, preferably from 1 to 7.

In a specific embodiment, the A ring is a benzene ring such that the compound of the invention is as shown in Formula II below:

Wherein B, R ¹ , R ² and R ^{3 are} as defined above; and m is 0-5, preferably any integer of 1-5.

In a preferred embodiment, the above benzene ring in the compound of the present invention may be substituted or unsubstituted, for example, the compound of the present invention may be as shown in the following formula III:

In the formula,

R ^{2 is} selected from

R ³ is selected from H, or C ₁ -C ₆ alkyl, preferably methyl or isopropyl;

In a further embodiment, the above benzene rings in the compounds of the invention may be substituted as ortho, meta and/or para. In a preferred embodiment, the above benzene rings in the compounds of the invention are ortho-, meta- and para-substituted. In a further preferred embodiment, R ³ in the above formula III is selected from C ₁ -C ₆ alkyl, preferably methyl or isopropyl; R ⁵ , R ⁷ and R ⁸ are H; R ⁴ and R ⁶ Independent from the following group:

The present inventors synthesized a series of 5,8-dihydropteridin-6,7-dione compounds which have not been reported in the literature, and the specific compounds are as follows:

In a preferred embodiment, the compound of the invention is a compound as shown below:

Based on the compounds of the present invention, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient .

Examples of pharmaceutically acceptable salts of the compounds of the invention include, but are not limited to, inorganic and organic acid salts such as the hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate salts. , fumarate, mandelate and oxalate; and inorganic and formed with bases such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methyl glucosamine Organic base salt.

While the needs of each individual vary, one skilled in the art can determine the optimal dosage of each active ingredient in the pharmaceutical compositions of the present invention. In general, the compound of the present invention or a pharmaceutically acceptable salt thereof is orally administered to a mammal daily in an amount of from about 0.0025 to 50 mg/kg body weight. Preferably, however, it is about 0.01 to 10 mg per kilogram of oral administration. For example, a unit oral dose can include from about 0.01 to 50 mg, preferably from about 0.1 to 10 mg, of a compound of the invention. The unit dose may be administered one or more times per day in one or more tablets, each tablet containing from about 0.1 to 50 mg, conveniently from about 0.25 to 10 mg of the compound of the invention or a solvate thereof.

The pharmaceutical composition of the present invention can be formulated into a form suitable for various administration routes, including but not limited to being formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial A form of administration, intranasal or topical, for the treatment of tumors and other diseases. The amount administered is an amount effective to ameliorate or eliminate one or more conditions. For the treatment of a particular disease, an effective amount is an amount sufficient to ameliorate or in some way alleviate the symptoms associated with the disease. Such doses can be administered as a single dose or can be administered according to an effective therapeutic regimen. The amount administered may cure the disease, but administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms. The dosage of the drug will be determined by the age of the patient, the health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

The pharmaceutical preparation of the present invention can be administered to any mammal as long as they can obtain the therapeutic effect of the compound of the present invention. The most important of these mammals is humans.

The compounds of the invention or pharmaceutical compositions thereof are useful in the treatment of a variety of diseases mediated by epidermal growth factor receptor kinase (EGFR). Herein, the disease mediated by EGFR is various cancers. The cancer includes, but is not limited to, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophagus Cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumors.

The pharmaceutical preparations of the invention can be made in a known manner. For example, it is manufactured by a conventional mixing, granulating, tableting, dissolving, or freeze drying process. In the manufacture of oral formulations, the mixture can be selectively milled by combining the solid adjuvant with the active compound. If necessary or necessary, after adding an appropriate amount of auxiliary agent, the mixture of particles is processed to obtain a tablet or tablet core.

Suitable excipients are, in particular, fillers, such as sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; and binders, such as starch pastes, including corn starch. , wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, or polyvinylpyrrolidone. If necessary, a disintegrating agent such as the above-mentioned starch, and carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added. Adjuvants are especially flow regulators and lubricants, for example, silica, talc, stearates such as calcium magnesium stearate, stearic acid or polyethylene glycol. If desired, the tablet core can be provided with a suitable coating that is resistant to gastric juice. For this purpose, a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, a lacquer solution and a suitable organic solvent or solvent mixture. For the preparation of a gastric juice resistant coating, a suitable cellulose solution such as cellulose acetate phthalic acid or hydroxypropyl methylcellulose phthalic acid can be used. A dye or pigment can be added to the coating of the tablet or tablet core. For example, a combination for identifying or for characterizing the dosage of an active ingredient.

One skilled in the art can prepare the pharmaceutical compositions of the present invention into any dosage form based on their expertise and actual needs. For example, in particular embodiments, the pharmaceutical compositions of the present invention are those suitable for oral administration, including, but not limited to, tablets, solutions, suspensions, capsules, granules, powders.

Based on the above compounds and pharmaceutical compositions, the invention further provides a method of treating an EGFR mediated disease, the method comprising administering to a subject in need thereof a compound or pharmaceutical composition of the invention.

Methods of administration include, but are not limited to, various methods of administration well known in the art, which can be determined based on the actual circumstances of the patient. These methods include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal or topical routes of administration.

The invention also encompasses the use of a compound of the invention in the manufacture of a medicament for preventing or treating an EGFR mediated disease or inhibiting EGFR activity.

Advantages of the invention:

1. The compound provided by the present invention is a novel structure of 5,8-dihydropteridin-6,7-dione;

2. The compound provided by the present invention has excellent inhibitory activity against mutant EGFR or EGFR mutant cancer cells;

3. The compound provided by the present invention lays a foundation for the development of an EGFR-targeted drug capable of selectively inhibiting T790M mutation and can overcome clinical drug resistance, and has great industrialization and commercialization prospects as well as market value and significant economic benefits.

The technical solutions of the present invention are further described below in conjunction with the specific embodiments, but the following embodiments are not intended to limit the present invention, and all the application methods according to the principles and technical means of the present invention are within the scope of the present invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Materials and Methods

The synthesis of the 5,8-dihydropteridin-6,7-dione compounds of the present invention is as follows:

Reagents and conditions: (a) (Boc) ₂ O, Et ₃ N, CH ₃ OH, room temperature, 24 h; (b) 2,4-dichloro-5-nitropyrimidine, Na ₂ CO ₃ , DMF, -70 °C, 1h; (c) arylamine, DIPEA, THF, room temperature, overnight; (d) H ₂ , Pd / C, MeOH, room temperature, 10 h; (e) diethyl oxalate, triethylamine, EtOH, reflux 30h; (f) alkyl halide, Cs ₂ CO ₃ , DMF, room temperature, overnight; (g) trifluoroacetic acid, CH ₂ Cl ₂ , room temperature, 5 h; (h) acryloyl chloride, Et ₃ N, CH ₂ Cl ₂ , 0 ° C to room temperature, overnight.

Example 1

The specific synthesis method of the above steps a-h is as follows:

Synthesis of 1.(3-Aminophenyl)carbamic acid tert-butyl ester

1,3-phenylenediamine (10.800 g, 100 mmol) and triethylamine (10.100 g, 100 mmol) were weighed in a 250 mL single-necked flask, dissolved in 100 mL of methanol, and stirred for 15 minutes under ice bath. Further, Boc-anhydride (21.800 g, 100 mmol) was dissolved in 40 mL of methanol, and added dropwise to the above reaction mixture. After the dropwise addition was completed, the mixture was stirred at room temperature for 24 hours. The TLC was used to carry out the conversion of the starting material, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography ( petroleum ether/ethyl acetate=4:1, v/v) to give (3-aminophenyl)carbamic acid tert-butyl ester as white solid. 13.312 g, yield 64%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.03 (t, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.55 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.43 (s) , 1H), 6.36 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 3.54 (s, 2H), 1.51 (s, 9H). LC-MS: m/z: 209.1 (M+H) ⁺ .

2. Synthesis of tert-butyl (3-(2-chloro-5-nitropyrimidin-4-amino)phenyl)carbamate

Weigh 2,4-dichloro-5-nitropyrimidine (1.940 g, 10 mmol), N,N-diisopropylethylamine (2.580 g, 20 mmol) in a 100 mL round bottom flask, add 30 mL DMF to dissolve, ice Stir for 10 minutes under bath conditions. Further, (3-aminophenyl)carbamic acid tert-butyl ester (2.080 g, 10.0 mmol) was dissolved in 20 mL of DMF, and the mixture was slowly added dropwise to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. The TLC traces the complete conversion of the raw materials, and ice water is added to the reaction liquid to precipitate a solid, which is suction filtered, washed with water, and dried. The crude product was recrystallized from CH _₂ Cl ₂ to give (3- (2-chloro-5-nitro-4-ylamino) phenyl) carbamate as an orange solid 2.950g, 81% yield. ^{_{1 H NMR (400MHz, CDCl 3}} ) δ10.19 (s, 1H), 9.20 (s, 1H), 7.84 (s, 1H), 7.36 (d, J = 5.2Hz, 2H), 7.21-7.18 (m, 1H), 6.63 (s, 1H), 1.56 (s, 9H). LC-MS: m/z: 366.1 (M+H) ⁺ .

3. (3-((2-(2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)phenyl)amino Synthesis of tert-butyl formate

(3-(2-Chloro-5-nitropyrimidin-4-amino)phenyl)carbamic acid tert-butyl ester (2.920 g, 8 mmol), N,N-diisopropylethylamine (2.064 g, 16 mmol) In a 100 mL three-necked flask, 30 mL of tetrahydrofuran was added to dissolve. Further, 2-methoxy-4-(4-methylpiperazinyl)aniline (1.768 g, 8 mmol) was dissolved in 15 mL of tetrahydrofuran, and slowly dropped into the above reaction solution under argon gas protection conditions. Reflux overnight. The TLC was used to trace the conversion of the starting material, and a part of the solvent was removed by rotary evaporation to precipitate a solid, which was filtered, washed with THF and dried. The crude product was recrystallized from CH ₂ Cl ₂ to give (3-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5-nitropyrimidine-4- Tert-butyl)amino)phenyl)carbamic acid tert-butyl ester reddish brown solid 3.340 g, yield 76%. ^{1 H NMR (400MHz, DMSO-} d 6) δ10.24 (s, 1H), 9.42 (s, 1H), 9.19 (s, 1H), 9.03 (s, 1H), 7.54 (s, 1H), 7.39 ( d, J = 8.4 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 7.09 (t, J = 8.0 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 6.34 (d, J = 8.8 Hz, 1H), 3.76 (s, 3H), 3.15 (t, J = 4.4 Hz, 4H), 2.47 (t, J = 4.4 Hz, 4H), 2.24 (s, 3H), 1.47 (s, 9H). LC-MS: m/z: 551.4 (M+H) ⁺ .

4. (3-((5-Amino-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)carbamic acid Synthesis of tert-butyl ester

Weighing (3-((2-(2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)phenyl)amino Tert-butyl formate (3.300 g, 6 mmol), palladium-carbon catalyst (0.318 g, 0.3 mmol, 10% Pd) was dissolved in a 250 mL thick-walled pressure bottle, added with 80 mL of methanol, and hydrogen gas was introduced and reacted at room temperature for 6 hours. The TLC was used to track the conversion of the starting material, suction filtration, the filtrate was spun dry, and the crude product was recrystallized from ethanol to give (3-((5-amino-4-((2-methoxy-4-(4-methylpiperazinyl))benzene) Tert-butyl)amino)pyrimidin-4-yl)amino)phenyl)carbamic acid tert-butyl ester 2.775 g of white solid, yield 89%. ^{1 H NMR (400MHz, DMSO-} d 6) δ9.32 (s, 1H), 8.17 (s, 1H), 7.99 (d, J = 8.8Hz, 1H), 7.96 (s, 1H), 7.59 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 6.60 (d, J=2.4 Hz, 1H), 6.38 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 4.39 (s, 2H), 3.82 (s, 3H), 3.07 (t, J = 4.4 Hz, 4H) , 2.48 (t, J = 4.4 Hz, 4H), 2.25 (s, 3H), 1.48 (s, 9H). LC-MS: m/z: 521.3 (M+H) ⁺ .

5. (3-(2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydropteridine- Synthesis of 8(5H))phenyl)carbamic acid tert-butyl ester

Weighing (3-((5-amino-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)carbamic acid tert-Butyl ester (2.600 g, 5 mmol) and triethylamine (1.010 g, 10 mmol) were dissolved in a 100 mL flask and added with 40 mL of ethanol. Diethyl oxalate (2.190 g, 15 mmol) was added to the reaction mixture, and the mixture was refluxed for 48 hours. The TLC tracks the conversion of the raw materials, suction filtration, washing the filter cake with ethanol, and drying. (3-(2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydropterin-8 (5H)) tert-butyl phenyl)carbamate yellow solid 2.15 g, yield 75%. ^{1 H NMR (400MHz, DMSO-} d 6) δ9.64 (s, 1H), 8.14 (s, 1H), 7.59 (s, 1H), 7.57 (s, 1H), 7.55 (d, J = 8.0Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H) , 6.06 (d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.03 (t, J = 4.4 Hz, 4H), 2.44 (t, J = 4.4 Hz, 4H), 2.22 (s, 3H) , 1.45 (s, 9H). HRMS (ESI): calcd for C ₂₉ H ₃₅ N ₈ O ₅ (M+H) ⁺ 575.2730, calc. 575.2725.

6. N-(3-(2-(2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydro butterfly Synthesis of pyridine-8-(5H))phenyl)acrylamide (Compound 001)

Weighing (3-(2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydropteridine- 8(5H)) tert-butyl phenyl)carbamate (0.861 g, 1.5 mmol) was dissolved in a 50 mL round-bottomed flask, 10 mL of dichloromethane was added, and 2 mL of trifluoroacetic acid was slowly dropped, and stirred at room temperature overnight. The TLC was used to track the conversion of the starting material, neutralized to a basic state by adding a saturated NaHCO ₃ solution, extracted with dichloromethane, and the organic layer was collected. The solvent was removed by rotary evaporation to give 8-(3-aminophenyl)-2-((2-methoxy) 4-(4-Methylpiperazinyl)phenyl)amino)-5,8-dihydropteridin-6,7-dione 491 mg, yield 69%, product was used in the next step without purification. .

Weigh 8-(3-aminophenyl)-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5,8-dihydropteridin-6, 7-Diketone (0.474 g, 1 mmol), Et ₃ N (0.152 g, 1.5 mmol) was dissolved in a 25 mL flask, 10 mL of dichloromethane, and stirred for 10 min. Further, acryloyl chloride (105 μL, 1.3 mmol) was dissolved in 2 mL of dichloromethane, and the mixture was slowly added to the mixture, and stirred at room temperature overnight. The TLC was used to carry out the conversion of the starting material, and the mixture was extracted with ice water, and extracted with dichloromethane. The organic layer was collected, evaporated, evaporated, evaporated, and evaporated to silica gel column chromatography (DCM/CH ₃ OH = 15:1, v/v). N-(3-(2-(2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydropteridine 8-(5H))phenyl)acrylamide 124 mg, yield 23%. ^{1 H NMR (400MHz, DMSO-} d 6) δ10.69 (s, 1H), 8.22 (s, 1H), 7.51 (d, J = 8.0Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.59 - 6.52 (m, 2H), 6.26 ( Dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.07 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.78 (s, 3H) , 3.31 (t, J = 4.4 Hz, 4H), 3.20 (t, J = 4.4 Hz, 4H), 2.74 (s, 3H). HRMS (ESI): calculated C ₃₁ H ₃₉ N ₈ O ₅ (M+ H) ⁺ 529.2312, experimental value 529.2232.

7. (3-(5-Ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7- Synthesis of tert-butyl ester of dihydropteridine-8(5H))phenyl)carbamate

Weighing (3-(2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-dihydropteridine- 8(5H)) tert-butyl phenyl)carbamate (0.861g, 1.5mmol), Cs ₂ CO ₃ (0.587g, 1.8mmol) in 25mL flask, dissolved in 10mL DMF, added dropwise ethyl iodide (180μL, 2.25mmol) ), stirring at room temperature overnight. TLC was used to trace the conversion of the starting material, water was added, and the mixture was extracted with methylene chloride. The organic layer was collected, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (DCM/CH ₃ OH = 20:1, v/v). (3-(5-ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7-di Hydrogen pteridine-8(5H))phenyl)carbamic acid tert-butyl ester 414 mg, yield 46%. ^{_{1 H NMR (400MHz, CDCl 3}} ) δ8.59 (s, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 7.54 (d, J = 8.0Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.44 (s, 1H), 6.99 (d, J = 7.2 Hz, 1H), 6.72 (s, 1H), 6.45 (d, J = 2.4 Hz, 1H), 6.17 (d, J = 6.8 Hz, 1H), 4.55 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.14 (t, J = 4.4 Hz, 4H), 2.62 (t, J = 4.4 Hz, 4H), 2.38 (s, 3H), 1.51 (t, J = 6.8 Hz, 3H), 1.48 (s, 9H). HRMS (ESI): Calculated C ₃₁ H ₃₉ N ₈ O ₅ (M+H) ⁺ 603.3043 603.3043.

8. N-(3-(5-ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6, Synthesis of 7-Dihydropteridine-8-(5H))phenyl)acrylamide (Compound 003)

Weighing (3-(5-ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7- Dihydropteridine-8(5H))phenyl)carbamic acid tert-butyl ester (0.400 g, 0.66 mmol) was dissolved in a 50 mL round-bottomed flask, 10 mL of dichloromethane was added, and 2 mL of trifluoroacetic acid was slowly added dropwise, and stirred at room temperature overnight. The TLC was used to carry out the conversion of the starting material, and the mixture was neutralized with a saturated NaHCO ₃ solution to be basic, and extracted with dichloromethane. The organic layer was collected, and the solvent was removed by rotary evaporation. The crude product was recrystallized from dichloromethane to give 8-(3-aminophenyl)-5. -ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5,8-dihydropteridine-6,7-dione 185 mg, yield 55%, the product was used directly in the next step.

Weigh 8-(3-aminophenyl)-5-ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-5,8-dihydro The pteridine-6,7-dione (0.180 g, 0.36 mmol), Et ₃ N (0.055 g, 0.54 mmol) was dissolved in a 25 mL flask, 10 mL of dichloromethane, and stirred for 10 min. Further, acryloyl chloride (38 μL, 0.47 mmol) was dissolved in 1 mL of dichloromethane, and the mixture was slowly added to the mixture and stirred at room temperature overnight. The TLC was used to carry out the conversion of the starting material, and the mixture was extracted with ice water, and extracted with dichloromethane. The organic layer was collected, evaporated, evaporated, evaporated, and evaporated to silica gel column chromatography (DCM/CH ₃ OH = 15:1, v/v). N-(3-(5-ethyl-2-((2-methoxy-4-(4-methylpiperazinyl)phenyl)amino)-6,7-dioxo-6,7 - Dihydropteridine-8-(5H))phenyl)acrylamide 98 mg, yield 49%. ^{1 H NMR (400MHz, DMSO-} d 6) δ10.42 (s, 1H), 8.57 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.53 ( t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.06 - 6.04 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 3.02 (t, J = 4.4 Hz, 4H), 2.45 (t, J = 4.4 Hz, 4H), 2.23 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). HRMS (ESI): calcd for C ₂₉ H ₃₃ N ₈ O ₄ (M+H) ⁺ 557.2625.

The following compounds were synthesized according to the above procedures a-g:

N-(3-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-6,7-dioxo-6 ,7-Dihydropteridine-8-(5H)-yl)phenyl)acrylamide (Compound 002)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.46 (s, 1H), 8.48 (s, 1H), 7.92 (d, J = 8.0Hz, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 1.6 Hz, 1H) , 6.46 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 6.02 (d, J = 8.4 Hz, 1H), 5.77 (dd , J = 10.0 Hz, J = 1.2 Hz, 1H), 3.77 (s, 3H), 3.55 - 3.53 (m, 4H), 3.06 - 3.02 (m, 4H), 2.57 (s, 3H), 2.32 (s, 3H) .HRMS (ESI): calcd for _{_{_{_{C 28 H 31 N 8 O 4}}}} (M + H) + 543.2468, Found 543.2470.

N-(3-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo-5-propyl-6 ,7-Dihydropteridine-8-(5H)-yl)phenyl)acrylamide (Compound 004)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.70 (s, 1H), 8.58 (s, 1H), 7.97 (s, 1H), 7.91 (d, J = 8.0Hz, 1H), 7.77 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.59 - 6.52 (m, 2H), 6.26 ( Dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.11-6.09 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H), 3.78 (s, 3H), 3.31-3.29 (m, 4H), 3.16-3.14 (m, 4H), 2.72 (s, 3H), 1.85-1.77 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). HRMS (ESI): calcd for C ₃₀ H ₃₅ N ₈ O ₄ (M+H) ⁺ 571.278.

N-(3-(5-isopropyl-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo- 6,7-Dihydropteridine-8-(5H)-yl)phenyl)acrylamide (Compound 005)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.63 (s, 1H), 8.57 (s, 1H), 7.96 (s, 1H), 7.90 (d, J = 8.4Hz, 1H), 7.75 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H) , 6.54 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.12-6.10 (m, 1H), 5.77 (dd, J = 10.0 Hz, J=1.6 Hz, 1H), 5.38-5.30 (m, 1H), 3.79 (s, 3H), 3.31-3.29 (m, 4H), 3.26-3.24 (m, 4H), 2.79 (s, 3H) ), 1.40 (d, J = 6.4 Hz, 6H). HRMS (ESI): calcd for C ₃₀ H ₃₅ N ₈ O ₄ (M+H) ⁺ 571.278.

N-(3-(2-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-isopropyl-6,7-dioxo-6 ,7-Dihydropteridine-8-(5H)-yl)phenyl)acrylamide (Compound 006)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.54 (s, 1H), 9.61 (s, 1H), 8.61 (s, 1H), 7.91 (d, J = 8.0Hz, 1H), 7.76 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.8 Hz, 1H), 6.50 (dd, J=17.2Hz, J=10.4Hz, 1H), 6.26 (dd, J=17.2Hz, J=2.0Hz, 1H), 5.76 (dd, J=10.0Hz, J=1.6Hz, 1H), 5.37-5.31 (m, 1H), 2.94-2.91 (m, 4H), 2.85-2.82 (m, 4H), 2.58 (s, 3H), 1.99 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). HRMS (ESI): calcd for C ₃₀ H ₃₅ N ₈ O ₃ (M+H) ⁺ 555.2832.

N-(3-(2-((3-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-isopropyl-6,7-dioxo- 6,7-Dihydropteridine-8-(5H)-yl)phenyl)acrylamide (Compound 007)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.59 (s, 1H), 9.57 (s, 1H), 8.62 (s, 1H), 7.90 (d, J = 8.4Hz, 1H), 7.75 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.04-6.99 (m, 2H), 6.55-6.49 (m, 2H), 6.27 (dd, J =17.2 Hz, J=2.0 Hz, 1H), 5.77 (dd, J=10.0 Hz, J=1.6 Hz, 1H), 5.37-5.31 (m, 1H), 3.57 (s, 3H), 3.19-3.07 (m) , 8H), 2.74 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). HRMS (ESI): Calculated C ₃₀ H ₃₅ N ₈ O ₄ (M+H) ⁺ 571.278.

N-(3-(2-((4-(2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)amino)-5-isopropyl-6 ,7-dioxo-6,7-dihydropteridin-8-(5H)-yl)phenyl)acrylamide (Compound 008)

^{_{1 H NMR (400MHz, CDCl 3}} ) δ8.69 (s, 1H), 8.57 (s, 1H), 7.75 (s, 1H), 7.71 (d, J = 8.0Hz, 1H), 7.61 (s, 1H) , 7.44 (t, J = 8.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.35 (dd, J = 16.4 Hz, J = 0.8 Hz, 1H), 6.26-6.19 (m, 2H) , 5.92-5.90 (m, 1H), 5.64 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 5.49 - 5.43 (m, 1H), 3.80 (s, 3H), 3.40 (t, J = 7.6 Hz, 2H), 2.87 (s, 3H), 2.50 (t, J = 7.6 Hz, 2H), 2.35 (s, 6H), 1.48 (d, J = 6.0 Hz, 6H). HRMS (ESI): calculated value C ₃₀ H ₃₇ N ₈ O ₄ (M+H) ⁺ 573.2938, experimental value 573.2939.

N-(3-(2-(4-(2-(dimethylamino)ethoxy)-2-methoxyphenyl)amino)-5-isopropyl-6,7-dioxo -6,7-dihydropteridin-8-(5H)-yl)phenyl)acrylamide (compound 009)

^{1 H NMR (400MHz, DMSO-} d 6) δ10.64 (s, 1H), 8.57 (s, 1H), 8.01 (s, 1H), 7.87 (d, J = 8.4Hz, 1H), 7.78 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 2.4 Hz, 1H) , 6.54 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 6.16-6.14 (m, 1H), 5.76 (dd, J = 10.0 Hz, J=1.6 Hz, 1H), 5.37-5.31 (m, 1H), 4.21 (t, J = 4.8 Hz, 2H), 3.78 (s, 3H), 3.26 (t, J = 4.8 Hz, 2H) , 2.69 (s, 6H), 1.40 (d, J = 6.0 Hz, 6H). HRMS (ESI): calcd for C ₂₉ H ₃₄ N ₇ O ₅ (M+H) ⁺ 560.2621.

N-(3-(5-isopropyl-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino) )-6,7-dioxo-6,7-dihydropteridin-8(5H)-yl)phenyl)acrylamide (compound 010)

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.58 (s, 1H), 8.56 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.55-6.48 (m, 2H), 6.27 ( Dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 6.08 - 6.06 (m, 1H), 5.78 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.36 - 5.30 (m, 1H), 3.77 (s, 3H), 3.62-3.60 (m, 2H), 3.00-2.92 (m, 4H), 2.69-2.67 (m, 2H), 2.56 (t, J = 11.6 Hz, 3H), 2.42 - 2.39 ( m, 2H), 1.82-1.81 (m, 2H), 1.54-1.49 (m, 1H), 1.39 (d, J = 6.4 Hz, 6H). HRMS (ESI): Calculated C ₃₅ H ₄₄ N ₉ O ₄ (M+H) ⁺ 654.3516, experimental value 654.3512.

Example 2. Biological activity test

The in vitro inhibitory effect of the compounds provided by the present invention on EGFR kinase activity was as follows:

In vitro enzyme activity analysis: Wild type and various mutant types (T790M, L858/T790M) EGFR were purchased from Yingjie Company. Ten concentration gradients from 5.1 x 10 ^-11 mol/L to 1.0 x 10 ^-6 mol/L were set for all compounds to be tested.

The concentration of different kinases was determined by optimization experiments. The corresponding concentrations were: EGFR (PV3872, Invitrogen) 0.287 μg/μL, EGFR-T790M (PV4803, Invitrogen) 0.174 μg/μL, EGFR-L858R/T790M (PV4879, Yingjie Company) ) 0.055 μg / μL. The compound was diluted three times from 5.1 x 10 ^-9 M to 1 x 10 ^-4 M in DMSO. 4 μL of the compound was dissolved in 96 μL of water to obtain a 4x compound solution. 40 μM ATP was dissolved in 1.33 x kinase buffer and the kinase/peptide mixture contained 2x kinase, 4 μM tyrosine 4 peptide ready for use. The 10 μL kinase reaction included 2.5 μL of compound solution, 5 μL of kinase/peptide mixture, and 2.5 μL of ATP solution. 5 μL of phosphorylated peptide solution was used instead of the kinase/peptide mixture as a 100% phosphorylation control. 2.5 μL of 1.33x kinase buffer was used instead of the ATP solution as a 100% inhibition control, and 2.5 μL of 4% DMSO was used as a 0% inhibition control instead of the compound solution. The solution in the plate was thoroughly mixed and incubated at room temperature for 1.5 hours. After 5 μL of Development Solution was added to each well, incubation was continued for 1 hour at room temperature, and the non-phosphorylated peptide was cleaved during this time. Finally, the reaction was terminated by the addition of 5 μL of Stop Reagent. The plates were measured with EnVision Multilabel Reader (Perkin Elmer). Experimental data was calculated using GraphPad Prism version 4.0. Repeat 3 times or more for each experiment.

Cell proliferation and growth inhibition assay: H1975 (non-small cell lung cancer cells, EGFR ^L858R/T790M ), A431 (non-small cell lung cancer cells, EGFR wild type), cells were obtained from ATCC. Cell proliferation activity was assessed using MTS assay. The cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each cell of each cell was adjusted according to the absorbance value (absorbance value at 490 nm of 1.3-2.2). Six concentration gradients (0.1 nM - 10 [mu]M) were set for the compounds to be tested, and at least 6 sets of parallel controls were used for each concentration value.

H1975, A431 cells were cultured in the corresponding medium, and the cells were passaged at least twice after resuscitation, and then used for experimental use. The log phase cells were trypsinized and resuspended in culture. H1975 (1000 cells per well), A431 (2000 cells per well) were seeded in 96-well plates in a volume of 100 μL; 6 sets of parallel and 7 columns were set. The plates were placed in an incubator at 37 ° C in a 5% carbon dioxide overnight. The compound was dissolved in DMSO to a concentration of 10 μM per liter, and then the compound concentration was gradually diluted to obtain a compound concentration of 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM per liter, respectively. 2 μL of the compound solution was added to 998 μL of the medium, and the mixture was thoroughly mixed. 100 μL of the mixture was added to a 96-well plate. 2 μL of DMSO was used instead of the compound solution as a 0% inhibition control. After incubation for 68 hours, 20 μL of MTT (5 mg/mL) was added. At 4 hours, the supernatant was discarded and 150 μL of DMSO was added. After shaking for 10 minutes, the well plates were read with Synergy HT (Bio TeK) (OD490). Data using GraphPad Prism version 4.0 calculated nonlinear regression model adjusting IC ₅₀ value by using the dose response curves obtained.

The test results are shown in Table 1 below.

Table 1

>10.0 indicates that the inhibition of cells is weak.

discuss:

After extensive and in-depth research, the inventors designed and synthesized a series of 5,8-dihydropteridin-6,7-dione compounds that have not been reported in the literature. The obtained compounds were subjected to molecular and cellular levels. The activity assay yielded a batch of compounds that selectively inhibited the EGFR T790M mutation. The present inventors have further found that the difference in proliferation inhibition ability of the compound of the present invention against EGFR mutant cancer cells (H1975) and EGFR wild type cancer cells (A431) is higher than that of mutant EGFR and wild type EGFR kinase activity. Thus, suggesting that the compounds of the present invention have better differential toxicity in vivo, may be a third-generation EGFR-targeted drug that selectively inhibits T790M mutations, overcomes clinical resistance, or is more active as a further modification and/or The basis for compounds that differ in toxicity.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

a compound of formula I or a pharmaceutically acceptable salt thereof:

Wherein A is a benzene ring, a five- or six-membered heterocyclic ring, or a C 3 -C 8 cycloalkyl group;

R 1 is each independently selected from the group consisting of hydrogen, halogen, C 1 -C 3 alkoxy, C 1 -C 3 alkyl, C 1 -C 4 alkylamido, substituted piperazinyl, substituted homopiperazinyl, substituted? Lolinyl, substituted thiomorpholinyl, 4-N-methylpiperazinyl, 4-N-acetylpiperazinyl, 4-N,N-dimethylpiperidinyl, substituted piperidinyl, N, N-dimethylaminoethylamino, N,N-dimethylaminoethoxy, 4-(4-N-methylpiperazinyl)piperidinyl, -NR a R b , wherein R a And R b may be selected from the group consisting of alkyl and nitrogen-containing alkyl groups;

B is selected from the following group:

R 2 is each independently selected from the group consisting of:

R 3 is selected from the group consisting of hydrogen, C 1 -C 10 alkyl, substituted C 1 -C 10 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted benzyl, optionally substituted Heterocyclic group;

m is any integer from 0 to 7, preferably from 1 to 7.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is as shown in Formula II:

Wherein B, R 1 , R 2 and R 3 are as defined in claim 1;

m is an integer from 0 to 5, preferably from 1 to 5.
The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein the compound is as shown in Formula III:

In the formula,

R 2 is selected from

R 3 is selected from H, or C 1 -C 6 alkyl, preferably methyl or isopropyl;

R 4 , R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of
The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein

R 3 is selected from C 1 -C 6 alkyl, preferably methyl or isopropyl;

R 5 , R 7 and R 8 are H;

R 4 and R 6 are independently selected from the group consisting of:
a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof:
A pharmaceutical composition comprising the compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
Use of a compound according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment or prevention of an EGFR mediated disease, or inhibition of EGFR.
The use according to claim 7, wherein the EGFR-mediated disease is cancer.
The use according to claim 8, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer, glioma, Ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumors.
A method of treating or preventing an EGFR-mediated disease, comprising administering a compound according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 to a subject in need thereof.