CN113307797B - Polysubstituted quinazoline compound and application thereof - Google Patents

Abstract

The invention discloses a polysubstituted quinazoline compound and application thereof, belonging to the field of chemical medicine. The substituted quinazoline compound shown as the general formula (I) and the pharmaceutically acceptable salt thereof have excellent brain barrier permeability, enhanced metabolic stability and longer metabolic half-life, show higher inhibition activity on an activated or drug-resistant mutant form EGFR than a wild type EGFR, and can effectively reduce side effects.

Description

Polysubstituted quinazoline compound and application thereof

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to a polysubstituted quinazoline compound and application thereof.

Background

Epidermal Growth Factor Receptor (EGFR) is one of the transmembrane protein tyrosine kinases of the erbB receptor family, which, when bound to growth factor ligands such as Epidermal Growth Factor (EGF), can homodimerize with additional EGFR molecules or heterodimerize with members of another family such as erbB2(HER2), erbB3(HER3) or erbB4(HER4), the homodimerization and/or heterodimerization of erbB receptors resulting in phosphorylation of key tyrosine residues within cells and in stimulation of many intracellular signaling pathways involved in cell proliferation and survival. Dysregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and survival of tumor cells, and is closely associated with human cancers such as lung cancer, head and neck cancer, colon cancer, and breast cancer.

Therefore, the erbB family is an ideal target for the development of anticancer drugs. Specific protein tyrosine kinase inhibitors are of great interest as potential anticancer drugs. In 2004, cases based on this target drug were reported (Science [2004] 304, 1497-1500 and New England Journal of Medicine [2004] 350, 2129-2139). Typical representatives of currently marketed reversible inhibitors of EGFR include Gefitinib (Gefitinib), Erlotinib (Erlotinib), the structures of which are shown below, for inhibiting EGFR wild-type and activating mutant (e.g., exon19 deletion activating mutation or L858R activating mutation).

Clinical researches prove that gefitinib and erlotinib have good therapeutic effect on non-small cell lung cancer patients with EGFR exon deletion or L858R point mutation. However, with the emergence of resistance, which limits the further clinical use of such inhibitors, studies have shown that 50% of gefitinib, erlotinib resistance after treatment is associated with a secondary mutation in EGFR (T790M). Therefore, studies to overcome the drug resistance caused by the T790M mutation are also being conducted, and irreversible inhibitors have become one of the directions of research.

Irreversible EGFR inhibitors have certain advantages over reversible EGFR inhibitors. Irreversible EGFR inhibitors can inhibit EGFR for long periods of time, limited only by the normal rate of receptor recombination. It has been found that the irreversible EGFR inhibitor can overcome the drug resistance caused by the T790M mutation to some extent by covalently binding to a cysteine residue (Cys797) on EGFR through Michael Addition reaction to enlarge the binding site of the irreversible EGFR inhibitor to ATP (Oncogene [2008], 27: 4702-. Irreversible EGFR inhibitors currently on the market or under investigation include Afatinib (Afatinib), Neratinib (Neratinib), EKB-569(Pelitinib), PF00299804 (dacitinib), etc., and the structures thereof are shown below. However, the irreversible EGFR inhibitor has a great inhibition effect on wild EGFR, and can bring about great toxic and side effects such as diarrhea, nausea, rash and the like, thereby limiting the clinical application of the irreversible EGFR inhibitor.

International patent WO2012/061299a1 applied to west build averamelis research corporation (Avila Therapeutics) discloses a class of pyrimidine compounds, wherein a representative compound is CO1686(Rociletinib), and the structure is as follows. The literature reports that CO1686 can selectively act on EGFR activating mutation and T790M drug-resistant mutation, but has weak inhibition effect on wild-type EGFR (Cancer Discovery, 2013,2 (12): 1404-1415). However, CO1686 was rejected by the FDA for pre-marketing due to a lower than expected response rate and side effects of hyperglycemia and QT wave prolongation.

International patent WO2013/014448a1 filed by AstraZeneca also discloses a series of pyrimidines, wherein representative pyrimidine compounds are ositinib (osimertinib), the structural formula of the pyrimidine compounds is shown in the specification, and the pyrimidine compounds have better inhibitory effects on EGFR activating type mutation and T760M drug-resistant type mutation compared with wild-type EGFR, and the drugs are approved to be sold in the market at present. The most common adverse reactions (not less than 25%) of the drug are diarrhea, rash, dry skin and nail toxicity.

Furthermore, several generations of inhibitors have been marketed that show limited efficacy in treating non-small cell lung cancer patients with brain metastases, such as: gefitinib, erlotinib, afatinib, Oseitinib, etc., because none of them can effectively cross the Blood Brain Barrier (BBB) (Journal of Clinical Oncology of Clinical Journal of the American Society of Clinical Oncology,2006,24(27): 4517-. Meanwhile, several reports have shown that brain metastasis of lung cancer occurs as an unmet Clinical need (Journal of neuro-Oncology,2005,75(1): 5-14; Journal of Clinical Oncology,2004,22(14): 2865-.

Juxtamembrane metastasis occurs when cancer spreads to the cerebrospinal fluid (the tissue layer covering the brain and spinal cord). Metastases may spread through the blood to the meninges or they may travel from brain metastases carried by cerebrospinal fluid (CSF) that flows through the meninges. If brain tumors enter the CFS and survive, they can travel throughout the central nervous system, which leads to neurological problems (Surgical neurology International,2013,4(Suppl 4): S265-S288). The incidence of leptomeningeal metastases is increasing, partly because cancer patients live longer, and because many chemotherapeutics and molecular targeted therapies do not reach concentrations in the cerebrospinal fluid sufficient to kill tumor cells.

Meanwhile, some of the current quinazoline ring drugs have poor curative effects, and further improvement of brain barrier permeability, enhancement of metabolic stability, improvement of pharmacokinetic properties and drug potential are still needed.

Disclosure of Invention

The problems to be solved by the invention are as follows:

the novel polysubstituted quinazoline compound or the pharmaceutically acceptable salt thereof has unexpected excellent brain barrier permeability, enhanced metabolic stability and longer metabolic half-life, shows higher inhibitory activity on an activated or drug-resistant mutant form EGFR than a wild type EGFR, and can effectively reduce side effects.

Solution to the problem:

the invention firstly provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof,

wherein:

when the Y is O, the reaction is carried out,

R ¹ selected from 2-alkenoyl, C4-C8 linear or branched alkyl, C3-C6 cycloalkyl, substituted benzyl or aryl;

R ² selected from methoxy or H; the substituted group is halogenated C1-C4 alkyl; halo includes fluoro, chloro, bromo or iodo;

R ³ selected from H, F, Cl;

x is selected from NH or O;

z is selected from

When the Y is NH, the reaction mixture is,

R ¹ selected from 2-enoyl, deuterated methyl, C1-C8 straight or branched chain alkyl, C3-C6 cycloalkyl, substituted benzyl or aryl; the substituted group is halogenated C1-C4 alkyl; halo includes fluoro, chloro, bromo or iodo;

R ² selected from methoxy or H;

R ³ selected from H, F, Cl;

x is selected from NH or O;

z is selected from

In one embodiment of the invention, R ¹ Preferably from acryloylMethyl, deuterated methyl, cyclopropanemethyl, 2-ethylbutyl, 2-methylpentyl and 4-trifluoromethyl benzyl. Further, in some embodiments R ¹ Preferably from acryloyl, deuterated methyl, cyclopropanemethyl.

In one embodiment of the invention, Z is preferably selected from

In one embodiment of the invention, the compound is selected from:

in one embodiment of the present invention, the compound is further preferably a compound:

in one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate. Still further, the pharmaceutically acceptable salt is selected from the hydrochloride, sulfate, succinate or mesylate salt.

The present invention also provides a process for the preparation of a compound of formula (I) comprising:

the first reaction scheme is as follows:

as shown in a reaction formula 1, performing nucleophilic substitution on an intermediate A and an intermediate B to obtain an intermediate C; removing Boc protecting group from the intermediate C by trifluoroacetic acid to obtain an intermediate D; finally, the intermediate D and the corresponding aldehyde are subjected to reduction ammoniation to obtain a compound E, wherein R ₁ 、R ₂ X and R ₃ Is as defined in formula (I);

alternatively, the first and second electrodes may be,

the second reaction scheme is as follows:

as shown in reaction formula 2, intermediate D and acryloyl chloride are subjected to nucleophilic substitution to obtain compound F, wherein, Y, R ₂ X and R ₃ Is as defined in formula (I);

alternatively, the first and second electrodes may be,

the reaction scheme III:

reacting the intermediate G with phenyl chloroformate to obtain an intermediate H as shown in a reaction formula 3; performing amine ester exchange on the intermediate H and (R) -4-Boc-2-methylpiperazine to obtain an intermediate I; removing Boc protecting group from the intermediate I by trifluoroacetic acid to obtain an intermediate J; finally, the intermediate J and the corresponding aldehyde are subjected to reduction ammoniation to obtain a compound K; wherein R is ₁ 、R ₂ X and R ₃ Is as defined in formula (I);

alternatively, the first and second electrodes may be,

the reaction scheme is four:

as shown in a reaction formula 4, performing amine ester exchange on the intermediate G and diethyl squarate to obtain an intermediate L; intermediates L and (R)Carrying out amine ester exchange on the-4-Boc-2-methylpiperazine to obtain an intermediate M; removing Boc protecting group from the intermediate M by trifluoroacetic acid to obtain an intermediate N; and finally, reducing and ammoniating the intermediate N and formaldehyde to obtain a compound O. Wherein R is ₂ And R ₃ Are as defined in formula (I).

A third object of the present invention is to provide a pharmaceutical composition comprising a compound of formula (I) as described above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

The fourth object of the present invention is to provide the use of a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disease mediated by an EGFR-activating or drug-resistant mutant in a mammal.

In one embodiment of the invention, the disease mediated by EGFR-activating or drug-resistant mutants is cancer, and specifically includes: non-small cell lung cancer or metastatic non-small cell lung cancer.

The fifth object of the present invention is to provide an antitumor agent, comprising: a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

In one embodiment of the present invention, the anti-tumor drug may further include the following anti-tumor components:

(i) antineoplastic drugs acting on the DNA structure;

(ii) antineoplastic agents that affect nucleic acid synthesis;

(iii) anti-tumor drugs that affect nucleic acid transcription;

(iv) tubulin synthesized antineoplastic drugs;

(v) cell signaling pathway inhibitors such as epidermal growth factor receptor inhibitors;

(vi) an anti-tumor monoclonal antibody.

The invention has the beneficial effects that:

the present invention provides novel quinazoline inhibitors of activated mutant forms of epidermal growth factor receptor which have unexpectedly superior brain barrier permeability properties which allow their use in the treatment of cancers which have metastasized to the CNS, particularly brain metastases and leptomeningeal metastases, and which have better pharmacodynamic properties, are more metabolically stable, exhibit greater inhibitory activity against activated or drug-resistant mutant forms of EGFR than wild-type EGFR, and are effective in reducing side effects such as skin rash and diarrhea.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples.

The term "disease" as used herein refers to any condition or disorder that impairs or interferes with the normal function of a cell, organ or tissue.

The term "inhibitor" as used herein refers to a compound or agent that has the ability to inhibit a biological function of a targeted protein or polypeptide, for example by inhibiting the activity or expression of the protein or polypeptide.

The term "antineoplastic agent" as used herein refers to any agent useful in the treatment of neoplastic disorders.

The term "pharmaceutically acceptable" as used herein, means those ingredients which are, within the scope of sound medicine, suitable for use in contact with the tissues of human beings and other mammals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. "pharmaceutically acceptable salt" refers to any non-toxic salt that, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the present invention or a prodrug of a compound.

The term "effective amount" or "therapeutically effective amount" as used herein means that the amount of a compound or pharmaceutical composition described herein is sufficient for the intended use, including, but not limited to, the treatment of disease. In some embodiments, the amount is detected to be effective for killing or inhibiting cancer cell growth or spread; the size or number of tumors; or the severity level, stage and progression of the cancer. The therapeutically effective amount may vary depending on the intended application, e.g., in vitro or in vivo, the condition and severity of the disease, the age, weight, or mode of administration of the subject, etc. The term also applies to a particular response in which the dose will induce the target cell, e.g., reduce cell migration. The specific dosage will depend, for example, on the particular compound chosen, the species of the subject and their age/existing health or risk of health, the route of administration, the severity of the disease, administration in combination with other agents, the time of administration, the tissue to which it is administered, and the administration device, among other things.

In the present invention "administering" or "administering" an individual compound means providing a compound of the invention to an individual in need of treatment.

The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of the invention may also exhibit multiple tautomeric forms, in which case the invention expressly includes all tautomeric forms of the compounds described herein. All such isomeric forms of such compounds are included in the present invention. All crystalline forms of the compounds described herein are expressly included in the present invention.

< Compound or pharmaceutically acceptable salt thereof >

The invention provides a novel quinazoline compound in an activated mutation form of an epidermal growth factor receptor or a pharmaceutically acceptable salt thereof, wherein the structural formula of the quinazoline compound is shown as a general formula (I):

the compounds of formula (I) include pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate. Preferably, from the pharmaceutical viewpoint, the salt of the present invention is a hydrochloride, sulfate, succinate or methanesulfonate.

It will be appreciated that certain compounds of formula (I) or pharmaceutically acceptable salts thereof may be in the form of solvated as well as unsolvated forms such as, for example, water and forms. It is to be understood that the invention encompasses all such solvate forms possessing inhibitory activity against activated mutant EGFR.

The synthesis of the compounds of general formula (I) according to the invention can be carried out by the person skilled in the art of synthetic chemistry. The documents mentioned in the background of the invention are hereby incorporated by reference in their entirety. The preparation method is described in detail in the examples.

< pharmaceutical composition >

The invention provides a pharmaceutical composition comprising a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like. In these solid dosage forms, the compounds of general formula (I) according to the invention as active ingredient are mixed with at least one customary inert excipient (or carrier), for example with sodium citrate or dicalcium phosphate. Or mixing with the following components: (1) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, and the like; (2) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic and the like; (3) humectants, such as glycerol and the like; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and the like; (5) a slow solvent such as paraffin and the like; (6) absorption accelerators such as quaternary ammonium compounds and the like; (7) wetting agents such as cetyl alcohol and glyceryl monostearate and the like; (8) adsorbents, for example, kaolin, and the like; (9) lubricants, for example, talc, calcium stearate, solid polyethylene glycols, sodium lauryl sulfate, and the like, or mixtures thereof. Capsules, tablets, pills, etc. may also contain buffering agents.

The solid dosage forms, e.g., tablets, dragees, capsules, pills, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other crystalline forms of materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active ingredient may also be in microencapsulated form with one or more of the above excipients.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated in liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, and the like. In addition to the compounds of formula (I) or pharmaceutically acceptable salts thereof as active ingredients, the liquid dosage forms may contain inert diluents conventionally employed in the art, such as water and other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil and the like or mixtures of such materials and the like. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, perfuming agents and the like.

Such suspending agents include, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar, and the like, or mixtures of these materials.

The compounds of the present invention and pharmaceutically acceptable salts thereof may be formulated for parenteral injection in dosage forms including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated into dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like. The compounds of the general formula (I) according to the invention or their pharmaceutically acceptable salts as active ingredients are mixed under sterile conditions with physiologically acceptable carriers and optionally preservatives, buffers and, if desired, propellants.

The compounds of formula (I) of the present invention or a pharmaceutically acceptable salt thereof will be administered to a mammal in a unit dose in the range of 0.01 to 2000mg/kg, particularly 2.5 to 1000mg/kg, particularly 5 to 500mg/kg and this should provide an effective dose. However, the daily dosage will necessarily vary depending upon the host treated, the particular route of administration, and the severity of the disease being treated. Thus, the optimum dosage may be determined by the practitioner who is treating any particular patient.

< use >

The present invention provides the use of a compound of formula (I) as defined above and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the treatment of diseases mediated by EGFR-activated or drug-resistant mutants, particularly cancer, in mammals, especially humans.

In the present invention, the activating mutant form of EGFR, the drug-resistant mutant form of EGFR may be, for example, an L858R activating mutant, an Exon19 deletion activating mutant, and/or a T790M resistant mutant. Thus, the disease, disorder or condition mediated by an EGFR-activating or drug-resistant mutant may be, for example, a disease, disorder or condition mediated by an L858R activating mutant, an Exon19 deletion activating mutant and/or a T790M resistance mutant, and the invention is particularly applicable to EGFR-activating mutant-mediated diseases, disorders or conditions such as an L858R activating mutant, an Exon19 deletion activating mutant. Types of cancer that may be susceptible to treatment with a compound of formula (I) or a pharmaceutically acceptable salt thereof include, but are not limited to: ovarian cancer, cervical cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-hodgkin lymphoma, lung cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumors, thyroid cancer, cholangiocarcinoma, endometrial cancer, kidney cancer, anaplastic large cell lymphoma, acute myelogenous leukemia, multiple myeloma, melanoma, and mesothelioma. Preferably, the cancer comprises non-small cell lung cancer, metastatic non-small cell lung cancer.

The treatment of cancer as described herein, a compound of formula (I) or a pharmaceutically acceptable salt thereof, is administered to a mammal, more particularly a human.

Certain non-small cell lung cancer patients with CNS metastases (particularly brain metastases and/or leptomeningeal metastases) exhibit CNS symptoms such as headache and vomiting. For these patients, Whole Brain Radiation Therapy (WBRT) can be used to ameliorate these symptoms, and when used in combination with WBRT, the compounds of the present invention, or pharmaceutically acceptable salts thereof, can enhance the anti-tumor effects of WBRT and further ameliorate CNS symptoms.

The treatment of EGFR activity in the form of an activated mutant, or a drug-resistant mutant, of the present invention may be applied as a sole therapy or in addition to a compound of the present invention, which may involve conventional surgical or radiation therapy (e.g., WBRT as described herein), may be administered in combination with other pharmaceutically acceptable therapeutic agents, and in combination with other anti-tumor drugs, and such combination therapy may be effected by the simultaneous, sequential or separate use of the individual components of the therapy. The therapeutic oncology agents include, but are not limited to: antitumor agents acting on the chemical structure of DNA, such as cisplatin, antitumor agents affecting nucleotide synthesis such as methotrexate, 5-fluorouracil and the like, antitumor agents affecting nucleic acid transcription such as doxorubicin, epirubicin, aclarubicin and the like, antitumor agents affecting tubulin synthesis such as taxol, vinorelbine and the like, aromatase inhibitors such as aminoglutethimide, letrozole, rening and the like, cell signaling pathway inhibitors such as epidermal growth factor receptor inhibitor imatinib, gefitinib, erlotinib, afatinib, ocitinib and the like, 6- (4-bromo-2-chloro-phenylamino) -7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy) -amide or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts thereof, 1- [ (1S) -1- (imidazo [1,2-a ] pyridin-6-yl) ethyl ] -6- (1-methyl-1H-pyrazol-4-yl) -1H [1,2,3] triazolo [4,5-b ] pyrazine or a pharmaceutically acceptable salt thereof. Anti-tumor mabs, e.g., anti-CTLA-4 antibodies, immunosuppressive agents PD-1, PD-L1, OX40 agonist antibodies, and the like, the components to be combined may be administered simultaneously or sequentially, in a single formulation or in different formulations. Such combinations include not only combinations of one or other active agents of the compounds of the present invention, but also combinations of two or more other active agents of the compounds of the present invention.

The following examples illustrate, but do not limit, the synthesis of the compounds of formula (I). The temperatures are given in degrees Celsius. All evaporation was performed under reduced pressure if not otherwise stated. Reagents were purchased from commercial suppliers and used without further purification if not otherwise stated. The structure of the final products, intermediates and starting materials is confirmed by standard analytical methods, such as elemental analysis, spectroscopic characterization, e.g., MS, NMR. Abbreviations used are those conventional in the art.

The following intermediate substances are involved in the specific examples and can be synthesized by the following process routes:

intermediate 6: preparation of 4-tert-butyl-1-4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (2R) -2-methylpiperazine-1, 4-dicarboxylate

Step a preparation of 4-hydroxy-7-methoxyquinazolin-6-ylacetate (intermediate 1):

a mixture of 7-methoxyquinazoline-4, 6-diol (10.0g,52mmol) and pyridine (8.2g,104mmol) in acetic anhydride (50mL) was heated to 80 ℃ for 1h and the reaction was complete by TLC. The reaction was cooled and distilled under reduced pressure, the residue was poured into 200mL of water, filtered and the filter cake was dried under vacuum to give intermediate 1(12.1g, yield 99%) which was used in the next reaction without further purification. MS-ESI (M/z) 235.05[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:12.21(s,1H),8.09(s,1H),7.75(s,1H),7.28(s,1H),3.92(s,3H),2.30(s,3H).

Step b preparation of 4-chloro-7-methoxyquinazolin-6-ylacetate (intermediate 2):

1.0mol/L thionyl chloride (205mL) was added dropwise to a mixture of intermediate 1(12.0g,51mmol) in acetonitrile (100mL) under ice bath conditions, followed by 0.5mL of N, N-dimethylformamide, and after completion of the addition, the mixture was refluxed at 80 ℃ and the reaction was checked by TLC. The solvent was evaporated under reduced pressure, the residue was poured into 150mL of water, saturated aqueous sodium bicarbonate was slowly added dropwise to the residue in ice bath, the pH was adjusted to 8.0-9.0, filtered, the filter cake was washed with water, and the filter cake was dried under vacuum to give intermediate 2(12.1g, yield 93.7%) which was used in the next reaction without further purification. MS-ESI (M/z) 253.05[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.56(s,1H),7.82(s,1H),7.38(s,1H),3.93(s,3H),2.31(s,3H).

Step c preparation of 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl acetate (intermediate 3):

to a suspension of intermediate 2(10.0g,40mmol) in acetonitrile (100mL) was added 2-fluoro-3-chloroaniline (6.3g,43mmol), the mixture was refluxed at 80 ℃ overnight and the reaction was stopped by TLC. The solvent was distilled off under reduced pressure to give intermediate 3(14.3g, yield 99%) which was used in the next reaction without further purification. MS-ESI (M/z) 362.15[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:11.58(s,1H),8.88(s,1H),8.64(s,1H),7.64(t,J＝7.5Hz,1H),7.54(t,J＝7.4Hz,1H),7.50(s,1H),7.37(t,J＝8.2Hz,1H),4.02(s,3H),2.40(s,3H).

Step d preparation of 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-ol (intermediate 4):

to a mixture of intermediate 3(14.3g,40mmol) in methanol (150mL) was added anhydrous potassium carbonate (13.7g,100mmol), the mixture was stirred at room temperature overnight and the reaction was complete by TLC. The solvent was evaporated under reduced pressure and the residue was poured into 300mL of water, filtered and the filter cake was dried under vacuum to give intermediate 4(9.8g, yield 77.5%) which was used in the next reaction without further purification. MS-ESI (M/z) 320.15[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.38(s,1H),8.30(s,1H),7.61(s,1H),7.51(t,J＝7.5Hz,1H),7.3(t,J＝7.4Hz,1H),7.24(t,J＝8.1Hz,1H),7.17(s,1H),3.95(s,3H).

Step e preparation of 4-tert-butyl-1- {4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl } (2R) -2-methylpiperazine-1, 4-dicarboxylate (intermediate 5):

to a mixture of intermediate 4(9.8g,31mmol) and tert-butyl (3R) -4- (chlorocarbonyl) -3-methylpiperazine-1-carboxylate (16.1g,62mmol) in N, N-dimethylformamide (150mL) was added anhydrous potassium carbonate (8.5g,62mmol), and the mixture was stirred at room temperature overnight to terminate the reaction by TLC. The mixture was poured into 150mL of water, filtered and the filter cake was dried in vacuo to give intermediate 5(15.4g, 92% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 546.10[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.75(s,1H),8.48(s,1H),8.23(s,1H),7.50(q,J＝7.6Hz,2H),7.34(s,1H),7.28(t,J＝8.1Hz,1H),4.50～4.20(m,1H),3.98～4.04(m,1H),3.95(s,3H),3.82(d,J＝13.7Hz,2H),3.24～3.04(m,2H),2.98～2.82(m,1H),1.44(s,9H),1.23(s,3H).

Step f preparation of 4-tert-butyl-1-4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (2R) -2-methylpiperazine-1, 4-dicarboxylate (intermediate 6):

intermediate 5(15.0g,2mmol) was dissolved in a mixed solution of dichloromethane (160mL) and trifluoroacetic acid (40mL) and reacted at room temperature for 1h with TLC detection of the end of the reaction. The solvent was evaporated under reduced pressure, 150mL of water was added to the residue, the pH was adjusted to 8.0-9.0 with saturated aqueous sodium bicarbonate, the layers were separated, the aqueous layer was extracted with 100mL of dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give intermediate 6(11.4g, yield 93%), which was used in the next reaction without further purification. MS-ESI (M/z):446.8[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.74(s,1H),8.47(s,1H),8.22(s,1H),7.50(q,J＝7.9Hz,2H),7.33(s,1H),7.28(t,J＝8.1Hz,1H),4.35～4.10(m,1H),3.95(s,3H),3.85～3.60(m,1H),3.20～3.05(m,1H),2.99～2.88(m,1H),2.83～2.73(m,2H),2.60～2.54(m,1H),1.32(s,3H).

Intermediate 13: preparation of 4- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] carbamoyl } - (R) -3-methylpiperazine

Step a preparation of 4-chloro-7-methoxy-6-nitroquinazoline (intermediate 7):

1.0mol/L thionyl chloride (80mL) was slowly added dropwise under ice bath conditions to a mixture of 7-methoxy-6-nitroquinazolin-4-one (4.5g,20.3mmol) and acetonitrile (60mL), followed by 2 drops of N, N-dimethylformamide, and the reaction was complete by TLC at 80 ℃ under reflux for 1 h. The solvent was evaporated under reduced pressure to give intermediate 7(4.8g, 99% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 234.05[ M + H] ⁺ .

Step b preparation of 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxy-6-nitroquinazoline (intermediate 8):

to a mixture of intermediate 7(4.8g,20mmol) and acetonitrile (60mL) was added 3-chloro 2-fluoroaniline (3.1g,21mmol) dropwise, refluxed at 80 ℃ for 30min and the reaction was complete by TLC. Cooled to room temperature, filtered, the filter cake washed with dichloromethane and dried in vacuo to afford intermediate 8(6.9g, 99% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 349.00[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d6)δ:11.87(s,1H),9.54(s,1H),8.89(s,1H),7.64(s,2H),7.54(t,J＝6.6Hz,1H),7.37(t,J＝8.2Hz,1H),4.12(s,3H).

Step c preparation of 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxy-6-aminoquinazoline (intermediate 9):

intermediate 8(6.8g,19.5mmol) was added to a mixed solution of water (40mL) and ethanol (120mL), iron powder (5.5g,97.5mmol) and ammonium chloride (7.3g,136.5mmol) were added, and the reaction was refluxed at 90 ℃ for 1h and checked by TLC to completion. Cooling to room temperature, adjusting the pH value to 8-9 with saturated sodium bicarbonate aqueous solution, adding 200mL of ethanol, 100mL of dichloromethane and 100mL of ethyl acetate into the system, stirring for 1h at room temperature, assisting in filtering with diatomite, washing a filter cake with ethanol, collecting a filtrate, and reducing the pressureThe solvent was evaporated, 100mL water was added to the residue, filtered and the filter cake was dried in vacuo to give intermediate 9(6.1g, 98% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 319.00[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:10.04(s,1H),8.43(s,1H),7.94～6.78(m,5H),5.71(s,2H),4.00(s,3H).

Step d preparation of phenyl 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-ylcarbamate (intermediate 10):

pyridine (2.7g,33.8mmol) and phenyl chloroformate (3g,18.8mmol) were added to a solution of intermediate 9(6g,18.8mmol) in N, N-dimethylformamide (20mL), and the mixture was reacted at room temperature for 2 hours to complete the reaction by TLC. The reaction was added to 100mL of water, extracted with ethyl acetate, the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to give intermediate 10(6.8g, yield 82%) which was used in the next reaction without further purification. MS-ESI (M/z) 439.00[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.95(s,1H),9.65(s,1H),8.64(s,1H),8.45(s,1H),7.51～7.41(m,4H),7.33～7.22(m,5H),4.03(s,3H).

Step e preparation of tert-butyl 4- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] carbamoyl } - (R) -3-methylpiperazine-1-carboxylate (intermediate 11):

to a solution of intermediate 10(6.2g,14mmol) in N, N-dimethylformamide (20mL) was added (R) -4-Boc-2-methylpiperazine (5.7g,28mmol), the temperature was raised to 60 ℃ for reaction overnight, and the reaction was terminated by TLC. The reaction was added dropwise to 50mL of ice water, filtered, and the filter cake was dried under vacuum to give intermediate 11(5.7g, 75% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 545.10[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.78(s,1H),8.54(s,1H),8.40(s,1H),8.03(s,1H),7.47(q,J＝6.7Hz,2H),7.25(d,J＝8.7Hz,2H),4.43～4.30(m,1H),4.00(s,3H),3.91～3.73(m,3H),3.10(t,J＝12.6Hz,2H),1.43(s,9H),1.14(d,J＝6.7Hz,3H).

Preparation of 4- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] carbamoyl } - (R) -3-methylpiperazine (intermediate 12):

intermediate 11(5.5g,10.1mmol) was dissolved in a mixed solution of dichloromethane (40mL) and trifluoroacetic acid (20mL) and reacted at room temperature for 1h with TLC detection. And (3) distilling the solvent under reduced pressure, adding 60mL of water into the residue, adjusting the pH value to 8.0-9.0 by using a saturated sodium bicarbonate aqueous solution, separating the solution, extracting the water layer by using ethyl acetate, combining the organic layers, drying the organic layers by using anhydrous sodium sulfate, and distilling the solvent under reduced pressure to obtain an intermediate 12(4.2g, the yield is 94%), wherein the intermediate can be used for the next reaction without further purification. MS-ESI (M/z) 445.15[ M + H] ⁺ .

Intermediate 13: preparation of 4- { [4- ((3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl ] carbamoyl } - (R) -3-methylpiperazine

The preparation method refers to the synthesis of intermediate 12, except that 3-chloro-2-fluorophenol is substituted for 3-chloro-2-fluoroaniline.

MS-ESI(m/z):446.15[M+H] ⁺ . ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.78(s,1H),8.59(s,1H),8.07(s,1H),7.60(t,J＝8.0Hz,1H),7.53(t,J＝8.0Hz,1H),7.48(s,1H),7.37(t,J＝8.0Hz,1H),4.33～4.29(m,1H),4.08(s,3H),3.84～3.78(m,1H),3.20～3.13(m,1H),2.79(d,J＝12.0Hz,1H),2.66(d,J＝8.0Hz,1H),2.36～2.29(m,1H),2.07(d,J＝8.0Hz,1H),1.23(s,3H).

Intermediate 14: preparation of 4- { [4- ((3-chloro-2-fluorophenyl) amino) quinazolin-6-yl ] carbamoyl } - (R) -3-methylpiperazine

The preparation method was referenced to the synthesis of intermediate 12, except that 6-nitroquinazolin-4-one was substituted for 7-methoxy-6-nitroquinazolin-4-one.

MS-ESI(m/z):415.15[M+H] ⁺ . ¹ H-NMR(400MHz,DMSO-d ₆ )δ：9.85(s,1H),8.88(s,1H),8.47(s,1H),8.42(s,1H),7.83(d,J＝8.0Hz,1H),7.73(d,J＝8.0Hz,1H),7.54～7.44(m,2H),7.27(t,J＝8.0Hz,1H),4.46～4.37(m,1H),3.93(d,J＝12.0Hz,2H),3.82～3.7(m,2H),3.14～3.05(m,3H),1.14(d,J＝8.0Hz,3H).

Intermediate 17: preparation of 3- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] amino } - (R) -4- (2-methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione

Step a preparation of 3- { [ (4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] amino } -4-ethoxycyclobut-3-ene-1, 2-dione (intermediate 15):

intermediate 9(2.5g,7.8mmol) was added to ethanol (30mL), diethyl squarate (5.3g,31.4mmol) and zinc trifluoromethanesulfonate (8.6g,23.5mmol) were added and the reaction was allowed to react overnight at 90 ℃ with TLC detection complete. Concentration under reduced pressure, addition of water to the residue, beating, suction filtration, and vacuum drying of the filter cake gave intermediate 15(1.9g, 54.7% yield), which was used in the next reaction without further purification. MS-ESI (M/z) 443.10[ M + H] ⁺ .

Step b preparation of 3- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] amino } - (R) -4- (4-Boc-2-methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione (intermediate 16):

intermediate 15(1.8g,3.0mmol) was added to ethanol (30mL), and (R) -4-Boc-2-methylpiperazine (1.2g,6.0mmol) and zinc trifluoromethanesulfonate (3.3g,9.0mmol) were added and reacted at 90 ℃ overnight. The reaction was cooled to room temperature, filtered with suction, and the filtrate was concentrated under reduced pressure to remove the solvent by evaporation and purified by silica gel column chromatography (DCM: MeOH ═ 20:1) to give intermediate 16(0.65g, yield 26.8%). MS-ESI (M/z) 597.20[ M + H] ⁺ .

Step c preparation of 3- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] amino } - (R) -4- (2-methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione (intermediate 17):

intermediate 16(0.6g,1.0mmol) was added to a mixture of trifluoroacetic acid (5mL) and dichloromethane (10mL)The reaction solution is reacted for 1h at room temperature, and the reaction is finished by TLC detection. And (3) distilling the solvent under reduced pressure, adding 20mL of water into the residue, adjusting the pH value to 8.0-9.0 by using a saturated sodium bicarbonate aqueous solution, separating the solution, extracting the water layer by using ethyl acetate, combining the organic layers, drying the organic layers by using anhydrous sodium sulfate, and distilling the solvent under reduced pressure to obtain an intermediate 17(0.46g, the yield is 92.1%), wherein the intermediate can be used for the next reaction without further purification. MS-ESI (M/z) 497.15[ M + H] ⁺ .

Intermediate 18: preparation of 3- { [4- ((3-chloro-2-fluorophenyl) amino) -quinazolin-6-yl ] amino } - (R) -4- (2-methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione

The preparation method was referenced to the synthesis of intermediate 17, except that 6-nitroquinazolin-4-one was substituted for 7-methoxy-6-nitroquinazolin-4-one.

Example 1: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4- (cyclopropylmethyl) piperazine-1-carboxylate

To a solution of intermediate 6(200mg,0.45mmol) in dichloromethane (15mL) was added cyclopropylformaldehyde (47.24mg,0.67mmol), and the reaction was stirred at room temperature under nitrogen for 0.5 h. To the reaction system was added a mixture of sodium cyanoborohydride (113mg,1.8mmol), acetic acid (2mL) and methanol (10mL), and the mixture was stirred at room temperature for 2h, after which the reaction was terminated by TLC. Adding water into the reaction system, adjusting the pH value to 8.0-9.0 by using saturated sodium bicarbonate aqueous solution, separating, extracting by dichloromethane, combining organic layers, drying by anhydrous sodium sulfate, evaporating the solvent under reduced pressure, and separating and purifying by silica gel column chromatography (dichloromethane-methanol with the volume ratio of 20:1) to obtain the target product, namely, the example (197mg, yield 88%). MS-ESI (M/z) 500.2[ M + H] ⁺ 。 ¹ H-NMR(400MHz,DMSO-d6)δ:9.73(s,1H),8.46(s,1H),8.21(s,1H),7.49(q,J＝8.0Hz,2H),7.32(s,1H),7.27(t,J＝8.0Hz,1H),4.32(s,1H),3.94(s,3H),3.30～3.22(m,1H),2.99(d,J＝12.0Hz,1H),2.87(d,J＝12.0Hz,1H),2.21(d,J＝8.0Hz,2H),2.17(d,J＝16.0Hz,1H),2.03～1.93(m,1H),1.35(s,3H),0.90～0.79(m,1H),0.53～0.48(m,2H),0.13～0.09(m,2H).

Example 2: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4- (2-ethylbutyl) -piperazine-1-carboxylate

The synthesis was as in example 1, with 2-ethylbutyraldehyde replacing cyclopropylformaldehyde.

MS-ESI(m/z):530.30[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.73(s,1H),8.47(s,1H),8.22(s,1H),7.56～7.45(m,2H),7.33(s,1H),7.28(t,J＝7.7Hz,1H),4.41～4.20(m,1H),3.95(s,3H),3.86～3.64(m,1H),3.31～2.27(m,1H),2.85(d,J＝11.2Hz,1H),2.73(d,J＝11.4Hz,1H),2.17(dd,J＝12.3,7.6Hz,1H),2.10(dd,J＝12.1,6.6Hz,2H),2.00～1.92(m,1H),1.48～1.43(m,1H),1.40～1.28(m,7H),0.86(t,J＝7.4Hz,6H).

Example 3: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4- (2-methylpentyl) piperazine-1-carboxylate

The synthesis was as in example 1, with 2-methylpentanal replacing cyclopropylformaldehyde.

MS-ESI(m/z):530.30[M+H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.73(s,1H),8.47(s,1H),8.22(s,1H),7.54～7.46(m,2H),7.33(s,1H),7.28(t,J＝7.7Hz,1H),4.42～4.25(m,1H),3.95(s,3H),3.86～3.72(m,1H),3.28～3.15(m,1H),2.83(t,J＝12.0Hz,1H),2.73(t,J＝10.6Hz,1H),2.18～1.92(m,4H),1.67(s,1H),1.45～1.28(m,6H),1.13～1.01(m,1H),0.90～0.86(m,6H).

Example 4: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4- [4- (trifluoromethyl) benzyl ] piperazine-1-carboxylate

The synthesis was as in example 1, with 4-trifluoromethylbenzaldehyde replacing cyclopropylformaldehyde.

MS-ESI(m/z):604.25[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.73(s,1H),8.47(s,1H),8.22(s,1H),7.73(d,J＝8.1Hz,2H),7.60(d,J＝8.0Hz,2H),7.55～7.50(m,2H),7.34(s,1H),7.28(t,J＝8.1Hz,1H),4.48～4.06(m,2H),3.95(s,3H),3.91～3.77(m,1H),3.68(d,J＝14.0Hz,1H),3.58(d,J＝14.0Hz,1H),2.87(d,J＝11.3Hz,1H),2.69(d,J＝11.6Hz,1H),2.23(d,J＝10.8Hz,1H),2.10(t,J＝11.6Hz,1H),1.38(s,3H).

Example 5: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4-acryloylpiperazine-1-carboxylate

Under nitrogen atmosphere and at 0 ℃, triethylamine (91mg,0.9mmol) is slowly added dropwise into a dichloromethane solution (5mL) of intermediate 6(200mg,0.45mmol), after dropwise addition, a dichloromethane (1mL) solution of acryloyl chloride (41mg,0.45mmol) is slowly added dropwise at 0 ℃, after dropwise addition, the mixture is moved to room temperature overnight, and the reaction is detected to be complete by TLC. Water was added to the reaction system, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography to give the desired product, example (188mg, yield 84%). MS-ESI (M/z) 500.05[ M + H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.73(s,1H),8.45(s,1H),7.66(s,1H),7.52(s,1H),7.32(s,1H),7.16(d,J＝7.1Hz,2H),6.59(s,1H),6.39(d,J＝16.7Hz,1H),5.79(d,J＝10.4Hz,1H),4.75～4.43(m,2H),4.20～4.06(m,1H),3.94(s,3H),3.88～3.79(m,1H),3.60～3.46(m,1H),3.40～3.14(m,2H),2.95(s,1H),1.33(s,3H).

Example 6: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4-methyl- (R) -2-methylpiperazine-1-carboxamide

To a solution of intermediate 12(200mg,0.45mmol) in dichloromethane (10mL) was added paraformaldehyde (20mg,0.68mmol), and the reaction was stirred at room temperature under nitrogen for 0.5 h. To the reaction system was added a mixture of sodium cyanoborohydride (113mg,1.8mmol), acetic acid (2mL) and methanol (10mL), and the mixture was stirred at room temperature for 2h, after which the reaction was terminated by TLC. Water was added to the reaction system, the pH was adjusted to 8.0 to 9.0 with a saturated aqueous sodium bicarbonate solution, the mixture was separated, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the mixture was separated and purified by silica gel column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain the objective product, example (185mg, yield 90%). MS-ESI (M/z) 459.10[ M + H] ⁺ 。 ¹ H-NMR(400MHz,DMSO-d6)δ:9.76(s,1H),8.56(s,1H),8.40(s,1H),7.93(s,1H),7.47(q,J＝7.7Hz,2H),7.26(t,J＝9.2Hz,2H),4.00(s,3H),3.85(d,J＝13.2Hz,1H),3.20～3.06(m,2H),2.79(d,J＝10.3Hz,1H),2.67(d,J＝10.4Hz,1H),2.36～2.28(m,1H),2.19(s,3H),2.10～2.04(m,1H),1.27(s,3H)。

Example 7: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4- (cyclopropylmethyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis method refers to example 6, wherein cyclopropylformaldehyde replaces paraformaldehyde.

MS-ESI(m/z):499.15[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.78(s,1H),8.56(s,1H),8.39(s,1H),7.93(s,1H),7.47(q,J＝6.8Hz,2H),7.27(t,J＝9.2Hz,2H),4.37～4.29(m,1H),4.00(s,3H),3.86(d,J＝12.7Hz,1H),3.18～3.08(m,1H),2.98(d,J＝11.1Hz,1H),2.86(d,J＝11.2Hz,1H),2.20(d,J＝6.2Hz,2H),2.13(dd,J＝11.1,3.8Hz,1H),1.97～1.89(m,1H),1.27(d,J＝6.6Hz,3H),1.17(t,J＝7.1Hz,1H),0.57～0.41(m,2H),0.12～0.09(m,2H).

Example 8: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4- (2-ethylbutyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with 2-ethylbutyraldehyde replacing paraformaldehyde.

MS-ESI(m/z):529.25[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.78(s,1H),8.55(s,1H),8.39(s,1H),7.92(s,1H),7.47(q,J＝6.4Hz,2H),7.27(t,J＝9.3Hz,2H),4.34～4.28(m,1H),3.99(s,3H),3.85(d,J＝12.7Hz,1H),3.12(t,J＝12.2Hz,1H),2.82(d,J＝11.2Hz,1H),2.71(d,J＝11.1Hz,1H),2.19～2.09(m,2H),2.08～2.02(m,1H),1.94～1.86(m,1H),1.28～1.24(m,5H),0.85(d,J＝7.4Hz,6H).

Example 9: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4- (2-methylpentyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis procedure is as in example 6, with 2-methylpentanal replacing paraformaldehyde.

MS-ESI(m/z):529.25[M+H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.90(s,1H),8.65(s,1H),8.16(t,J＝7.5Hz,1H),7.97(s,1H),7.45(s,1H),7.37(s,1H),7.20(t,J＝8.0Hz,1H),7.13(t,J＝8.1Hz,1H),4.32～4.23(m,1H),4.08(s,3H),3.83(d,J＝12.7Hz,1H),3.44～3.30(m,1H),2.91(d,J＝11.1Hz,1H),2.77(t,J＝12.9Hz,1H),2.32～2.12(m,5H),1.42(d,J＝7.0Hz,5H),1.18～1.04(m,J＝8.0Hz,2H),0.95～0.92(m,3H),0.91～0.88(m,3H).

Example 10: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4- [4- (trifluoromethyl) benzyl ] - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with 4-trifluoromethylbenzaldehyde replacing paraformaldehyde.

MS-ESI(m/z):603.25[M+H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.99(s,1H),8.62(s,1H),7.84(s,1H),7.62(d,J＝7.8Hz,2H),7.54(s,1H),7.51(d,J＝10.1Hz,2H),7.33(t,J＝7.4Hz,1H),7.18(t,J＝8.1Hz,1H),4.36～4.24(m,1H),4.12(s,3H),3.87(d,J＝12.7Hz,1H),3.74～3.54(m,2H),3.46～3.34(m,1H),3.04～2.92(m,1H),2.75(d,J＝11.4Hz,1H),2.44～2.32(m,1H),2.04～1.98(m,1H),1.43(d,J＝6.6Hz,3H).

Example 11: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

Triethylamine (91mg,0.9mmol) was slowly added dropwise to a dichloromethane solution (5mL) of intermediate 12(200g,0.45mmol) under nitrogen at 0 ℃, after completion of the addition, a dichloromethane solution (1mL) of acryloyl chloride (41mg,0.45mmol) was slowly added dropwise at 0 ℃, after completion of the addition, the reaction was allowed to warm to room temperature overnight and the reaction was detected by TLC. Water was added to the reaction system, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the product was separated and purified by silica gel column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain the desired product, example (194mg, yield 87%). MS-ESI (M/z) 499.15[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.76(s,1H),8.53(s,1H),8.40(s,1H),8.07(s,1H),7.48(q,J＝7.3,6.8Hz,2H),7.31～7.20(m,2H),6.86(d,J＝13.3Hz,1H),6.18(d,J＝17.0Hz,1H),5.74(d,J＝12.5Hz,1H),4.30～4.14(m,1H),4.00(s,3H),3.94(d,J＝12.7Hz,1H),3.48～3.38(m,1H),3.27～3.04(m,3H),2.92～2.82(m,1H),1.13(d,J＝8.0Hz,3H).

Example 12: 4- [ (3-chloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

To a solution of intermediate 12(1g,2.2mmol) in acetonitrile (10mL) was added anhydrous potassium carbonate (0.62g,4.5mmol), and D was slowly added dropwise to the reaction system at 0 ℃ under a nitrogen atmosphere ₃ -tosylmethyl ester (0.43g,2.3mmol) in acetonitrile (2mL) and transferred to room temperature overnight with complete titration and TLC check reaction. Water was added to the reaction system, followed by extraction with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure to remove the solvent, and separated and purified by silica gel column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain the objective product, example (0.78g, yield 75%). MS-ESI (M/z) 462.15[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ9.79(s,1H),8.55(s,1H),8.39(s,1H),7.95(s,1H),7.47(t,J＝7.6Hz,2H),7.27(d,J＝9.8Hz,2H),4.35～4.29(m,1H),3.99(s,3H),3.87～3.81(m,1H),3.17～3.08(m,1H),2.78(d,J＝10.8Hz,1H),2.66(d,J＝11.2Hz,1H),2.06(d,J＝8.8Hz,1H),1.91～1.82(m,1H),1.24(d,J＝3.7Hz,3H).

Example 13: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4-methyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with intermediate 13 replacing intermediate 12.

MS-ESI(m/z):460.10[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.78(s,1H),8.59(s,1H),8.07(s,1H),7.60(t,J＝7.3Hz,1H),7.53(t,J＝7.5Hz,1H),7.48(s,1H),7.37(t,J＝8.2Hz,1H),4.33～4.29(m,1H),4.08(s,3H),3.84～3.78(m,1H),3.20～3.13(m,1H),2.79(d,J＝11.0Hz,1H),2.66(d,J＝10.4Hz,1H),2.36～2.29(m,1H),2.18(s,3H),2.07(d,J＝8.1Hz,1H),1.23(s,3H).

Example 14: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4- (cyclopropylmethyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with intermediate 13 replacing intermediate 12 and cyclopropylformaldehyde replacing paraformaldehyde.

MS-ESI(m/z):500.15[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.79(s,1H),8.59(s,1H),8.06(s,1H),7.60(t,J＝7.3Hz,1H),7.54(t,J＝7.6Hz,1H),7.48(s,1H),7.40～7.34(m,1H),4.37～4.27(m,1H),4.08(s,3H),3.83(d,J＝12.7Hz,1H),3.17(t,J＝11.7Hz,1H),2.99(d,J＝11.3Hz,1H),2.86(d,J＝11.2Hz,1H),2.20(d,J＝6.6Hz,2H),2.13(d,J＝15.2Hz,1H),1.99～1.91(m,1H),1.26(d,J＝11.2Hz,3H),0.88～0.79(m,1H),0.51～0.46(m,2H),0.16～0.11(m,2H).

Example 15: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4- (2-ethylbutyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, wherein intermediate 13 was substituted for intermediate 12, and 2-ethylbutanal was substituted for paraformaldehyde.

MS-ESI(m/z):530.25[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.79(s,1H),8.58(s,1H),8.02(s,1H),7.59(t,J＝7.0Hz,1H),7.55～7.50(m,1H),7.48(s,1H),7.37(t,J＝8.3Hz,1H),4.32～4.26(m,1H),4.08(s,3H),3.81(d,J＝12.6Hz,1H),3.20～3.11(m,1H),2.83(d,J＝11.2Hz,1H),2.71(d,J＝11.2Hz,1H),2.15(dd,J＝12.2,7.5Hz,1H),2.08(dd,J＝11.5,7.5Hz,2H),1.97～1.87(m,1H),1.45(q,J＝6.4Hz,1H),1.38～1.28(m,4H),1.25(d,J＝6.8Hz,3H),0.85(t,J＝8.0Hz,6H).

Example 16: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4- (2-methylpentyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis procedure is as in example 6, wherein intermediate 13 replaces intermediate 12, 2-methylpentanal instead of paraformaldehyde.

MS-ESI(m/z):530.20[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.79(s,1H),8.58(s,1H),8.02(s,1H),7.59(t,J＝8.2Hz,1H),7.53(t,J＝6.9Hz,1H),7.48(s,1H),7.37(t,J＝8.6Hz,1H),4.33～4.27(m,1H),4.08(s,3H),3.81(d,J＝12.7Hz,1H),3.21～3.11(m,1H),2.86～2.78(m,1H),2.74～2.66(m,1H),2.14～2.06(m,2H),2.04～1.91(m,2H),1.70～1.64(m,1H),1.43～1.32(m,2H),1.29～1.19(m,5H),0.90～0.86(m,6H).

Example 17: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4- [4- (trifluoromethyl) benzyl ] - (R) -2-methylpiperazine-1-carboxamide

The synthesis procedure is as in example 6, wherein intermediate 13 replaces intermediate 12, 4-trifluoromethylbenzaldehyde instead of paraformaldehyde.

MS-ESI(m/z):604.20[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.77(s,1H),8.59(s,1H),8.08(s,1H),7.72(d,J＝8.4Hz,2H),7.59(d,J＝7.9Hz,3H),7.48(s,1H),7.37(t,J＝7.4Hz,1H),4.36～4.29(m,1H),4.08(s,3H),3.85(d,J＝12.8Hz,1H),3.66(d,J＝14.0Hz,1H),3.56(d,J＝12.0Hz,1H),3.21(t,J＝12.3Hz,1H),2.84(d,J＝11.1Hz,1H),2.67(d,J＝10.0Hz,1H),2.20(d,J＝7.5Hz,1H),2.07(t,J＝10.8Hz,1H),1.28(d,J＝6.6Hz,3H).

Example 18: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4-deuterated methyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 12, with intermediate 14 replacing intermediate 13.

MS-ESI(m/z):463.15[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.78(s,1H),8.59(s,1H),8.05(s,1H),7.60(t,J＝7.6Hz,1H),7.53(t,J＝8.0Hz,1H),7.48(s,1H),7.37(t,J＝8.1Hz,1H),4.36–4.27(m,1H),4.09(s,3H),3.86–3.77(m,1H),3.21–3.12(m,1H),2.82–2.74(m,1H),2.66(d,J＝10.7Hz,1H),2.12–2.04(m,1H),1.92–1.84(m,1H),1.24(s,3H).

Example 19: 4- (3-chloro-2-fluorophenoxy) -7-methoxyquinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 11, with intermediate 13 replacing intermediate 12.

MS-ESI(m/z):500.15[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.75(s,1H),8.60(s,1H),8.20(s,1H),7.60(t,J＝6.7Hz,1H),7.54(t,J＝7.9Hz,1H),7.49(s,1H),7.37(t,J＝7.5Hz,1H),6.83(dd,J＝16.8,10.5Hz,1H),6.18(dd,J＝16.6,7.2Hz,1H),5.74(d,J＝12.7,1H),4.44–4.37(m,1H),4.09(s,3H),3.90(d,J＝12.0Hz,1H),3.43(d,J＝12.2Hz,1H),3.14(t,J＝12.1Hz,1H),3.18–3.10(m,1H),3.04(d,J＝12.7Hz,1H),2.87(t,J＝12.1Hz,1H),1.10(d,J＝6.8Hz,3H).

Example 20: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4-methyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with intermediate 14 replacing intermediate 12.

MS-ESI(m/z):429.05[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.83(s,1H),8.80(s,1H),8.48(s,1H),8.41(s,1H),7.83(d,J＝6.8Hz,1H),7.72(d,J＝9.0Hz,1H),7.49(dt,J＝14.5,7.5Hz,2H),7.27(t,J＝8.1Hz,1H),4.34(t,J＝5.1Hz,1H),3.91(d,J＝13.2Hz,1H),3.48–3.40(m,1H),3.17(d,J＝5.3Hz,1H),3.13–3.03(m,1H),2.80(d,J＝11.2Hz,1H),2.67(d,J＝10.7Hz,1H),2.19(s,3H),1.25(d,J＝6.6Hz,3H).

Example 21: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4- (cyclopropylmethyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, with intermediate 14 replacing intermediate 12 and cyclopropylformaldehyde replacing paraformaldehyde. .

MS-ESI(m/z):469.10[M+H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.68(s,1H),8.41(s,1H),8.30(s,1H),7.81(d,J＝8.9Hz,1H),7.59(d,J＝8.9Hz,1H),7.16(q,J＝7.8Hz,2H),7.02(s,1H),4.45–4.25(m,1H),3.91(d,J＝13.0Hz,1H),3.45–3.31(m,1H),3.09(d,J＝11.4Hz,1H),2.99(d,J＝11.6Hz,1H),2.42–2.24(m,4H),1.26(s,3H),0.89(s,1H),0.63-0.54(m,2H),0.15-0.12(m,2H).

Example 22: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4- (2-ethylbutyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, wherein intermediate 14 replaced intermediate 12, 2-ethylbutyraldehyde instead of paraformaldehyde.

MS-ESI(m/z):499.30[M+H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.66(s,1H),8.43(s,1H),8.22(s,1H),7.79(d,J＝8.9Hz,1H),7.59(d,J＝8.7Hz,1H),7.19–7.09(m,2H),7.05(s,1H),4.36–4.26(m,1H),3.87(d,J＝12.9Hz,1H),3.31(d,J＝12.8Hz,1H),2.87(d,J＝11.3Hz,1H),2.73(d,J＝11.5Hz,1H),2.30–2.20(m,2H),2.18–2.14(m,1H),2.08–2.02(m,1H),1.38(s,3H),1.35–1.24(m,5H),0.88(t,J＝7.4Hz,6H).

Example 23: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4- (2-methylpentyl) - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, wherein intermediate 14 replaced intermediate 12, 2-methylpentanal instead of paraformaldehyde.

MS-ESI(m/z):499.20[M+H] ⁺ ； ¹ H-NMR(400MHz,CDCl ₃ -d ₆ )δ:8.66(s,1H),8.46(s,1H),8.22(s,1H),7.83(d,J＝8.9Hz,1H),7.66(s,1H),7.21～7.09(m,3H),4.42～4.31(m,1H),3.97～3.87(s,1H),3.44～3.34(m,1H),2.99～2.89(m,1H),2.84～2.75(m,1H),2.34～2.18(m,4H),1.38～1.35(m,1H),1.29～1.22(m,5H),1.18～1.00(m,2H),0.94～0.88(m,6H).

Example 24: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4- [4- (trifluoromethyl) benzyl ] - (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 6, wherein intermediate 14 was substituted for intermediate 12, 4-trifluoromethylbenzaldehyde instead of paraformaldehyde.

MS-ESI(m/z):573.30[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:8.65(s,1H),8.49(s,1H),8.15(s,1H),7.85(d,J＝8.9Hz,1H),7.66(d,J＝9.1Hz,1H),7.60(d,J＝8.0Hz,2H),7.49(d,J＝7.8Hz,2H),7.16(dq,J＝16.4,8.2Hz,4H),4.39～4.30(m,1H),3.94(d,J＝12.8Hz,1H),3.64(d,J＝13.7Hz,1H),3.52(d,J＝13.6Hz,1H),3.36(t,J＝12.3Hz,1H),2.90(d,J＝11.2Hz,2H),2.70(d,J＝11.4Hz,2H),2.31(d,J＝11.3Hz,2H),2.19(t,J＝11.0Hz,2H),1.26(s,3H).

Example 25: 4- [ (3-chloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was as in example 11, with intermediate 14 replacing intermediate 12.

MS-ESI(m/z):469.10[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.87(s,1H),8.93(s,1H),8.48(s,1H),8.42(s,1H),7.84(d,J＝9.1Hz,1H),7.74(d,J＝9.0Hz,1H),7.49(q,J＝8.0Hz,2H),7.27(t,J＝8.1Hz,1H),6.96～6.79(m,1H),6.23～6.13(m,1H),5.74(d,J＝7.9Hz,1H),4.50～4.42(m,1H),4.00～3.94(m,1H),3.43～3.37(m,1H),3.24～3.13(m,1H),3.12～3.02(m,1H),3.00(d,J＝13.9Hz,1H),2.82(t,J＝12.4Hz,1H),1.23(s,3H).

Example 26: 3- { [4- ((3-chloro-2-fluorophenyl) amino) -7-methoxyquinazolin-6-yl ] amino } -4- (4-methyl-2- (R) -methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione

To a solution of intermediate 17(200mg,0.40mmol) in dichloromethane (10mL) was added paraformaldehyde (18mg,0.60mmol), and the reaction was stirred at room temperature under nitrogen for 0.5 h. To the reaction was added a mixture of sodium cyanoborohydride (101mg,1.6mmol), acetic acid (2mL) and methanol (10mL), and the mixture was stirred at room temperature for 2h, after which the reaction was terminated by TLC. Water was added to the reaction system, the pH was adjusted to 8.0 to 9.0 with a saturated aqueous sodium bicarbonate solution, the mixture was separated, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the mixture was separated and purified by silica gel column chromatography (dichloromethane-methanol, volume ratio 20:1) to obtain the objective product, example (179mg, yield 87%). MS-ESI (M/z) 511.20[ M + H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.60(s,1H),8.42(s,1H),7.89(s,1H),7.49(q,J＝7.0,5.8Hz,2H),7.33～7.18(m,2H),3.99(s,3H),3.67(s,1H),3.44(t,J＝12.4Hz,1H),2.74(d,J＝11.3Hz,1H),2.66(d,J＝11.2Hz,1H),2.16(s,3H),1.98(d,J＝12.1Hz,1H),1.90(d,J＝11.2Hz,1H),1.35(d,J＝6.7Hz,3H).

Example 27: 3- { [4- ((3-chloro-2-fluorophenyl) amino) -quinazolin-6-yl ] amino } -4- (4-methyl-2- (R) -methylpiperazin-1-yl) cyclobut-3-ene-1, 2-dione

The synthesis was as in example 26, with intermediate 18 replacing intermediate 17.

MS-ESI(m/z):481.20[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.75(s,1H),8.45(s,1H),7.87(s,1H),7.81–7.65(m,3H),7.55–7.47(m,2H),7.29(t,J＝8.1Hz,1H),4.14(dd,J＝5.5,3.3Hz,1H),3.49(d,J＝13.0Hz,2H),2.76(d,J＝12.0Hz,1H),2.67(d,J＝4.6Hz,1H),2.33(s,1H),2.21(d,J＝4.2Hz,1H),2.17(s,3H),1.36(d,J＝6.8Hz,3H).

Example 28: 4- [ (3, 4-dichloro-2-fluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4-acryloylpiperazine-1-carboxylate

The synthesis procedure is as in example 5, with 3, 4-dichloro-2-fluoroaniline replacing 3-chloro-2-fluoroaniline.

MS-ESI(m/z):534.20[M+H] ⁺ ； ¹ H NMR(400MHz,Chloroform-d)δ8.70(s,1H),8.52(t,J＝8.6Hz,1H),7.65(s,1H),7.35(s,3H),6.60(s,1H),6.43–6.39(m,1H),5.83–5.79(m,1H),4.74～4.41(m,2H),4.19～4.08(m,1H),3.99(s,3H),3.88～3.79(m,1H),3.61～3.46(m,1H),3.41～3.14(m,2H),2.94(s,1H),1.62(s,3H).

Example 29: 4- [ (3-chloro-2, 4-difluorophenyl) amino ] -7-methoxyquinazolin-6-yl (R) -2-methyl-4-acryloylpiperazine-1-carboxylate

The synthesis was as in example 5, wherein 3-chloro-2, 4-difluoroaniline was substituted for 3-chloro-2-fluoroaniline.

MS-ESI(m/z):518.10[M+H] ⁺ ； ¹ H NMR(400MHz,Chloroform-d)δ8.69(s,1H),8.51(t,J＝8.8Hz,1H),7.65(s,1H),7.45-7.31(m,3H),6.61(s,1H),6.43–6.38(m,1H),5.83–5.76(m,1H),4.74～4.41(m,2H),4.19～4.08(m,1H),3.99(s,3H),3.89～3.79(m,1H),3.63～3.46(m,1H),3.41～3.13(m,2H),2.94(s,1H),1.61(s,3H).

Example 30: 4- [ (3, 4-dichloro-2-fluorophenyl) amino ] -quinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis procedure is as in example 25, with 3, 4-dichloro-2-fluoroaniline replacing 3-chloro-2-fluoroaniline.

MS-ESI(m/z):503.10[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.87(s,1H),8.94(s,1H),8.53(s,1H),8.45(s,1H),7.88(d,J＝9.1Hz,1H),7.74(d,J＝9.0Hz,1H),7.51(q,J＝8.0Hz,1H),7.27(t,J＝8.1Hz,1H),6.96～6.79(m,1H),6.23～6.12(m,1H),5.73(d,J＝7.9Hz,1H),4.50～4.42(m,1H),4.00～3.94(m,1H),3.43～3.37(m,1H),3.24～3.13(m,1H),3.12～3.02(m,1H),3.00(d,J＝13.9Hz,1H),2.82(t,J＝12.4Hz,1H),1.23(s,3H).

Example 31: 4- [ (3-chloro-2, 4-difluorophenyl) amino ] -quinazolin-6-yl-4-acryloyl- (R) -2-methylpiperazine-1-carboxamide

The synthesis was carried out according to example 25, in which 3-chloro-2, 4-difluoroaniline was used instead of 3-chloro-2-fluoroaniline.

MS-ESI(m/z):487.20[M+H] ⁺ ； ¹ H-NMR(400MHz,DMSO-d ₆ )δ:9.86(s,1H),8.93(s,1H),8.52(s,1H),8.43(s,1H),7.92-7.74(m,2H),7.51(q,J＝8.0Hz,1H),7.27(t,J＝8.1Hz,1H),6.93～6.78(m,1H),6.26～6.16(m,1H),5.72(d,J＝7.9Hz,1H),4.51～4.42(m,1H),4.00～3.92(m,1H),3.43～3.35(m,1H),3.26～3.15(m,1H),3.12～3.02(m,1H),3.00(d,J＝13.9Hz,1H),2.82(t,J＝12.4Hz,1H),1.23(s,3H).

Example 32: biological activity assay

NCI-H3255(L858R) cells were maintained in BEBM medium supplemented with BEGM Bulletkit (CC-4175) containing 10% Fetal Bovine Serum (FBS) (Gibco). PC-9 (exon 19 deleted for EGFR) cells were maintained in RPMI1640 (Gibco) containing 10% fetal bovine serum. NCI-H838(EGFR wild-type) cells were maintained in RPMI1640 (Gibco) containing 10% fetal bovine serum.

All cells were allowed to have 5% CO at 37 deg.C ₂ Grown in a humidified incubator. Cellular phosphorylation of endogenous p-EGFR in cell lysates was measured according to the protocol described in the Phospho-EGF Receptor (Tyr1068) Sandwich ELISA Kit (phosphorylation-EGF Receptor (Tyr1068) Sandwich ELISA Kit).

100 μ L of cells (32000 cells/well) were seeded in RPMI1640 + 1% fetal bovine serum in 96-well cell culture plates and 5% CO at 37 deg.C ₂ Incubate overnight. Using Tecan, cells were dosed with compounds serially diluted in 100% DMSO. After addition of these compounds, the cell plates were incubated for an additional 4H, (for NCI-H838: rhEGF was added to the cell plates at a final concentration of 100ng/mL rhEGF for 5 minutes of stimulation), then medium was withdrawn and 110. mu.L of IP lysis buffer was added to each well (IP lysis buffer: 1: 100 phosphatase inhibitor cocktail to IP lysis buffer 2)&3. 1: 100 protease inhibitor cocktail 2). The plates were placed at 4 ℃ while spinning at 300rpm for 0.5-1 hour. 100 μ L/well of cell lysate was transferred to coated plates (cell signaling kit) and incubated overnight at 4 ℃ while spinning at 300 rpm. The plates were brought from 4 ℃ to 37 ℃ while spinning at 300rpm for 1 hour. After aspiration and washing of the plates with 1-fold wash buffer, 100 μ L of detection antibody was added to each well. The plates were sealed with tape and incubated at 37 ℃ for 2 hours while spinning at 300 rpm. After aspiration and washing of the plates with 1-fold washing buffer, 100. mu.L of HRP-labeled secondary antibody (cell signaling kit) was added to each well. The plate was sealed with tape and incubated at 37 ℃ for 1 hour while spinning at 300 rpm. After aspiration and washing of the plates with 1-fold wash buffer, 100 μ l of TMB substrate (cell signaling kit) was added to each well. The plate was sealed with tape and incubated at 37 ℃ for 30 minutes while spinning at 300 rpm. To these plates 100 μ L of stop solution (cell signaling kit) was added and the absorbance read at 450nm over 30 minutes on a SpectraMax M5e microplate reader. The data obtained with each compound was exported to a suitable software package (e.g., H-BASE) for curve-fitting analysis, from which data the IC was determined ₅₀ The value is obtained.

The activity of compounds in selectively inhibiting EGFR exon 20 insertion mutations can be assessed using the murine progenitor B cell line Ba/F3 cells that have been transduced with EGFR exon 20 insertion. The expression vector pLVX-IRES puro (Clontech) encoding human EGFR exon 20 insertion a763_ Y764insFQEA was transfected into HEK293 cells by the trans lentiviral ORF assembly system (Thermo Scientific) to generate a virus encoding EGFR exon 20 insertion. Infection by EGFR exon 20 virus was maintained in a medium supplemented with 10% fetal bovine serum, 200. mu. M L-glutamine/200. mu.g/mL penicillin/200. mu.g/mL streptomycin (Life Technology), and 10ng/mL IL-3 (R)&D system) in RPMI1640 medium and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing an EGFR exon 20 insertion (designated Ba/F3-EGFR-A763_ Y764insFQEA) can be propagated in the absence of IL-3. Method for determining the antiproliferative activity of a compound: BaF3-EGFR exon 20 cells (A763_ Y764insFQEA) (2500 cells/well) seeded in 96-well plates were treated with test compounds (dissolved in DMSO) at a range of concentrations (4-fold dilution, maximum concentration: 10,000 nM). Incubate at 37 ℃ in 5% CO ₂ Plates were incubated for 72 hours and passed

Aqueous single solution cell proliferation assay (Promega) to indirectly measure the number of viable cells in each well. This assay is an enzyme used to convert blue formazan derivatives by detecting tetrazolium saltsColorimetric methods for determining the number of viable cells by measuring the metabolic activity of viable cells. Reagents (20 μ L) were added to each well and the plates were returned to the incubator for 2 hours. The absorbance in each well was then measured at 490nm using an Envision plate reader (PerkinElmer). IC was calculated by determining the concentration of compound required to reduce the MTS signal by 50% compared to DMSO control in a best-fit curve using Microsoft XLFit software or Accelrys Pipeline Pilot ₅₀ The value is obtained.

Data (nM) for the activity assays for the examples of the invention and the reference compounds are shown in table 1:

TABLE 1 Activity measurement data (nM) of the compounds obtained in the examples

Where n is the number of experimental replicates.

This shows that examples 1, 5, 6, 7, 11, 12, 25, 28, 29, 30, 31 have a potency superior to AZD3759, and that examples 1, 5, 6, 7, 11, 12, 25, 28, 29, 30, 31 are selective over AZD3759, have the potential to lower the therapeutic dose and reduce adverse effects.

Example 33: blood brain barrier penetration assay

According to the literature (Journal of Medicinal Chemistry,2013,56(1):2-12) K _{p, uu brain} And K _{p，uu CSF} Both are the main parameters measured and optimized during CNS drug discovery. Relationship K between the concentration of unbound drug in brain and blood _p，uu Brain prediction of drug on brain Leptomeningeal Metastasis (LM) caused by metastatic spread of cancer to leptomeningeal, metastatic tumor caused central nervous system dysfunction. K is _{p，uu CSF} Indicating the distribution of the drug in the CSF compared to the distribution of the drug in the blood,it drives the drug response during leptomeningeal transfer therapy.

In vitro blood and brain binding assays were performed on HT dialysis plates with semipermeable membranes. Diluted blood (with DPBS 1:1, pH7.4) and brain homogenate (with DPBS 1:3, pH7.4) were spiked with 5 μ M test compound (in triplicate) and dialyzed in 150 μ L of an equal volume of 100mM PBS buffer (pH7.4) at 37 ℃ for 4 hours in a slowly rotating plate. At the end of the incubation, 50 μ L aliquots from the receiver side and 5 μ L aliquots from the donor compartment were taken. mu.L of the sample was further diluted with 45. mu.L of blank blood or brain homogenate. Paired samples were matrix matched with buffer or blank blood/brain homogenates and mixed for 2min and then precipitated with 150 μ L cold acetonitrile with 100ng/mL tolbutamide as an internal standard. After centrifugation at 4000rpm for 20min, the supernatant was diluted with 0.1% aqueous formic acid and analyzed for LC/MS. The unbound fraction (fu) of the test compound in the brain homogenate and diluted blood was calculated by the ratio of the buffer side reaction to the brain homogenate/blood side reaction, and the following equation f was used _u，bl (f _u，br )＝(1/D)/[(1/fu-1)+1/D)]Calculating the unbound fraction of test compound in undiluted blood and tissue from the measured fu in the homogenate and diluted blood (f) _u，bl And f _u，br ). D is the dilution factor.

The short term oral absorption (SOA) model is an in vivo screening model for identifying brain penetration of a compound. Six male wistar rats were dosed orally with 2mg/kg of compound in 1% methylcellulose. After 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours and 7 hours of administration, cerebrospinal fluid (CSF) was collected from the cisterna magna and blood samples (> 60 μ L/time point/each site) were collected via cardiac puncture into individual EDTA coagulation tubes and then immediately diluted with 3 volumes of water. Brain tissue was harvested and homogenized in 3 volumes of phosphate buffered saline (pH 7.4). All samples were stored at about-70 ℃ prior to LC/MS analysis.

Standards were prepared by labeling blank blood, brain homogenate, and artificial CSF from 0.2ng/mL to 500 ng/mL. Homogenized brain tissue along with blood samples were precipitated by adding 3 volumes of cold acetonitrile containing an internal standard (40ng/mL dexamethasone and 40ng/mL diclofenac), and 10 μ L of CSF sample was precipitated with 100 μ L of cold acetonitrile containing an internal standard. After vortexing for 2min and centrifugation at 14,000rpm for 5min, the supernatant was analyzed by LC/MS/MS. Two sets of standard curves were run at the beginning and end of each batch from the analysis of the blood samples. Standard curves were made for brain and CSF samples, along with the test samples.

Following oral administration, the AUC brain/AUC blood measurements in rodents are expressed as the brain/blood ratio (K) _p Brain) total brain levels. The free fraction of the test compound in the biological matrix is determined by an in vitro blood and brain binding assay. K is calculated by the following equation _{p, uu brain} And K _{p，uu CSF} ∶K _{p, uu brain} ＝AUC _Brain /AUC _{Blood, blood-enriching agent and method for producing the same} ×(f _{u, brain} /f _{u. blood} ) And K _{p，uu CSF} ＝AUC _CSF /(AUC _{Blood, blood-enriching agent and method for producing the same} ×f _{u. blood} )。

The data determined for the examples according to the invention and for AZD3759 are shown in the table below:

TABLE 2 examples of the invention and experimental data on brain barrier permeability of AZD3759

Compound (I)	K p, uu brain	K p，uu CSF
			1	1.65	1.68
5	1.31	1.36
			6	1.33	1.35
7	1.52	1.57
			11	1.30	1.33
12	1.43	1.46
			25	1.33	1.38
28	1.31	1.35
			29	1.30	1.35
30	1.32	1.36
			AZD3759	1.31	1.35

Examples 1, 7, 12 of the invention have unexpectedly superior brain barrier permeability characteristics when compared to AZD3759, and examples 5, 6, 11, 25 exhibit brain barrier permeability characteristics comparable to AZD3759 with superior potency and selectivity.

Example 34: evaluation of Compound stability Using human liver microsomes

Determination of liver microsomal enzyme stability of the example compounds:

measurement System: the metabolic stability of the compound of the present invention was tested using 1mM NADPH for liver microparticles mixed in men and women. The samples were analyzed using a mass spectrometer. HRMS was used to determine peak area response ratios (peak area corresponding to test compound or control divided by peak area of the analytical internal standard) without running a standard curve. In order to detect all possible metabolites, HRMS scans were performed at the appropriate m/z range.

The measurement conditions were as follows: the assay was performed with one incubation (N ═ 1). Test compounds were incubated at 37 ℃ in buffer containing 0.5 mg/ml liver microsomal protein. Reactions were initiated by addition of cofactors and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours, positive controls (5 μ M testosterone) were incubated in parallel and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours.

And (3) measuring quality control: the control compound testosterone was performed in parallel to confirm the enzymatic activity of the (liver) microsomes. After the final time point, the addition of NADPH to the reaction mixture was confirmed using fluorimetry. The T1/2 of the control met acceptable internal standards.

The analysis method comprises the following steps:

liquid chromatography column: thermo BDS Hypersil c 1830 x2.0mm, 3 μm, with guard column m.p., buffer: 25mM formic acid buffered solution, pH 3.5;

aqueous phase (a): 90% water, 10% buffer; organic phase (B): 90% acetonitrile, 10% buffer; flow rate: 300 microliters/min autosampler: injection volume was 5 microliters. See table 3 for gradient program.

TABLE 3 gradient program

Time (minutes)	％A	％B
			0.0	100	0
1.5	0	100
			2.0	0	100
2.1	100	0
			3.5	100	0

By using human liver microsomes, examples 1, 7, 12, 25, 28, 30 as described in the present invention show a metabolic half-life of more than 24 hours, significantly more than the 20 hour metabolic half-life of AZD 3759. The relatively long metabolic half-life allows them the potential to reduce therapeutic doses and extend the time interval between administrations.

Claims (9)

1. A compound having the general formula (I) or a pharmaceutically acceptable salt thereof,

the following compounds are specifically selected:

2. the compound of claim 1, wherein said pharmaceutically acceptable salt is an inorganic salt or an organic salt; wherein the inorganic salt is selected from any one or more of the following: hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from any one or more of: formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.

3. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

4. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a disease mediated by an EGFR-activating or drug-resistant mutant in a mammal.

5. The use of claim 4, wherein the EGFR-activating or drug-resistant mutant-mediated disease is non-small cell lung cancer.

6. The use of claim 4, wherein the EGFR-activating or drug-resistant mutant-mediated disease is metastatic non-small cell lung cancer.

7. An anti-tumor drug, comprising: a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

8. The antitumor agent as claimed in claim 7, further comprising any one of the following antitumor components:

(i) antineoplastic drugs acting on the DNA structure;

(ii) antineoplastic agents that affect nucleic acid synthesis;

(iii) anti-tumor drugs that affect nucleic acid transcription;

(iv) tubulin synthesized antineoplastic drugs;

(v) inhibitors of cellular signaling pathways;

(vi) an anti-tumor monoclonal antibody.

9. The antitumor agent as claimed in claim 8, wherein said cell signaling pathway inhibitor is an epidermal growth factor receptor inhibitor.