CN105985342B - As the pyrimido-pyrimidine derovatives of EGFR inhibitor and its application - Google Patents

Abstract

The present invention relates to pyrimidopyrimidinedione derivatives as EGFR inhibitors and their application. Specifically, the present invention relates to a compound represented by formula I, a pharmaceutical composition containing a compound of formula I and the use of said compound in the preparation of a drug for treating EGFR-related diseases or inhibiting EGFR:

Description

Pyrimidopyrimidinedione derivatives as EGFR inhibitors and applications thereof

technical field

The present invention relates to the field of medicinal chemistry; specifically, the present invention relates to a novel pyrimidopyrimidinedione derivative, its synthesis method and its application as an EGFR inhibitor in the preparation of drugs for tumor-related diseases.

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 mortality. It is one of the malignant diseases that threaten human health and cause death. According to research data, there were 12.7 million cancer patients in the world in 2008, of which more than 7 million died. In the world, 20% of new tumor patients are in China, and 24% of tumor death patients are in China. If effective prevention measures are not taken, or a better treatment plan is proposed, it is estimated that by 2030, there will be 26 million new cancer cases worldwide every year, and the number of cancer deaths will reach 17 million. Among existing cancers, lung cancer is the malignant tumor with the highest morbidity and mortality worldwide, and non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer patients. According to the forecast of the World Health Organization (WHO), by 2025, there will be more than 1 million new lung cancer cases in my country every year. Once diagnosed with lung cancer, patients have only a slim prospect of survival, with a 5-year survival rate of less than 15%.

Since the 1980s, with the in-depth study of tumor molecular biology, the molecular mechanism of tumor occurrence and development has become increasingly clear. Among many factors that induce cancer, the high expression of certain protein kinases caused by gene mutations in cancer cells is one of the main factors that lead to abnormal signal transduction pathways. Protein tyrosine kinase is an important factor in the signal transmission process, participates in a series of cellular activities, and is 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, phosphorylates the phenolic hydroxyl group, and transmits signals. Therefore, the development of selective protein kinase inhibitors to block or regulate diseases caused by the abnormality of these signaling pathways has been regarded as an effective research strategy for the development of anticancer drugs. Among the numerous tyrosine kinases, epidermal growth factor receptor tyrosine kinase (EGFR) is an indispensable and important component. EGFR consists of 1186 amino acids and encodes a 170-kDa transmembrane glycoprotein. EGFR can mediate multiple signal transduction pathways, transmit extracellular signals into cells, and 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 constitutively expressed component of many normal epithelial tissues (such as skin and hair follicles), while 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 mediated signal transduction pathway can achieve the purpose of treating lung cancer and open up a feasible way for targeted therapy of lung cancer.

In terms of clinical treatment, combined with traditional radiotherapy and 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 shows that most patients with non-small cell lung cancer will develop acquired drug resistance within 6-12 months after treatment with gefitinib or erlotinib. The drug resistance of about 50% of the cases is related to the mutation of an amino acid residue in the kinase domain of EGFR (threonine residue at position 790 is mutated to methionine, T790M) (The New England Journal of Medicine, 2005, 352, 786 -792). The T790M mutation causes steric hindrance when the inhibitor binds to EGFR or increases the affinity between EGFR and ATP, which greatly weakens the anticancer effect of such reversibly binding competitive inhibitors. The emergence of drug resistance not only reduces the sensitivity of patients to drugs, but also greatly reduces the quality of life of cancer patients. In order to overcome the drug resistance caused by the T790M mutation, a series of irreversible ATP competitive inhibitors (such as CI-1033, HKI-272, PF00299804, etc.) have entered the clinical research stage. Irreversible inhibitors contain a Michael receptor fragment, which can form a covalent bond with a conserved amino acid residue (Cys797) in the ATP binding site of EGFR, thereby obtaining a stronger EGFR binding affinity than reversible inhibitors. However, due to the poor selectivity of these drugs to wild-type and mutant EGFR, their maximum tolerated dose (MTD) is low, and the effect of clinical trials is not obvious.

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

Contents of the invention

The object of the present invention is to provide pyrimidopyrimidinedione derivatives capable of selectively inhibiting T790M mutation as EGFR inhibitors.

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

Another object of the present invention is to provide the use of the above compound in the preparation of medicaments for treating EGFR-related diseases or inhibiting EGFR.

In a first aspect, the present invention provides a compound represented by general formula I or a pharmaceutically acceptable salt thereof:

In the formula, A is a benzene ring, a five- or six-membered heterocycle, and a C ₃ -C ₈ cycloalkyl group;

^Each R is independently selected from hydrogen, halogen, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkyl, C ₁ -C ₄ alkylamido, substituted piperazinyl, substituted homopiperazinyl, Substituted morpholinyl, substituted thiomorpholinyl, 4-N-methylpiperazinyl, 4-N-acetylpiperazinyl, 4-N,N-dimethylpiperidinyl, substituted piperazinyl Adyl group, -NR _a R _b , wherein, R _a and R _b can be independently selected from alkyl groups and nitrogen-containing alkyl groups;

R ² are each independently selected from the following groups:

R ³ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl, substituted C ₁ -C ₁₀ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, optionally substituted C ₃ -C _Cycloalkyl , optionally substituted aryl, optionally substituted benzyl, optionally substituted heterocyclic, optionally substituted aromatic heterocyclic;

B is selected from the following group:

m is an integer of 0-7.

In a specific embodiment, the compound is shown in general formula II:

In the formula, B, R ¹ , R ² , R ³ are as defined above;

m is an integer of 0-5.

In a specific embodiment, the compound is shown in general formula III:

In the formula,

^R2 is selected from

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

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

In a specific embodiment, ^R5 and ^R8 are H; alternatively, ^R5 , ^R7 and ^R8 are H.

In a specific embodiment, the present invention provides a compound or a pharmaceutically acceptable salt thereof selected from the group consisting of:

In a second aspect, the present invention provides a pharmaceutical composition, which contains the compound or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier or excipient.

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

In the third aspect, the present invention provides the use of the compound described in the first aspect of the present invention in the preparation of a drug for treating or preventing EGFR-mediated diseases, or inhibiting 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 cancer 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 fourth aspect, the present invention provides a method for treating or preventing EGFR-mediated diseases using the compound described in the first aspect of the present invention.

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 tumors .

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered a group of novel pyrimidopyrimidine diketone derivatives, which can selectively inhibit the EGFR T790M mutation, and have an IC ₅₀ value for EGFR ^T790M/L858R kinase inhibitory activity Reach the nM level; the inhibitory activity IC ₅₀ value of the proliferation of cancer cells (EGFR ^L858R/T790M mutation) reaches the μM level. The present invention has been accomplished on this basis.

The present inventors synthesized candidate compounds having EGFR inhibitory activity. The structure of the obtained candidate compounds was optimized, and a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compounds that have not been reported in the literature were designed and synthesized, and their structures were characterized . Molecular and cellular activity tests were carried out on this series of compounds, and a batch of compounds with selective inhibition of EGFR T790M mutation were obtained. Among them, compound 007 has an IC ₅₀ inhibitory activity of EGFR ^T790M/L858 kinase of 30.0 nM, and an IC ₅₀ inhibitory activity of H1975 (non-small cell lung cancer cells, EGFR ^L858R/T790M ) cell proliferation of 0.23 μM.

Definition of Terms

Some of the groups involved in this paper are defined as follows:

Herein, "alkyl" refers to a saturated branched or linear alkyl group with a carbon chain length of 1-10 carbon atoms, and preferred alkyl groups include 2-8, 1-6, 1-4, Alkyl groups with 3-8, 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. Alkyl groups may be substituted by one or more substituents, such as halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1-4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.

Herein, "alkoxy" refers to an oxy group substituted with an alkyl group. Preferred alkoxy groups are alkoxy groups of 1-6 carbon atoms in length, more preferably alkoxy groups of 1-4 carbon atoms in length, more preferably alkoxy groups of 1-3 carbon atoms in length. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, and the like. Alkoxy may be substituted by one or more substituents, such as halogen or haloalkyl. For example, an alkoxy group can be an alkyl group substituted with 1-4 fluorine atoms, or an alkyl group can be an alkyl group substituted with a fluoroalkyl group.

Herein, "alkenyl" generally means a monovalent hydrocarbon group having at least one double bond, usually containing 2-8 carbon atoms, preferably containing 2-6 carbon atoms, and may be straight or branched. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

As used herein, "alkynyl" generally means a monovalent hydrocarbon group having at least one triple bond, usually containing 2-8 carbon atoms, preferably containing 2-6 carbon atoms, more usually containing 2-4 carbon atoms, which may be a straight chain or branched. Examples of alkenyl groups include ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, hexynyl, and the like.

Herein, "halogen" refers to fluorine, chlorine, bromine or iodine.

Herein, "aryl" refers to a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, fenyl, tetralin base, indanyl, etc. Aryl can be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C _1-4 aldehyde, C _1-6 alkyl, cyano radical, nitro, amino, hydroxyl, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C _1-4 alkoxy , ethoxy formyl, N(CH ₃ ) and C _1-4 acyl, etc., heterocyclyl or heteroaryl, etc.

"Heterocyclic group" as used herein includes, but is not limited to, 5-membered or 6-membered heterocyclic groups containing 1-3 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, etc.

As used herein, "aromatic heterocyclyl" means containing 5-14 ring atoms and having 6, 10 or 14 electrons shared on the ring system. And the contained ring atoms are carbon atoms and optional 1-3 heteroatoms selected from oxygen, nitrogen and sulfur. Useful aromatic heterocyclic groups include piperazinyl, morpholinyl, piperidinyl, pyrrolidinyl, thienyl, furyl, pyryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, including but not limited to In 2-pyridyl, 3-pyridyl and 4-pyridyl, pyrazinyl, pyrimidinyl, etc.

The aromatic heterocyclic group can be optionally substituted by 1-5 (for example, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C _1-4 aldehyde, C _1-6 straight chain or branched chain alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (e.g. trifluoromethyl), halogen substituted alkoxy (e.g. trifluoromethoxy), carboxy, C _1-4 alkoxy, ethoxyformyl, N(CH ₃ ) and C _1-4 acyl.

Herein, "acyloxy" refers to a group with the structural formula "-O-C(O)-R", wherein R can be selected from alkyl, alkenyl and alkynyl. Said R can be optionally substituted.

Herein, "acylamino" refers to a group with the structural formula "-R'-NH-C(O)-R", wherein R' can be selected from hydrogen or alkyl, and R can be selected from alkyl, alkenyl , alkynyl, alkyl substituted by NR _c R _d , alkenyl substituted by NR _c R _d and alkynyl substituted by NR _c R _d , alkyl substituted by halogen, alkenyl substituted by cyano, Wherein, R _c and R _d can be selected from alkyl and alkenyl.

Herein, "optionally substituted" means that the substituent it modifies may be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C _1-4 aldehyde group, C _1-6 straight chain or branched chain alkyl, cyano, nitro, amino, hydroxyl, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxy, C _1-4 alkoxy, ethoxyformyl, N(CH ₃ ) and C _1-4 acyl.

Compounds of the invention

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

In the formula, A is a benzene ring, a five- or six-membered heterocycle, and a C ₃ -C ₈ cycloalkyl group;

^Each R is independently selected from hydrogen, halogen, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkyl, C ₁ -C ₄ alkylamido, substituted piperazinyl, substituted homopiperazinyl, Substituted morpholinyl, substituted thiomorpholinyl, 4-N-methylpiperazinyl, 4-N-acetylpiperazinyl, 4-N,N-dimethylpiperidinyl, substituted piperazinyl Adyl group, -NR _a R _b , wherein, R _a and R _b can be independently selected from alkyl groups and nitrogen-containing alkyl groups;

R ² are each independently selected from the following groups:

R ³ is selected from the group consisting of hydrogen, C ₁ -C ₁₀ alkyl, substituted C ₁ -C ₁₀ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, optionally substituted C ₃ -C _Cycloalkyl , optionally substituted aryl, optionally substituted benzyl, optionally substituted heterocyclic, optionally substituted aromatic heterocyclic;

B is selected from the following group:

m is an integer of 0-7.

In a specific embodiment, the A ring is a benzene ring, so that the compound of the present invention is shown in the following general formula II:

In the formula, B, R ¹ , R ² , R ³ are as described above; and m is an integer of 0-5.

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

In the formula,

^R2 is selected from

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

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

In a further embodiment, the above-mentioned benzene rings in the compounds of the present invention may be substituted in ortho, meta and/or para positions. In a preferred embodiment, the above-mentioned benzene rings in the compounds of the present invention are ortho-substituted and para-substituted. In another preferred embodiment, the above-mentioned benzene rings in the compounds of the present invention are ortho-substituted, meta-substituted and para-substituted. In a specific embodiment, R ⁵ and R ⁸ in the above general formula III are H; alternatively, R ⁵ , R ⁷ and R ⁸ are H.

The inventors synthesized a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compounds whose structures have not been reported in the literature, and the specific compounds are as follows:

On the basis of the compound of the present invention, the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the 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 this invention include, but are not limited to, inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate , fumarate, mandelate, and oxalate; and inorganic and Organic base salt.

Although the needs of each individual vary, those skilled in the art can determine the optimum dosage of each active ingredient in the pharmaceutical compositions of the present invention. Generally, the compound of the present invention or a pharmaceutically acceptable salt thereof is orally administered to mammals daily in an amount of about 0.0025 to 50 mg/kg body weight. Preferably, however, about 0.01 to 10 mg/kg is administered orally. For example, a unit oral dose may contain 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 as one or more tablets, each containing about 0.1 to 50 mg, conveniently about 0.25 to 10 mg of a compound of the present invention or a solvate thereof.

The pharmaceutical composition of the present invention can be formulated into formulations suitable for various routes of administration, including but not limited to being formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial Internal, nasal or external route of administration for the treatment of tumors and other diseases. The administered amount is an amount effective to ameliorate or eliminate one or more conditions. For the treatment of a particular disease, an effective amount is that amount sufficient to ameliorate or in some way alleviate symptoms associated with the disease. Such amounts may be administered as a single dose, or may be administered according to an effective treatment regimen. The amount administered may be curative, but usually the administration is to ameliorate the symptoms of the disease. Repeated dosing will generally be required to achieve the desired amelioration of symptoms. The dosage of the medicine will be determined according to the patient's age, 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 mammals as long as they can obtain the therapeutic effect of the compound of the present invention. The most important of these mammals is the human being.

The compound of the present invention or its pharmaceutical composition can be used to treat various diseases mediated by epidermal growth factor receptor kinase (EGFR). Herein, diseases mediated by EGFR are various cancers. Such cancers include, but are 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, esophageal cancer cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloid cancer and solid tumors.

The pharmaceutical preparations according to the invention can be manufactured in a known manner. For example, manufactured by conventional blending, granulating, tableting, dissolving, or freeze-drying processes. In the manufacture of oral dosage forms, solid excipients can be combined with the active compound and the mixture optionally milled. After adding appropriate amounts of auxiliaries, if desired or necessary, the mixture of granules is processed to obtain tablets or dragee cores.

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, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, a disintegrant, such as the above-mentioned starch, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added. Auxiliaries are especially flow regulators and lubricants, for example, silica, talc, stearates, such as magnesium calcium stearate, stearic acid or polyethylene glycol. Dragee cores may, if desired, be provided with a suitable coating resistant to gastric juices. For this purpose, concentrated sugar solutions can be used. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. To produce coatings resistant to gastric juices, suitable cellulose solutions, for example cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate, can be used. Dyestuffs or pigments may be added to the coating of the tablets or dragee cores. For example, for identification or to characterize combinations of active ingredient doses.

Based on the above compound and pharmaceutical composition, the present invention further provides a method for treating EGFR-mediated diseases, the method comprising administering the compound or pharmaceutical composition of the present invention to a subject in need.

The administration methods include but are not limited to various administration methods known in the art, which can be determined according to the actual situation 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 present invention also includes the use of the compound of the present invention in the preparation of drugs for preventing or treating EGFR-mediated diseases or inhibiting EGFR activity.

Advantages of the present invention:

1. The compound provided by the present invention is a new pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compound;

2. The compounds provided by the present invention have excellent inhibitory activity on cancer cells with mutant EGFR or EGFR mutation;

3. The compounds provided by the present invention lay the foundation for the development of EGFR-targeted drugs that can selectively inhibit the T790M mutation and overcome clinical drug resistance. They have great industrialization and commercialization prospects and market value, and significant economic benefits.

The technical solutions of the present invention are further described below in conjunction with specific examples of implementation, but the following examples of implementation do not constitute limitations to the present invention, and all various application methods adopted according to the principles and technical means of the present invention belong to the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated.

Materials and Methods

The synthesis of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compounds of the present invention is as follows:

Reagents and conditions: (a) DIPEA, CH ₃ CN, reflux; (b) ArNH ₂ , CH ₃ CN, reflux; (c) NaOH, THF, reflux; (d) R ³ NH ₂ , HATU, DIPEA, DMF; (e) K ₂ CO ₃ , CDI, THF, reflux; (f) trifluoroacetic acid, CH ₂ Cl ₂ , 0°C-room temperature; (g) acid chloride, Et ₃ N, CH ₂ Cl ₂ , 0°C-room temperature.

Example 1

The concrete synthetic method of above-mentioned steps a-g is as follows:

1. Synthesis of ethyl 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-chloropyrimidine-5-carboxylate

Weigh 2,4-dichloro-5-nitropyrimidine (2.210g, 10mmol) and DIPEA (1.290g, 10mmol) in a 50mL one-necked flask, add 15mL of acetonitrile to dissolve. Another tert-butyl (3-aminophenyl)carbamate (2.080 g, 10 mmol) was dissolved in 10 mL of acetonitrile, added dropwise to the above reaction solution, and refluxed for 2 h after the dropwise addition. TLC traced to the conversion of raw materials, cooled to room temperature, suction filtered, washed with acetonitrile, and dried the filter cake to obtain 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-chloropyrimidine-5 - Ethyl formate 3.371 g, yield 86%. ¹ H NMR (400MHz,DMSO-d ₆ )δ10.23(s,1H),9.50(s,1H),8.80(s,1H),7.70(s,1H),7.35(d,J＝8.0Hz, 1H), 7.29(t, J=8.0Hz, 1H), 7.23(d, J=8.0Hz, 1H), 4.38(q, J=7.2Hz, 2H), 1.49(s, 9H), 1.36(t, J=7.2Hz,3H).LC-MS: m/z:393.1(M+H) ⁺ .

2. Synthesis of ethyl 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-(phenylamino)pyrimidine-5-carboxylate

Weigh 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-chloropyrimidine-5-carboxylic acid ethyl ester (1.960g, 5mmol), aniline (0.558g, 6mmol) in 100mL Add 40mL of acetonitrile to the single-necked flask to dissolve, and heat up to reflux for 3h. TLC traced the conversion of raw materials, cooled to room temperature, suction filtered, washed with water, and dried the filter cake to obtain 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-(phenylamino)pyrimidine - 1.930 g of ethyl 5-carboxylate, yield 86%. ¹ H NMR (400MHz, CDCl ₃ ) δ10.46(s, 1H), 8.76(s, 1H), 7.57(d, J=8.0Hz, 2H), 7.36(t, J=7.6Hz, 3H), 7.23 (d, J=8.0Hz, 1H), 7.16(t, J=7.2Hz, 1H), 7.04(d, J=7.6Hz, 1H), 6.36(s, 1H), 4.37(q, J=7.2Hz , 2H), 1.54(s, 9H), 1.41(t, J=7.2Hz, 3H).LC-MS: m/z: 450.4(M+H) ⁺ .

3. Synthesis of 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-(phenylamino)pyrimidine-5-carboxylic acid

Weigh 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-(phenylamino)pyrimidine-5-carboxylic acid ethyl ester (1.796g, 4mmol) in a 100mL single-necked flask, add 30mL of tetrahydrofuran was dissolved, 10mL of 1M NaOH solution was added dropwise, the temperature was raised to 50°C, and stirred for 3h. TLC traced to the conversion of raw materials, cooled to room temperature, added dilute hydrochloric acid to adjust the pH to acidity, filtered with suction, washed with water, and dried the filter cake to obtain 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino) -2-(phenylamino)pyrimidine-5-carboxylic acid 1.499g, yield 89%. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.44(s,1H),9.82(s,1H),9.36(s,1H),8.70(s,1H),7.69(d,J＝8.0Hz, 2H),7.63(s,1H),7.43(s,1H),7.26-7.21(m,4H),6.98(t,J=6.8Hz,1H),1.47(s,9H).HRMS(ESI): Calculated for C ₂₂ H ₂₄ N ₅ O ₄ (M+H) ⁺ 422.1828, found 422.1823.

4. Synthesis of tert-butyl 3-(5-(methylcarbamoyl-2-(phenylamino)pyrimidine-4-amino)phenyl)carbamate

Weigh 4-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-2-(phenylamino)pyrimidine-5-carboxylic acid (1.263g, 3mmol), DIPEA (0.774g, 6mmol) In a 50mL flask, add 10mL of anhydrous DMF to dissolve. Another HATU (1.368g, 3.6mmol) was added to the above reaction solution in batches, and stirred at room temperature for 1h. Methylamine hydrochloride (0.396 g, 6 mmol) was added and stirred at room temperature overnight. TLC tracked the conversion of raw materials, added ice water, suction filtered, and washed with water to obtain 0.612 g of tert-butyl 3-(5-(methylcarbamoyl-2-(phenylamino)pyrimidine-4-amino)phenyl)carbamate , yield 47%. ¹ H NMR (400MHz, DMSO-d ₆ )δ11.33(s,1H),9.59(s,1H),9.31(s,1H),8.64(s,1H),8.50(d,J＝4.8Hz, 1H), 7.68(d, J=7.8Hz, 2H), 7.62(s, 1H), 7.49(br, 1H), 7.23(t, J=7.8Hz, 2H), 7.19(d, J=8.0Hz, 1H), 7.15(d, J=8.0Hz, 1H), 6.96(t, J=7.2Hz, 1H), 2.79(d, J=4.4Hz, 3H), 1.47(s, 9H).LC-MS: m/z:435.2(M+H) ⁺ .

5. (3-(3-Methyl-2,4-dioxo-7-(phenylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl ) phenyl) tert-butyl carbamate

Weigh tert-butyl 3-(5-(methylcarbamoyl-2-(phenylamino)pyrimidine-4-amino)phenyl)carbamate (0.434g, 1mmol), K ₂ CO ₃ (0.276g, 2mmol) into a 25mL flask, add 5mL of anhydrous tetrahydrofuran to dissolve, and heat up to reflux overnight. TLC traced the conversion of raw materials, cooled to room temperature, added ice water, extracted with EA, collected the organic layer, and removed the solvent by rotary evaporation. The crude product was separated by silica gel column chromatography (EA/PE=2:1, v/v) to obtain 193mg. Yield 42%. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.62(s,1H),8.92(s,1H),7.66(s,1H),7.52(d,J=8.0Hz,1H),7.45(t, J＝8.0Hz, 1H), 7.36-7.32(m, 2H), 7.02(d, J＝8.0Hz, 3H), 6.90(d, J＝7.6Hz, 1H), 3.29(s, 3H), 1.45( s,9H).HRMS(ESI): Calcd. for C ₂₄ H ₂₅ N ₆ O ₄ (M+H) ⁺ 461.1937, found 461.1939.

6. Synthesis of 1-(3-aminophenyl)-3-methyl-7-(phenylamino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

Weigh (3-(3-methyl-2,4-dioxo-7-(phenylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl ) phenyl) tert-butyl carbamate (0.193g, 0.42mmol) was dissolved in a 50mL flask by adding 5mL of dichloromethane, slowly added 1mL of trifluoroacetic acid dropwise, and stirred overnight at room temperature. TLC tracked the conversion of raw materials, added saturated NaHCO ₃ solution to neutralize, extracted with dichloromethane, collected the organic layer, and removed the solvent by rotary evaporation to obtain 1-(3-aminophenyl)-3-methyl-7-(phenylamino) Pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione 0.136g, yield 90%, the product was directly used in the next reaction without purification.

7. N-(3-(3-methyl-2,4-dioxo-7-(phenylamino)-3,4-dihydropyrimido[4,5-d]pyrimidine-1(2H) Synthesis of -yl)phenyl)acrylamide (Compound 001)

Weigh 1-(3-aminophenyl)-3-methyl-7-(phenylamino)pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (0.136g, 0.38mmol), DIPEA (0.097g, 0.76mmol) and a 25mL flask, add 3mL of dichloromethane to dissolve, and stir for 15 minutes under ice bath. Another acryloyl chloride (0.041 g, 0.46 mmol) was dissolved in 1 mL of dichloromethane, added dropwise to the above reaction solution, and stirred overnight at room temperature. TLC tracked the conversion of raw materials, spin-dried, and the crude product was separated by silica gel column chromatography (EA/PE=1.5:1, v/v) to obtain N-(3-(3-methyl-2,4-dioxo-7 -(Phenylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)acrylamide 83 mg, yield 53%. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.38(s, 1H), 8.93(s, 1H), 7.82(d, J=3.2Hz, 1H), 7.80(s, 1H), 7.53(t, J＝8.0Hz, 1H), 7.33-7.29(m, 2H), 7.15(d, J＝7.8Hz, 1H), 7.02-6.96(m, 2H), 6.89(t, J＝6.0Hz, 1H), 6.44(dd, J=17.2Hz, J=10.4Hz, 1H), 6.26(dd, J=17.2Hz, J=1.6Hz, 1H), 5.76(dd, J=10.4Hz, J=2.0Hz, 1H) ,3.30(s,3H).HRMS(ESI): Calcd. for C ₂₂ H ₁₉ N ₆ O ₃ (M+H) ⁺ 415.1519, found 415.1512.

The following compounds (compound 002-010) were all synthesized according to the above steps a-g:

N-(3-(7-((4-methoxyphenyl)amino)-3-methyl-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidine -1(2H))phenyl)acrylamide (compound 002)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.37(s,1H),10.23(s,1H),8.88(s,1H),7.82(d,J=8.0Hz,1H),7.79(s, 1H), 7.53(t, J=8.0Hz, 1H), 7.22(d, J=7.6Hz, 2H), 7.12(d, J=8.0Hz, 1H), 6.56(d, J=7.6Hz, 2H) ,6.44(dd,J=16.8Hz,J=10.0Hz,1H),6.24(dd,J=16.8Hz,J=1.6Hz,1H),5.76(dd,J=10.0Hz,J=1.6Hz,1H ), 3.65 (s, 3H), 3.29 (s, 3H). HRMS (ESI) calcd. for C ₂₃ H ₂₁ N ₆ O ₄ [M+H] ⁺ 445.1624, found 445.1627.

N-(3-(7-((2-methoxyphenyl)amino)-3-methyl-2,4-dioxo-3,4-dihydropyrimido[4,5-d]pyrimidine -1(2H))phenyl)acrylamide (compound 003)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.34(s,1H),8.89(s,1H),8.68(s,1H),7.80(s,1H),7.76(d,J＝8.0Hz, 1H), 7.50(t, J=8.0Hz, 1H), 7.41(d, J=8.0Hz, 1H), 7.12(d, J=7.6Hz, 1H), 6.95(d, J=7.6Hz, 2H) ,6.44(dd,J=16.8Hz,J=10.0Hz,1H),6.25(dd,J=17.2Hz,J=2.0Hz,1H),5.76(dd,J=10.4Hz,J=2.0Hz,1H ), 3.78 (s, 3H), 3.29 (s, 3H). HRMS (ESI) calculated for C ₂₃ H ₂₁ N ₆ O ₄ [M+H] ⁺ 445.1624, found 445.1631.

N-(3-(7-((2-methoxy-4-morpholinophenyl)amino)-3-methyl-2,4-dioxo-3,4-dihydropyrimido[4 ,5-d] pyrimidine-1 (2H)) phenyl) acrylamide (compound 004)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.36(s,1H),8.80(s,1H),8.44(s,1H),8.03(s,1H),7.85(d,J＝8.0Hz, 1H), 7.69(t, J=2.0Hz, 1H), 7.52(t, J=8.4Hz, 1H), 7.31(d, J=8.8Hz, 1H), 7.09(d, J=7.6Hz, 1H) ,6.57(d,J=2.0Hz,1H),6.45(dd,J=16.8Hz,J=10.0Hz,1H),6.26(dd,J=17.2Hz,J=2.0Hz,1H),5.77(dd ,J=10.4Hz,J=2.0Hz,1H),3.77(s,3H),3.58-3.54(m,4H),3.06-2.99(m,4H),2.05(s,3H).HRMS(ESI) _Calcd . for _C27H28N7O5 [M+H] ⁺ _528.1995 , found 528.1993 _.

N-(3-(7-((2-methoxy-4-thiomorpholinophenyl)amino)-3-methyl-2,4-dioxo-3,4-dihydropyrimido [4,5-d]pyrimidine-1(2H))phenyl)acrylamide (compound 005)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.35(s,1H),8.84(s,1H),8.60(s,1H),8.85(s,1H),7.70(s,1H),7.48( t, J=8.0Hz, 1H), 7.17(d, J=8.4Hz, 1H), 7.09(d, J=7.6Hz, 1H), 6.44(dd, J=17.2Hz, J=10.0Hz, 1H) ,6.27(dd,J=16.8Hz,J=2.0Hz,1H),5.97(s,1H),5.77(dd,J=10.0Hz,J=1.6Hz,1H),3.75(s,3H),3.42 -3.39(m,4H),3.28(s,3H),2.66-2.63(m,4H).HRMS(ESI) calculated value C ₂₇ H ₂₈ N ₇ O ₄ S[M+H] ⁺ 546.1923, experimental value 546.1924 .

N-(3-(7-((4-(4-acetyl-3-)-2-methoxyphenyl)amino)-3-methyl-2,4-dioxo-3,4- Dihydropyrimido[4,5-d]pyrimidine-1(2H))phenyl)acrylamide (compound 006)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.34(s,1H),8.83(s,1H),8.59(s,1H),7.86(s,1H),7.69(s,1H),7.48( t,J=8.4Hz,1H),7.17(d,J=8.8Hz,1H),7.09(d,J=8.0Hz,1H),6.55(s,1H),6.44(dd,J=17.2Hz, J＝10.4Hz, 1H), 6.26(dd, J＝17.2Hz, J＝1.6Hz, 1H), 5.99(s, 1H), 5.76(dd, J＝10.0Hz, J＝1.2Hz, 1H), 3.76 (s,3H),3.58-3.54(m,4H),3.28(s,3H),3.05-2.99(m,4H),2.05(s,3H).HRMS(ESI)C ₂₉ H ₃₁ N ₈ O ₅ [M+H] ⁺ 571.2417, found 571.2417.

N-(3-(7-((4-(4-(dimethylamino)piperidine)-2-methoxyphenyl)amino)-3-methyl-2,4-dioxo-3, 4-Dihydropyrimido[4,5-d]pyrimidine-1(2H))phenyl)acrylamide (compound 007)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.36(s,1H),8.83(s,1H),8.54(s,1H),7.88-7.86(m,1H),7.70(s,1H), 7.47(t,J=8.0Hz,1H),7.16(d,J=8.4Hz,1H),7.08(d,J=8.0Hz,1H),6.50-6.43(m,2H),6.26(dd,J ＝16.8Hz, J＝1.6Hz, 1H), 5.98(s, 1H), 5.76(dd, J＝10.0Hz, J＝1.2Hz, 1H), 3.75(s, 3H), 3.59-3.58(m, 2H ), 3.28(s,3H), 2.60-2.55(m,2H), 2.22(s,6H), 1.82-1.79(m,2H), 1.47-1.40(m,2H). HRMS(ESI) calculated value C ₃₀ H ₃₅ N ₈ O ₄ [M+H] ⁺ 571.2781, found 571.2780.

N-(3-(7-((4-(4-acetylpiperazinyl)-2-methoxyphenyl)amino)-3-ethyl-2,4-dioxo-3,4-di Hydropyrimido[4,5-d]pyrimidine-1(2H))phenyl)acrylamide (compound 008)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.38(s,1H),8.85(s,1H),8.64(s,1H),7.88(d,J=4.0Hz,1H),7.69(s, 1H), 7.49(t, J=8.0Hz, 1H), 7.18(d, J=8.4Hz, 1H), 7.12(d, J=7.6Hz, 1H), 6.55(s, 1H), 6.46(dd, J＝17.2Hz, J＝10.0Hz, 1H), 6.27(dd, J＝16.8Hz, J＝1.6Hz, 1H), 6.00-5.97(m, 1H), 5.78(dd, J＝10.0Hz, J＝ 2.0Hz,1H),3.94(q,J＝6.4Hz,2H),3.77(s,3H),3.58-3.54(m,4H),3.05-2.98(m,4H),2.06(s,3H), 1.19 (t, J=6.8Hz, 3H). HRMS (ESI) calculated for C ₃₀ H ₃₃ N ₈ O ₅ [M+H] ⁺ 585.2574, found 585.2572.

N-(3-(7-((4-(4-acetylpiperazinyl)-2-methoxyphenyl)amino)-3-isopropyl-2,4-dioxo-3,4- Dihydropyrimido[4,5-d]pyrimidine-1(2H))phenyl)acrylamide (compound 009)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.37(s,1H),8.83(s,1H),8.61(s,1H),7.86(d,J=8.0Hz,1H),7.67(s, 1H), 7.48(t, J=8.0Hz, 1H), 7.17(d, J=7.6Hz, 1H), 7.10(d, J=7.6Hz, 1H), 6.55(s, 1H), 6.45(dd, J＝16.8Hz, J＝10.0Hz, 1H), 6.27(dd, J＝16.8Hz, J＝1.6Hz, 1H), 6.00-5.97(m, 1H), 5.78(dd, J＝10.0Hz, J＝ 2.0Hz, 1H), 5.15-5.11(m, 1H), 3.76(s, 3H), 3.57-3.54(m, 4H), 3.05-2.97(m, 4H), 2.05(s, 3H), 1.44(d , J = 6.4 Hz, 6H). HRMS (ESI) calcd. for C ₃₁ H ₃₅ N ₈ O ₅ [M+H] ⁺ 599.2730, found 599.2731.

N-(3-(7-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)amino)-3-benzyl-2,4-dioxo-3 ,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)acrylamide (compound 010)

¹ H NMR (400MHz,DMSO-d ₆ )δ10.36(s,1H),8.87(s,1H),8.69(s,1H),7.87(d,J=5.6Hz,1H),7.68(s, 1H), 7.48(t, J=8.0Hz, 1H), 7.38(d, J=6.8Hz, 2H), 7.33(t, J=7.2Hz, 2H), 7.26(t, J=7.2Hz, 1H) ,7.17(d,J=8.0Hz,1H),7.12(d,J=7.8Hz,1H),6.55(s,1H),6.44(dd,J=16.8Hz,J=10.0Hz,1H),6.26 (dd, J=17.2Hz, J=2.0Hz, 1H), 5.98(s, 1H), 5.77(dd, J=10.0Hz, J=1.6Hz, 1H), 5.09(s, 2H), 3.76(s ,3H), 3.58-3.55(m,4H), 3.05-2.97(m,4H), 2.05(s,3H). HRMS(ESI) calculated value C ₃₅ H ₃₅ N ₈ O ₅ [M+H] ⁺ 647.2730 , the experimental value is 647.2735.

The specific synthesis method of compound 011 is as follows:

Reagents and conditions: (a)-(e) as previously described; (f) N,N,N'-trimethylethylenediamine, K ₂ CO ₃ , DMSO; (n) Zn, NH ₄ Cl, CH ₃ OH; (o) acid chloride, Et ₃ N, CH ₂ Cl ₂ , 0°C - room temperature.

7-((4-((2-(dimethylamino)ethyl)(methyl)amino-2-methoxy-5-nitrophenyl)amino)-3-methyl-1-phenyl Synthesis of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

Weigh 7-((4-fluoro-2-methoxy-5-nitrophenyl)amino)-3-methyl-1-phenylpyrimido[4,5-d]pyrimidine-2,4( 1H,3H)-diketone (438mg, 1mmol), K ₂ CO ₃ (207mg, 1.5mmol), N,N,N'-trimethylethylenediamine (153mg, 1.5mmol) in a 10mL single-necked flask, add 4mL DMSO was dissolved, heated to 90°C, and stirred for 4h. TLC traced to the conversion of raw materials, cooled to room temperature, added ice water, extracted with ethyl acetate, collected the organic phase, spin-dried, and separated the crude product by silica gel column chromatography (DCM/CH ₃ OH=20:1, v/v) to obtain 7-((4-((2-(dimethylamino)ethyl)(methyl)amino-2-methoxy-5-nitrophenyl)amino)-3-methyl-1-phenyl Pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione 358 mg, yield 69%. ¹ HNMR (400MHz, CDCl ₃ ) δ8.97(s,1H), 7.88(s, 1H),7.63(s,1H),7.46-7.43(m,3H),7.19-7.16(m,2H),6.52(s,1H),3.83(s,3H),3.39(s,3H),3.08 (t, J=6.4Hz, 2H), 2.71(s, 3H), 2.41(t, J=6.8Hz, 2H), 2.17(s, 6H).LC-MS: m/z: 521.3(M+H ) ⁺ .

7-((5-amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)amino)-3-methyl-1-phenyl Synthesis of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione

Weigh 7-((4-((2-(dimethylamino)ethyl)(methyl)amino-2-methoxy-5-nitrophenyl)amino)-3-methyl-1- Phenylpyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (312mg, 0.6mmol) was dissolved in 50mL single-necked flask, dissolved in 8mL of methanol, dropped into 1mL of saturated ammonium chloride solution, added Zinc powder (390mg, 6mmol) was stirred overnight at room temperature. TLC traced to the conversion of raw materials, filtered off the zinc powder, and the filtrate was spin-dried. The crude product was separated by silica gel column chromatography (DCM/CH ₃ OH=15:1, v/v) to obtain 7-((5-amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)amino)-3-methyl-1-phenyl Pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione 180 mg, yield 61%. ¹ H NMR (400MHz, CDCl ₃ ) δ8.94(s,1H), 8.11(s ,1H),7.60(t,J=7.6Hz,2H),7.52(t,J=7.2Hz,1H),7.33(d,J=7.6Hz,2H),6.89(s,1H),6.51(s ,1H),3.70(s,3H),3.40(s,3H),2.81(t,J=6.4Hz,2H),2.52(s,3H),2.29(t,J=6.8Hz,2H),2.19 (s,6H).LC-MS: m/z:491.3(M+H) ⁺ .

N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((6-methyl-5,7-dioxo-8- Synthesis of phenyl-5,6,7,8-tetrahydropyrimido[4,5-d]pyrimidin-2-yl)amino)phenyl)acrylamide (compound 011)

Weigh 7-((5-amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)amino)-3-methyl-1- Phenylpyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione (147mg, 0.3mmol) and DIPEA (77mg, 0.6mmol) were dissolved in 25mL flask by adding 3mL dichloromethane, ice Stir in the bath for 15 minutes. Another acryloyl chloride (33 mg, 0.36 mmol) was dissolved in 1 mL of dichloromethane, added dropwise to the above reaction solution, and stirred overnight at room temperature. TLC followed the conversion of raw materials, spin-dried, and the crude product was separated by silica gel column chromatography (DCM/CH ₃ OH=15:1, v/v) to obtain N-(2-((2-(dimethylamino)ethyl) (Methyl)amino)-4-methoxy-5-((6-methyl-5,7-dioxo-8-phenyl-5,6,7,8-tetrahydropyrimido[4, 5-d]pyrimidin-2-yl)amino)phenyl)acrylamide 80 mg, yield 49%. ¹ H NMR (400MHz, CDCl ₃ )δ9.36(s, 1H), 9.08(s, 1H), 7.88(s, 1H), 7.50-7.46(m, 5H), 6.58(dd, J=17.2Hz, J＝10.0Hz, 1H), 6.48(dd, J＝17.2Hz, J＝1.6Hz, 1H), 5.83(dd, J＝10.0Hz, J＝2.0Hz, 1H), 3.83(s, 3H), 3.48 (s,3H),3.23(t,J=6.0Hz,2H),3.06(t,J=6.4Hz,2H),2.77(s,6H),2.68(s,3H).HRMS(ESI): calculation Value for C ₂₈ H ₃₃ N ₈ O ₄ (M+H) ⁺ 545.2625, found 545.2630.

Example 2. Biological activity test

The compound provided by the invention is carried out as follows to the in vitro inhibitory effect experiment of EGFR kinase activity:

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

The concentrations of different kinases are determined by optimization experiments, and the corresponding concentrations are: EGFR (PV3872, Invitrogen) 0.287 μg/μL, EGFR-T790M (PV4803, Invitrogen) 0.174 μg/μL, EGFR-L858R/T790M (PV4879, Invitrogen) 0.055 μg /μL. Compounds were diluted three-fold from 5.1x10 ^-9 M to 1x10 ^-4 M in DMSO. 4 μL of compound was dissolved in 96 μL of water to obtain a 4x compound solution. 40 μM ATP is dissolved in 1.33x Kinase Buffer, the Kinase/Peptide Mix contains 2x Kinase, 4 μM Tyrosine 4 Peptide is ready for use. A 10 μL kinase reaction consists of 2.5 μL compound solution, 5 μL kinase/peptide mix, 2.5 μL ATP solution. 5 μL of phosphorylated peptide solution was used instead of the kinase/peptide mix as a 100% phosphorylated control. 2.5 μL of 1.33x kinase buffer instead of ATP solution was used as 100% inhibition control, and 2.5 μL of 4% DMSO instead of compound solution was used as 0% inhibition control. The solution in the plate was mixed well and incubated at room temperature for 1.5 hours. Add 5 μL of Development Solution to each well and continue to incubate at room temperature for 1 hour, during which time the non-phosphorylated peptides are cleaved. Finally, 5 μL of stop reagent (StopReagent) was added to end the reaction. Well plates were measured with an EnVision Multilabel Reader (Perkin Elmer). Experimental data were calculated using GraphPad Prism version 4.0. Each experiment was repeated more than 3 times.

Cell proliferation and growth inhibition analysis: 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 by MTS assay. The cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each experiment of each cell line was adjusted according to the absorbance value (absorbance value at 490 nm was 1.3-2.2). Six concentration gradients (0.1 nM-10 μM) were set up for the compounds to be tested, and at least six groups of parallel controls were used for each concentration value.

H1975 and A431 cells were cultured in the corresponding medium, and the cells were passaged at least twice after recovery, and then used for experiments. Cells in log phase were trypsinized and resuspended in medium. H1975 (1000 cells per well) and A431 (2000 cells per well) were seeded in a 96-well plate with a volume of 100 μL; 6 groups of parallels and 7 columns were set up. The orifice plate was placed in an incubator with 5% carbon dioxide at 37°C overnight. The compounds were dissolved in DMSO to prepare a concentration of 10 μM per liter, and then the compound concentrations were gradually diluted to obtain compound concentrations of 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM per liter, respectively. 2 μL of compound solution was added to 998 μL of culture medium, and the mixture was mixed well. 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 culturing for 68 hours, 20 μL of MTT (5 mg/mL) was added. After 4 hours, discard the supernatant and add 150 μL DMSO. After shaking for 10 minutes, the plate was read with Synergy HT (Bio TeK) (OD490). Data were calculated using GraphPad Prism version 4.0, and _IC50 values were adjusted by non-linear regression models using dose-response curves.

The test results are shown in Table 1 below.

Table 1

*: >100 indicates that the inhibitory effect on cells is basically not detected.

discuss:

After extensive and in-depth research, the inventors have designed and synthesized a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione compounds that have not been reported in the literature. Activity tests at the molecular and cellular levels were carried out, and a batch of compounds capable of selectively inhibiting the EGFR T790M mutation were obtained. The inventors further found that the compound of the present invention has a higher inhibitory ability to inhibit the proliferation of EGFR mutant cancer cells (H1975) and EGFR wild-type cancer cells (A431) than the difference in the ability to inhibit the kinase activity of mutant EGFR and wild-type EGFR, thus It is suggested that the compound of the present invention has better differential toxicity in vivo, and may become a third-generation EGFR-targeted drug that selectively inhibits the T790M mutation and overcomes clinical drug resistance, or can be further modified to obtain better activity and/or differential toxicity. The basis for better compounds.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (9)

1. A compound represented by general formula III or a pharmaceutically acceptable salt thereof: In the formula, ^R2 is selected from R ³ is selected from C ₁ -C ₆ alkyl; or C ₁ -C ₆ alkyl substituted by phenyl; R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently selected from the group consisting of: 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein ^R is selected from methyl, isopropyl or benzyl. 3. The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R ⁵ and R ⁸ are H; or, R ⁵ , R ⁷ and R ⁸ are H. 4. A compound or a pharmaceutically acceptable salt thereof selected from the group consisting of: 5. A pharmaceutical composition, which contains the compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. 6. Use of the compound according to any one of claims 1-4 in the preparation of a drug for treating or preventing EGFR-mediated diseases, or inhibiting EGFR. 7. The use according to claim 6, characterized in that the EGFR-mediated disease is cancer. 8. The use according to claim 7, 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, ovarian cancer, Neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, gastric cancer, multiple myeloid cancer and solid tumors. 9. The use according to claim 8, wherein the solid tumor is glioma or lymphoma.