WO2019228330A1 - Substituted benzo[d]imidazole compound and pharmaceutical composition thereof - Google Patents

Abstract

The present invention provides a substituted benzo[d]imidazole compound and a pharmaceutical composition thereof, and uses thereof; the benzo[d]imidazole compound is a compound represented by the formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer, or isotopic variant of the described compound. The compound and composition of the present invention can be used for methods for treating illnesses related to cancer caused by EGFR (comprising cancer caused by EGFR mutations, for example, cancer having a T790M mutation, an L858R mutation, and a L858R/T790M double mutation).

Description

Substituted benzo [d] imidazole compounds and pharmaceutical compositions thereof Technical field

The invention belongs to the technical field of medicine, and particularly relates to a substituted benzo [d] imidazole compound, a pharmaceutical composition containing the compound, and use thereof. More specifically, the present invention relates to certain deuterated N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5-((4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [d] imidazol-6-yl) pyrimidin-2-yl) amino) phenyl) acrylamide, these deuterated compounds and The composition can be used to treat EGFR-related diseases, and these deuterium-substituted compounds have more excellent pharmacokinetic properties.

Background technique

The epidermal growth factor receptor (ie, EGFR, ErbB-1 or HER1) is a member of the ErbB receptor family. The ErbB receptor family includes four closely related members of the receptor tyrosine kinase: EGFR (ErbB-1), Her2 / c-neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). EGFR is a cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Mutations that affect EGFR expression or activity may cause cancer. EGFR has been reported to be dysregulated in most solid tumors such as lung, breast, and brain tumors. It is estimated that 30% of epithelial cancers are associated with mutations, amplifications or disorders of EGFR or family members.

Treatment methods based on the inhibition of EGFR by antibody drugs or small molecule inhibitor drugs such as gefitinib and erlotinib have been developed. In the case of non-small cell lung cancer (NSCLC), gefitinib and erlotinib are beneficial for 10% to 40% of patients. However, after a period of treatment, acquired resistance to gefitinib or erlotinib has become a major clinical problem. Studies have confirmed that one of the main causes of drug resistance is due to a new mutation in T790M, which is the "gatekeeper" of EGFR. Researchers then developed inhibitors targeting T790M, such as BIBW2992, and showed advantages in clinical trials. However, these inhibitors targeting the T790M mutation of EGFR also have considerable inhibitory activity against wild-type EGFR, which results in serious toxic side effects that limit its clinical application. Therefore, it is necessary to further develop more effective types of selective inhibitors of EGFR that target only mutant rather than wild type.

WO2018019204 discloses a compound T having the following structure, and the chemical name is N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5-((4- ( 4-fluoro-1-isopropyl-2-methyl-1H-benzo [d] imidazol-6-yl) pyrimidin-2-yl) amino) phenyl) acrylamide, compound T not only effectively inhibits the T790M mutation Moreover, it is highly selective for the T790M mutation compared to the wild type.

It is known that poor absorption, distribution, metabolism, and / or excretion (ADME) properties are the main reasons for the failure of clinical trials for many drug candidates. Many drugs currently on the market also limit their scope of application due to poor ADME properties. The rapid metabolism of drugs will cause many drugs that could be highly effective in treating diseases to be difficult to make because they are metabolized from the body too quickly. Although frequent or high-dose medication may solve the problem of rapid drug removal, this method will bring problems such as poor patient compliance, side effects caused by high-dose medication, and rising treatment costs. In addition, rapidly metabolizing drugs may expose patients to adverse toxic or reactive metabolites.

Therefore, it is still a challenging task to find new compounds that have the ability to treat EGFR-related diseases, have good oral bioavailability, and are druggable. Therefore, there remains a need in the art to develop compounds that have selective inhibitory activity and / or better pharmacodynamics / pharmacokinetics of mutant EGFR-mediated diseases suitable for use as therapeutic agents, and the present invention provides such compounds.

Summary of invention

In view of the above technical problems, the present invention discloses a new deuterium-substituted benzo [d] imidazole compound, as well as a composition and use thereof, which has better EGFR kinase inhibitory activity, and is resistant to drug-resistant mutations T790M, L858R, and Both of them are highly selective, have lower side effects, and better pharmacokinetic properties, and can be used to treat, prevent, and alleviate EGFR kinase-mediated diseases.

In this regard, the present invention adopts the following technical solutions:

In a first aspect of the invention, a compound of formula (I) is provided:

among them,

Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , Y ₈ and Y ₉ are each independently selected from hydrogen, deuterium, halogen, or trifluoromethyl;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from hydrogen or deuterium;

X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D;

The additional condition is that the above compounds contain at least one deuterium atom;

Or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, polymorph, stereoisomer or isotope variant thereof.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient. In a specific embodiment, a compound of the invention is provided in the pharmaceutical composition in an effective amount. In a specific embodiment, a compound of the invention is provided in a therapeutically effective amount. In a specific embodiment, a compound of the invention is provided in a prophylactically effective amount.

In another aspect, the present invention provides a method for preparing a pharmaceutical composition as described above, comprising the steps of: mixing a pharmaceutically acceptable excipient with a compound of the present invention to form a pharmaceutical composition.

In another aspect, the present invention provides treatment of EGFR-caused cancers (including cancers caused by EGFR mutations, eg, cancers with T790M mutation, L858R mutation, and L858R / T790M double mutation) -related disorders in a subject in need thereof. A method of: administering to a subject an effective amount of a compound of the invention. In a specific embodiment, the cancer caused by the EGFR is selected from the group consisting of: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, Gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma, etc. In specific embodiments, the compound is administered orally, subcutaneously, intravenously, or intramuscularly. In a specific embodiment, the compound is administered chronically.

Other objects and advantages of the invention will be apparent to those skilled in the art from the following detailed description, examples and claims.

definition

As used herein, unless otherwise specified, "deuterated" refers to the replacement of one or more hydrogens in a compound or group with deuterium; deuteration may be mono-, di-, poly- or fully substituted. The terms "one or more deuterated" and "one or more deuterated" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" refers to a compound containing a deuterium atomic proportion not higher than the natural deuterium isotope content (0.015%).

The term "pharmaceutically acceptable salt" means, within the scope of sound medical judgment, suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergies, etc., and with reasonable benefits / dangers Proportion of those salts. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., Pharmaceutically acceptable salts as described in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the invention include salts derived from suitable inorganic and organic acids and bases.

The compounds of the invention may be in amorphous or crystalline form. In addition, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "crystalline form" refers to different arrangements of chemical drug molecules, which generally appear as the existing form of the drug substance in a solid state. A drug can exist in multiple crystalline substance states, and different crystal forms of the same drug may have different dissolution and absorption in the body, which will affect the dissolution and release of the preparation.

The term "crystalline form" refers to different arrangements of chemical drug molecules, which generally appear as the existing form of the drug substance in a solid state. A drug can exist in multiple crystalline substance states, and different crystal forms of the same drug may have different dissolution and absorption in the body, which will affect the dissolution and release of the preparation.

As used herein, the term "subject" includes, but is not limited to: a human (ie, a male or female of any age group, for example, a pediatric subject (eg, infant, child, adolescent) or an adult subject (eg, Young adults, middle-aged adults or older adults)) and / or non-human animals, for example, mammals, for example, primates (for example, cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses , Sheep, goats, rodents, cats and / or dogs. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human animal.

"Disease", "disorder" and "disorder" are used interchangeably herein.

As used herein, unless otherwise stated, the term "treatment" includes the effect of a subject having a specific disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or delays or slows the disease, disorder, or condition. Or development of a condition ("therapeutic treatment"), and also includes effects that occur before a subject begins to suffer from a particular disease, disorder, or disease ("prophylactic treatment").

Generally, an "effective amount" of a compound refers to an amount sufficient to elicit a biological response of interest. As will be understood by one of ordinary skill in the art, the effective amount of a compound of the present invention can vary depending on factors such as the biological objective, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the age of the subject. Health conditions and symptoms. Effective amounts include therapeutically and prophylactically effective amounts.

Unless stated otherwise, a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit during the treatment of a disease, disorder, or condition, or one or more of which is associated with the disease, disorder, or condition. Symptoms are delayed or minimized. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent used alone or in combination with other therapies that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves the overall treatment, reduces or avoids the symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of other therapeutic agents.

Unless otherwise stated, a "prophylactically effective amount" of a compound used herein is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent a disease , The number of recurrences of a disorder or condition. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent used alone or in combination with other agents that provides a preventative benefit in the prevention of a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prevention, or an amount that enhances the preventive efficacy of other preventive agents.

"Combination" and related terms refer to the simultaneous or sequential administration of a therapeutic agent of the invention. For example, a compound of the invention can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or simultaneously in a single unit dosage form with another therapeutic agent.

Detailed ways

Compound

The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof:

among them,

Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , Y ₈ and Y ₉ are each independently selected from hydrogen, deuterium, halogen, or trifluoromethyl;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from hydrogen or deuterium;

X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D;

The additional condition is that the above compound contains at least one deuterium atom.

As a preferred embodiment of the present invention, the deuterium isotope content of deuterium at the deuterated position is at least 0.015% greater than the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably Ground is greater than 95%, and more preferably greater than 99%.

In a specific _{embodiment, "Y 1, Y 2,} Y 3, Y 4, Y 5, Y 6, Y 7, Y 8 and Y ₉ are each independently selected from hydrogen, deuterium, halogen or trifluoromethyl" comprising Y _{1 is} selected from hydrogen, deuterium, halogen, or trifluoromethyl, Y _{2 is} selected from hydrogen, deuterium, halogen, or trifluoromethyl, Y _{3 is} selected from hydrogen, deuterium, halogen, or trifluoromethyl, and so on, until Y _{9 is} selected from the group consisting of hydrogen, deuterium, halogen or trifluoromethyl. More specifically, Y ₁ is hydrogen, Y ₁ is deuterium, Y ₁ is halogen (F, Cl, Br, or I) or Y ₁ is trifluoromethyl, Y ₂ is hydrogen, Y ₂ is deuterium, and Y ₂ is Halogen (F, Cl, Br or I) or Y ₂ is trifluoromethyl, Y ₃ is hydrogen, Y ₃ is deuterium, Y ₃ is halogen (F, Cl, Br or I) or Y ₃ is trifluoromethyl And so on, until Y ₉ is hydrogen, Y ₉ is deuterium, Y ₉ is halogen (F, Cl, Br or I) or Y ₉ is trifluoromethyl.

In another specific _{embodiment, "R 1, R 2,} R 3, R 4 and R ₅ are each independently selected from hydrogen or deuterium" includes R ₁ is selected hydrogen or deuterium, R ₂ is selected from hydrogen or deuterium, R _{3 is} selected from hydrogen or deuterium, and so on until R _{5 is} selected from hydrogen or deuterium. More particularly, R ₁ is hydrogen comprising, R ₁ is deuterium, R ₂ is hydrogen, R ₂ is deuterium, R ₃ is hydrogen, R ₃ is deuterium, and so on, until R ₅ is hydrogen, deuterium, R ₅ is Technical solutions.

In another specific embodiment, "X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D" including X ₁ X _{3 is} selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D, X _{2 is} selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D, X _{3 is} selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D, and By analogy, until X _{7 is} selected from the technical schemes of CH ₃ , CD ₃ , CHD ₂ or CH ₂ D. More specifically, X ₁ is CH ₃ , X ₁ is CD ₃ , X ₁ is CHD ₂ or X ₁ is CH ₂ D, X ₂ is CH ₃ , X ₂ is CD ₃ , and X ₂ is CHD ₂ or X ₂ Is CH ₂ D, X ₃ is CH ₃ , X ₃ is CD ₃ , X ₃ is CHD ₂ or X ₃ is CH ₂ D, and so on, until X ₇ is CH ₃ , X ₇ is CD ₃ , and X ₇ is CHD ₂ or X ₇ is a technical solution of CH ₂ D.

In a preferred embodiment, the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof, wherein Y _{1 to} Y ₉ are each independently selected from hydrogen or deuterium, and R _{1 to} R ₅ and X _{1 to} X ₇ are as defined above, with the proviso that the above compound contains at least one deuterium atom.

In a preferred embodiment, the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof, wherein Y _{3 to} Y ₉ are hydrogen, Y ₁ and Y ₂ are each independently selected from hydrogen or deuterium, R _{1 to} R ₅ are each independently selected from hydrogen or deuterium, and X _{1 to} X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D.

In a preferred embodiment, Y ₁ is hydrogen.

In a preferred embodiment, X ₁ and X ₂ are the same.

In a preferred embodiment, X ₁ and X ₂ are each independently selected from CH ₃ or CD ₃ .

In a preferred embodiment, X ₃ and X ₄ are each independently selected from CH ₃ or CD ₃ .

In a preferred embodiment, X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ or CHD ₂ .

In a preferred embodiment, X ₅ is CH ₃ .

In a preferred embodiment, R ₂ , R ₃ , R ₄ and R ₅ are hydrogen.

In a preferred embodiment, R ₁ is deuterium.

In a preferred embodiment, R ₁ is hydrogen.

In a preferred embodiment, Y ₂ is deuterium.

In a preferred embodiment, Y ₂ is hydrogen.

As a preferred embodiment of the present invention, the compound has any one of the following structures, or a pharmaceutically acceptable salt thereof, but is not limited to the following structures:

The compounds of the invention may include one or more asymmetric centers, and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and / or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereomers or geometric isomers (such as cis and trans isomers), or may be in the form of a mixture of stereoisomers, This includes racemic mixtures and mixtures rich in one or more stereoisomers. Isomers can be separated from a mixture by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be obtained by Prepared by asymmetric synthesis.

Those skilled in the art will understand that organic compounds can form complexes with solvents that react in the solvent or precipitate or crystallize from the solvent. These complexes are called "solvates". When the solvent is water, the complex is called a "hydrate". The invention encompasses all solvates of the compounds of the invention.

The term "solvate" refers to the form of a compound or a salt thereof in combination with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be able to be separated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The "solvate" includes a solvate in a solution state and a separable solvate. Representative solvates include hydrates, ethanolates, and methanolates.

The term "hydrate" refers to a compound that is combined with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, for example, hydrates of the compounds may be used on behalf of the general formula Rx H ₂ O, where R is the compound, and x is a number greater than 0. A given compound can form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1, for example, hemihydrate (R0.5H ₂ O )) and a multi-hydrate (x is greater than 1, e.g., dihydrate (R2H ₂ O) and hexahydrate (R6H ₂ O)).

The compounds of the invention may be in an amorphous or crystalline form (polymorphic form). In addition, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form (or a salt, hydrate, or solvate) of a compound in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, photoelectric properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can lead to the predominance of a crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.

The present invention also includes isotopically-labeled compounds, which are equivalent to those described in formula (I), but one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹¹ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of the present invention. Certain isotopically-labeled compounds of the invention, such as those incorporating radioisotopes such as ³ H and ¹⁴ C, can be used for drug and / or substrate tissue distribution assays. Thallium, i.e. ³ H and carbon-14, i.e. ¹⁴ C isotopes, are particularly preferred because they are easy to prepare and detect. Furthermore, replacement with heavier isotopes, such as deuterium, ie, ² H, may be preferable in some cases because higher metabolic stability can provide therapeutic benefits, such as extending half-life in the body or reducing dosage requirements. An isotope-labeled compound of the formula (I) of the present invention and a prodrug thereof can generally be prepared in such a manner that, when performing the processes disclosed in the following schemes and / or examples and preparation examples, non-isotope-labeled reagents are replaced with readily available isotopically-labeled reagents Labeled reagent.

In addition, prodrugs are also included in the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted into its active form with a medical effect in vivo by, for example, hydrolysis in blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs Novel Delivery Systems, ACSSymposium Series Vol. 14, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D.Fleisher, S. Ramon, and H. Barbra, "Improved, or drug, delivery: solubility, limitation, overcome, and use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each article This article is for reference.

A prodrug is any covalently bonded compound of the invention, and when such a prodrug is administered to a patient, it releases the parent compound in vivo. Prodrugs are usually prepared by modifying functional groups, and the modification is performed in a manner such that the modification can be performed by routine manipulation or cleavage in vivo to produce the parent compound. Prodrugs include, for example, compounds of the invention in which a hydroxyl, amino, or thiol group is bonded to an arbitrary group, and when administered to a patient, can cleave to form a hydroxyl, amino, or thiol group. Thus, representative examples of prodrugs include, but are not limited to, acetate, amide, formate / amide, and benzoate / amide derivatives of hydroxyl, thiol, and amino functional groups of the compound of formula (I). In the case of a carboxylic acid (-COOH), an ester such as methyl ester, ethyl ester, or the like can be used. The esters themselves can be active and / or can be hydrolyzed under conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those groups that readily break down in the human body to release the parent acid or its salt.

synthesis

The compounds of the invention, including their salts, can be prepared using known organic synthesis techniques, and can be synthesized according to any of a number of possible synthetic pathways, such as those in the schemes below. The reaction for preparing the compound of the present invention can be performed in a suitable solvent, and those skilled in the art of organic synthesis can easily select a solvent. Suitable solvents may be substantially non-reactive with the starting material (reactant), intermediate, or product at the temperature at which the reaction is performed (eg, a temperature in the range of the solvent freezing temperature to the solvent boiling temperature). A given reaction may be performed in one solvent or a mixture of more than one solvent. The skilled person can select a solvent for a specific reaction step depending on the specific reaction step.

The preparation of the compounds of the invention may involve the protection and removal of different chemical groups. Those skilled in the art can easily determine whether protection and removal of protection are needed and the selection of an appropriate protecting group. The chemical properties of protecting groups can be found, for example, in Wuts and Greene, Protective Groups, Organic Synthesis, 4th Edition, John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.

The reaction can be monitored according to any suitable method known in the art. For example, spectroscopic means (such as nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹ H or ¹³ C), infrared (IR) spectroscopy, spectrophotometry (e.g., UV-visible light), mass spectrometry (MS)), or by chromatography Methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to monitor product formation.

Pharmaceutical compositions, preparations and kits

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of an active ingredient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active ingredient. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of an active ingredient.

A pharmaceutically acceptable excipient for use in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compounds formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin ), Buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, Sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substance, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-embedded Polymer, polyethylene glycol and lanolin.

The invention also includes kits (eg, pharmaceutical packaging). The provided kits can include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and / or dispersible packaging or other Suitable container). In some embodiments, the provided kit may also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and / or other therapeutic agent. In some embodiments, a compound of the invention and other therapeutic agents are provided in a first container and a second container to form a unit dosage form.

The pharmaceutical composition provided by the present invention can be administered by many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration Medication, implantation or other means of administration. For example, parenteral administration as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, and sternal administration , Cerebrospinal spinal membrane administration, intralesional administration, and intracranial injection or infusion techniques.

Generally, an effective amount of a compound provided herein is administered. Depending on the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc., the amount of compound actually administered can be determined by the physician .

When used to prevent a condition described in the present invention, a compound provided herein is administered to a subject at risk of developing the condition, typically based on and under the supervision of a physician, at a dosage level as described above. Subjects at risk for developing a particular disorder typically include subjects with a family history of the disorder, or those who are determined by genetic testing or screening to be particularly sensitive to the development of the disorder.

The pharmaceutical compositions provided herein can also be administered chronically ("long-term administration"). Long-term administration refers to administration of a compound or a pharmaceutical composition thereof over a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or the administration can be continued indefinitely, For example, the remainder of a subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a long period of time, for example, within a therapeutic window.

Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition may be administered by bolus, for example, to rapidly increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic level of the active ingredient, for example, an intramuscular or subcutaneous bolus dose allows for a slow release of the active ingredient, whereas a bolus delivered directly to a vein (e.g., by IV intravenous drip) can be more Rapid delivery allows the concentration of the active ingredient in the blood to rise quickly to an effective level. In other embodiments, the pharmaceutical composition may be administered in the form of a continuous infusion, for example, by IV infusion, to provide a steady state concentration of the active ingredient in the subject's body. Furthermore, in other embodiments, the bolus dose of the pharmaceutical composition may be administered first, followed by continuous infusion.

Oral compositions can take the form of a liquid solution or suspension in bulk or a powder in bulk. However, more generally, to facilitate accurate dosing, the composition is provided in unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dose for human patients and other mammals, each unit containing a predetermined number of active substances and suitable pharmaceutical excipients suitable for producing the desired therapeutic effect. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In such a composition, the compound is usually a minor component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), and the remainder is each useful for forming a desired administration form A carrier or excipient and processing aid.

For oral doses, a representative regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these dosage modes of administration, each dose provides about 0.01 to about 20 mg / kg of a compound of the invention, and preferred doses each provide about 0.1 to about 10 mg / kg, especially about 1 to about 5 mg / kg.

In order to provide a blood level similar to, or lower than, the injected dose, transdermal doses are usually selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 To about 10% by weight, and more preferably about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injection dose level ranges from about 0.1 mg / kg / hour to at least 10 mg / kg / hour. To obtain a sufficient steady state level, a preloaded bolus of about 0.1 mg / kg to about 10 mg / kg or more can also be given. For human patients from 40 to 80 kg, the maximum total dose cannot exceed about 2 g / day.

Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous vehicles and buffers, suspending and dispersing agents, coloring agents, flavoring agents, and the like. The solid form may include, for example, any of the following components, or compounds having similar properties: a binder, such as microcrystalline cellulose, tragacanth, or gelatin; an excipient, such as starch or lactose, a disintegrant, For example, alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as mint, water Methyl salicylate or orange flavor.

Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As mentioned earlier, in this composition, the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

Transdermal compositions are typically formulated as a topical ointment or cream containing an active ingredient. When formulated as an ointment, the active ingredient is typically combined with a paraffin or a water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and generally include other components for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope of the invention.

The compounds of the invention may also be administered via a transdermal device. Therefore, transdermal administration can be achieved using a reservoir or porous membrane type, or a variety of solid matrix patches.

The above components of the composition for oral administration, injection or topical administration are merely representative. Other materials and processing techniques are described in Section 8 of Remington's Pharmaceuticals, Science, 17th Edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

The compounds of the invention may also be administered in a sustained release form or from a sustained release delivery system. A description of representative sustained-release materials can be found in Remington's Pharmaceutical Sciences.

The invention also relates to a pharmaceutically acceptable formulation of a compound of the invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α-, β-, and γ-cyclodextrin consisting of 6, 7, and 8 α-1,4-linked glucose units, respectively, which optionally include one on the linked sugar moiety Or more substituents, including but not limited to: methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substituted. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, for example, a sulfobutyl ether β-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (eg, 10-50% in water).

Indication

The present invention provides a method for inhibiting a protein tyrosine kinase (such as EGFR kinase) or treating a disease (such as cancer, cell proliferative disease, inflammation, infection, immune disease, organ transplant, viral disease, cardiovascular disease or Method of metabolic disease), which comprises the step of administering to a subject in need of treatment a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, hydrate, crystalline form, prodrug thereof Or an isotope derivative, or a pharmaceutical composition according to the invention.

The compounds of the invention are useful for treating cancer caused by EGFR. In particular, the compounds are useful in the treatment of cancers caused by EGFR expressing EGFR mutants and in the treatment of cancers of EGFR refractory to RTKI therapy (eg, erlotinib or gefitinib).

The compounds of the present invention are inhibitors of at least one mutant of EGFR and are therefore suitable for use in treatment with one or more EGFR mutants (e.g., deletion mutations, activation mutations, resistance mutations, or combinations thereof, specific examples including T790M mutations, L858R mutation and L858R / T790M double mutation) activity related to one or more disorders. Therefore, in a specific embodiment, the present invention provides a method for treating a mutant EGFR-mediated disorder, which comprises administering to a patient in need thereof a compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, A solvate, a hydrate, a crystalline form, a prodrug or an isotope derivative, or a step of administering a pharmaceutical composition according to the present invention.

Cancers treatable by the compounds of the present invention include, but are not limited to, non-small cell lung cancer (NSCLS), small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, Gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, nasopharyngeal carcinoma and other hyperproliferative diseases. In addition, the compounds of the present invention are also useful for maintaining the prevention of cancer recurrence in patients in need of such treatment.

An effective amount of a compound of the present invention is usually administered in a single or multiple doses at an average daily dose of 0.01 to 50 mg of the compound per kg of the patient's body weight, preferably 0.1 to 25 mg of the compound per kg of the patient's body weight. Generally, the compound of the present invention can be administered to the patient in need of such treatment at a daily dose range of about 1 mg to about 3500 mg per patient, preferably 10 mg to 1000 mg. For example, the daily dose per patient may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000mg. Administration can be one or more times daily, weekly (or several days apart), or on an intermittent schedule. For example, the compound can be administered one or more times per day on an weekly basis (e.g., every Monday), indefinitely or for several weeks, such as 4-10 weeks. Alternatively, the compound can be administered daily for several days (e.g., 2-10 days), and then the compound is not administered for several days (e.g., 1-30 days), the cycle is repeated indefinitely, or a given number of times, such as 4-10 Cycles. For example, a compound of the invention may be administered daily for 5 days, then intermittently for 9 days, and then daily for 5 days, then intermittently for 9 days, and so on, the cycle may be repeated indefinitely or a total of 4-10 times.

When EGFR-TKI (eg, erlotinib or gefitinib) is used in combination with a compound of the invention, the components of the combination therapy can be administered at their monotherapy dose levels and schedules. For example, erlotinib has been administered orally at 150 mg per day for the treatment of non-small cell lung cancer, and it has been administered orally at 100 mg per day for pancreatic cancer. In another example, gefitinib has been administered orally at 250 mg per day for the treatment of non-small cell lung cancer.

Preferably, when EGFR-TKI (eg, erlotinib or gefitinib) is used in combination with a compound of the invention, the dosage level of one or both of its components is reduced compared to when used alone.

Examples

The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions or conditions recommended by the manufacturer. Unless stated otherwise, parts and percentages are parts by weight and percent by weight.

Generally, in the preparation process, each reaction is usually performed in an inert solvent at room temperature to reflux temperature (for example, 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

The abbreviations used in this article have the following meanings:

APCI	Atmospheric pressure chemical dissociation

TEA	Triethylamine
B 2 pin 2	Pinacol diborate
pTSA	p-Toluenesulfonic acid
PE	Petroleum ether
EA	Ethyl acetate
DMSO	Dimethyl sulfoxide
DMF	N, N-dimethylacetamide
DCM	Dichloromethane
XantPhos	4,5-bisdiphenylphosphine-9,9-dimethylxanthene
DIPEA	N, N-diisopropylethylamine
TFA	Trifluoroacetate

Example 1 N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -5-((4- (4-fluoro-2-methyl-1- (propane-2 -base -1,1,1,3,3,3-d ₆ ) -1H-benzo [d] imidazole-6-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide ( Preparation of compound I-1).

Use the following route for synthesis:

Step 1 Synthesis of Compound 1

Deuterated acetone (5.00 g, 78.0 mmol) was dissolved in 1,2-dichloroethane (100 mL), p-methoxybenzylamine (10.7 g, 78.0 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Add NaBH ₃ CN slowly and stir overnight at room temperature. Filtration, concentration of the filtrate, and column chromatography (PE / EA, V / V, 5/1) gave compound 69 as a colorless oily liquid 8.05 g.

Step 2 Synthesis of Compound 2

Compound 1 (8.05 g, 43.4 mmol) was dissolved in dichloromethane (80 mL), 95 mL of a saturated sodium bicarbonate solution was added, and a solution of acetyl chloride (5.1 g, 65.14 mmol) in dichloromethane (15 mL) was added dropwise under an ice water bath. The mixture was stirred with natural heating overnight, and the layers were separated. The aqueous phase was extracted with dichloromethane (30 mL × 3). The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration to obtain a brown oily liquid (9.842 g). A part of the above brown oily product (4.00 g, 17.6 mmol) was placed in a single-necked flask, 10 mL of water was added to disperse, 10 mL of concentrated hydrochloric acid was added, and the mixture was heated under reflux for 5 hours. Cooled to room temperature, extracted with ethyl acetate (10 mL × 3), washed the organic phase with saturated brine, dried over anhydrous sodium sulfate, and concentrated by filtration to obtain a colorless liquid (0.85 g).

Step 3 Synthesis of Compound 3

Take compound 2 (850 mg, 7.93 mmol) in toluene (10 mL), add compound 4-bromo-2,6-difluoroaniline (825 mg, 3.97 mmol), and add phosphorus oxychloride (1.13 mL, 12.1 mmol) in one portion. And TEA (1.64 mL, 11.9 mmol), heated to reflux for 5 hours. It was cooled to room temperature, diluted with EA (30 mL), filtered and concentrated to obtain 917 mg of a brown liquid. A brown liquid (917 mg, 3.03 mmol) was dissolved in THF (15 mL), tBuOK (680 mg, 6.06 mmol) was added at room temperature, and the mixture was heated under reflux overnight. Cool to room temperature, concentrate the reaction solution, add EA (5mL) and saturated brine (5mL) to separate the layers, extract the aqueous phase with EA (3mL × 3), combine the organic phases and wash with saturated brine, dry over anhydrous sodium sulfate, and filter Concentrated to give a pale yellow solid (658 mg, 2.37 mmol). The light yellow solid was placed in a single-necked flask, and pinacol diborate (904 mg, 3.56 mmol), tricyclohexyl phosphorus (113 mg, 0.04 mmol), palladium acetate (59 mg, 0.26 mol), and potassium acetate (699 mg, 7.12) were added in this order. mmol), replaced with nitrogen three times, and added anhydrous DMSO (11 mL) under nitrogen protection, and heated and stirred at 90 ° C for 45 minutes. The reaction was cooled to room temperature, poured into 50 mL of water, and extracted with EA-PE (v / v, 1/1, 10 mL * 2). The organic phases were combined and evaporated to dryness to obtain the crude product, which was directly used in the next step.

Step 4 Synthesis of Compound 4

The above crude compound 3 (485 mg, 0.57 mmol), 2,4-dichloropyrimidine (85 mg, 0.57 mmol), Pd (PPh ₃ ) ₂ Cl ₂ (24 mg, 0.034 mmol), 2M sodium carbonate solution (1.1 mL) Dissolved in DMF (5mL), replaced with nitrogen three times, and heated and stirred at 60 ° C for 2 hours. After cooling to room temperature, the reaction solution was poured into 20 mL of water and extracted with EA (5 mL × 3). The EA was combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography (PE / EA, V / V, 2 / 1) Compound 4 was obtained as a pale yellow solid (262 mg).

Step 5 Synthesis of Compound 5

Take compound 4 (262 mg, 0.84 mmol), 4-fluoro-2-methoxy-5-nitroaniline (188 mg, 1.01 mmol) and pTSA (641 mg, 3.37 mmol) into a single-mouth reaction flask under the protection of nitrogen. After that, anhydrous dioxane (10 mL) was added, and nitrogen was substituted three times. The reaction was heated and stirred at 100 ° C for 24 hours. The solvent was evaporated to dryness under reduced pressure and purified by preparative thin layer chromatography (PE / EA, V / V, 1/1) to obtain 220 mg of a gray solid. The gray solid (220 mg, 0.48 mmol) obtained above was dispersed uniformly by adding acetonitrile (5 mL), and then N ¹ , N ¹ , N ² -trimethylethane-1,2-diamine (59 mg, 0.57 mmol), and potassium carbonate were added. (132 mg, 0.96 mmol), heated to reflux at 90 ° C for 2 hours. The reaction solution was cooled to room temperature, and the insoluble solid was removed by filtration. The filtrate was evaporated to dryness to obtain Compound 5 as a red oil (126 mg).

Step 6 Synthesis of Compound I-1

The red oily compound 5 (126 mg) was dissolved in a mixed solution of ethanol-water (4 mL + 1 mL), and reduced iron powder (106 mg) and ammonium chloride (34 mg) were added, and the mixture was heated under reflux at 90 ° C for 5 hours to react. The reaction was cooled to room temperature, the insoluble solid was removed by filtration, and the filtrate was evaporated to dryness. The obtained oil was dissolved by adding 10 mL of DCM, 5 mL of a saturated aqueous sodium hydrogen carbonate solution was added, and the solution was cooled in an ice bath. To the above two-phase system, 0.15 M of acryloyl chloride methylene chloride (2.5 mL) was added dropwise, and the reaction was carried out under an ice bath for 30 minutes. The layers were separated and the organic phase was evaporated to dryness. Purification by preparative thin layer chromatography (DCM / MeOH, V / V, 20/1) gave compound I-1 as a white solid (59 mg). ¹ H NMR (400MHz, CDCl ₃ ) δ 9.65–9.37 (m, 2H), 8.51 (d, J = 5.3Hz, 1H), 8.03 (d, J = 1.3Hz, 1H), 7.81–7.71 (m, 1H), 7.67 (s, 1H), 7.14 (dd, J = 5.3, 2.4Hz, 1H), 6.72 (s, 1H), 6.54-6.07 (m, 2H), 5.90-5.40 (m, 1H), 4.72 (s, 1H), 3.90 (s, 3H), 3.20 (t, J = 6.0 Hz, 2H), 2.89 (d, J = 16.1 Hz, 2H), 2.71 (s, 3H), 2.68 (s, 3H) , 2.66 (s, 6H).

Example 2 N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -5-((4- (4-fluoro-2-methyl-1- (propane-2 -Yl-2-d) -1H-benzo [d] Preparation of imidazole-6-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (Compound I-2).

According to the synthesis method described in Example 1, step 1 in Example 1 is replaced by the following steps:

Take acetone (3.2g, 56mmol), add p-methoxybenzylamine (5.1g, 37mmol) to toluene (50mL) to dissolve it, heat and reflux in a 130 ° C oil bath for 3 hours, cool to room temperature, add 3.0 g of acetone continued to reflux for 4 hours. The reaction was cooled to room temperature, most of the toluene was distilled off under reduced pressure, 30 mL of tetrahydrofuran and 10 mL of MeOD were added, and the solution was cooled in an ice bath. 0.8 g of sodium deuterated borohydride was added in portions and the reaction was stirred at room temperature overnight. The solvent was evaporated to dryness under reduced pressure, 20 mL of water was added, and 20 mL of DCM was used for liquid separation. The organic phase was mixed with a saturated sodium bicarbonate aqueous solution (30 mL), stirred and dispersed uniformly, and cooled in an ice bath, and acetyl chloride (2.89 g, 37 mmol) was added dropwise. The reaction was continued for 1 hour after the dropwise addition. Separate the liquid and evaporate the organic phase through column chromatography (PE / EA, v / v, 8/1, 0.5% TEA) to obtain the compound N- (4-methoxyphenyl) propan-2-d-2-amine 3.04 g.

Use the compound N- (4-methoxyphenyl) propan-2-d-2-amine obtained in the above step to replace the compound 1 in step 2 in Example 1, and then repeat steps 2-6 in Example 1, Compound I-2 was prepared. Compound I-2 was an off-white solid (70 mg). ¹ H NMR (400MHz, CDCl ₃ ) δ 9.88 (s, 1H), 9.63 (s, 1H), 8.52 (d, J = 5.3 Hz, 1H), 8.03 (d, J = 1.3 Hz, 1H), 7.79 (d, J = 11.4 Hz, 1H), 7.67 (s, 1H), 7.13 (d, J = 5.3 Hz, 1H), 6.78 (s, 1H), 6.46 (s, 2H), 5.69 (d, J = 11.8Hz, 1H), 3.89 (s, 3H), 2.97 (s, 2H), 2.70 (s, 3H), 2.67 (s, 3H), 2.46 (s, 2H), 2.36 (s, 6H), 1.65 ( s, 6H).

Example 3 N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -5-((4- (4-fluoro-1-isopropyl-2- (methyl -d ₃ ) -1H-benzo [d] Preparation of imidazole-6-yl-5-d) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (Compound I-3).

Use the following route for synthesis:

Step 1 Synthesis of Compound 7

NaOD (40% heavy aqueous solution, catalytic amount) was added dropwise to a mixed solvent of compound 6 (1.50 g, 5.55 mmol) in heavy water (50 mL) and deuterated methanol-d ₄ (1 mL) at room temperature, and the reaction was performed at 140 ° C. The tube was sealed and allowed to react overnight. Cool to room temperature, extract with dichloromethane (50 mL x 3), combine the organic phases, wash with saturated brine (50 mL), dry over anhydrous sodium sulfate, remove the solvent, and concentrate the column for separation (eluent: petroleum ether / ethyl acetate Ester (v / v) = 3: 1), 1.45 g of a white solid was obtained in a yield of 97.0%. LC-MS (APCI): m / z = 275.0 (M + 1). ¹ H NMR (500MHz, DMSO-d ₆ ) δ7.73 (s, 1H), 4.87-4.60 (m, 1H), 1.51 (d, J = 6.9Hz, 6H).

Step 2 Synthesis of Compound 8

Palladium acetate (200 mg) and tricyclohexyl phosphorus (400 mg) were added to compound 7 (2.00 g, 7.30 mmol), pinacol diborate (2.80 g, 10.95 mmol), and potassium acetate (2.10 g, 21.90) under nitrogen. In a solution of anhydrous dimethyl sulfoxide (20 mL), the reaction was carried out at 100 ° C. for 2 hours under a nitrogen atmosphere. Cooled to room temperature, filtered through celite, washed the filter cake with ethyl acetate, washed the filtrate with saturated brine, dried over anhydrous sodium sulfate, concentrated the filtrate under reduced pressure, and concentrated the solution for column separation (eluent: petroleum ether / ethyl acetate (v / v) = 3: 1), 1.90 g of a white solid was obtained. Yield: 80.8%. LC-MS (APCI): m / z = 323.2 (M + 1) ⁺ .

Step 3 Synthesis of Compound 9

Acetonitrile (18 mL) and water (6 mL) were added to compound 8 (1.90 g, 5.88 mmol) and 2,4-dichloropyrimidine (1.10 g, 7.06 mmol), sodium carbonate (1.60 g, 14.70 mmol), In a mixture of bistriphenylphosphonium palladium dichloride (190 mg), the reaction was stirred at 80 ° C. for 2 hours under the protection of nitrogen. Cooled to room temperature, filtered through celite, washed the filter cake with dichloromethane, washed the filtrate with saturated brine, dried over anhydrous sodium sulfate, concentrated the filtrate under reduced pressure, and concentrated the solution for column separation (eluent: petroleum ether / ethyl acetate Ester (v / v) = 3: 1) gave 1.26 g of a white solid. The yield was 69.6%. LC-MS (APCI): m / z = 309.1 (M + 1) ⁺ .

Step 4 Synthesis of Compound 11

Under nitrogen, Pd (OAc) ₂ (120 mg) and Xantphos (200 mg) were added to compound 9 (1.26 g, 4.09 mmol), compound 10 (900 mg, 3.41 mmol), and cesium carbonate (2.70 g, 8.40 mmol). In water DMF (20mL), the reaction solution was reacted at 100 ° C overnight under the protection of nitrogen, cooled to room temperature, quenched by adding water, extracted with ethyl acetate (50mL x 3), combined with organic phases, washed with saturated brine (50mL), and anhydrous. After drying over sodium sulfate, the solvent was removed, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 10: 1) to obtain 1.40 g of a yellow solid. LC-MS (APCI): m / z = 541.2 (M + 1) ⁺ .

Step 5 Synthesis of Compound 12

Reduced iron powder (870 mg, 15.60 mmol) and ammonium chloride (416 mg, 7.80 mmol) were added to a mixed solution of compound 11 in ethanol and water (16 mL / 4 mL). The reaction was refluxed for 2 hrs, filtered through Celite, and the filtrate was decompressed It was concentrated, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 8: 1) to obtain 600 mg of a yellow solid with a yield of 34.5% in two steps. LC-MS (APCI): m / z = 511.2 (M + 1) ⁺ .

Step 6 Synthesis of Compound I-3

Under nitrogen protection, compound 12 (600 mg, 1.10 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and DIPEA (0.46 mL, 2.80 mmol) was added dropwise. After the dropwise addition was completed, the reaction system was cooled to -10 ° C. Acryloyl chloride (100 mg, 1.10 mmol) was slowly added at this temperature and stirred for 1.0 hrs. Water was added, and dichloromethane (50 mL x 3) was extracted. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 10: 1) 350 mg was obtained with a yield of 56.3%. HPLC: 95.92%. LC-MS (APCI): m / z = 565.2 (M + 1) ⁺ , ¹ H NMR (400MHz, DMSO-d6) δ 10.03 (s, 1H), 8.91 (s, 1H), 8.49 (d, J = 5.3Hz, 1H), 8.22 (d, J = 3.0Hz, 2H), 7.57 (d, J = 5.3Hz, 1H), 7.02 (s, 1H), 6.51 (br, 1H), 6.29–6.22 (m , 1H), 5.76--5.72 (m, 1H), 4.87--4.78 (m, 1H), 3.86 (s, 3H), 3.04--2.88 (m, 2H), 2.69 (s, 3H), 2.63--2.56 (m , 2H), 2.27 (s, 6H), 1.58 (d, J = 6.9Hz, 6H).

Example 4 N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -5-((4- (4-fluoro-1-isopropyl-2- (methyl -d ₃ ) -1H-benzo [d] Preparation of imidazole-6-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (Compound I-4).

According to the synthesis method described in Example 3, the following steps are used to replace Step 1 in Example 3:

5-Bromo-N ¹ -isopropylbenzene-1,2-diamine (3.00m, 13.10mmol) was added to glacial acetic acid-d ₄ (15mL), and the reaction was carried out at reflux for 2hrs. Cool to room temperature, remove acetic acid under reduced pressure, adjust the residue to pH 7 with saturated sodium bicarbonate solution, extract with dichloromethane (50ml x 3), wash the combined organic layers with saturated brine, dry over anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 20: 1) to obtain 2.68 g of a 6-bromo-1-isopropyl-2- (methyl-d) compound as a brown-red oil. ₃ ) -1H-benzo [d] imidazole, yield: 80.22%, LC-MS (APCI): m / z = 256.1 (M + 1) ⁺ .

Use the compound 6-bromo-1-isopropyl-2- (methyl-d ₃ ) -1H-benzo [d] imidazole obtained in the above step to replace the compound 7 in step 2 in Example 3, and repeat the example. Step 2-6 in 3, 100 mg of compound I-4 was prepared, and the yield was 68.3%. HPLC: 95.63%. LC-MS (APCI): m / z = 564.0 (M + 1) ⁺ , ¹ H NMR (400MHz, CDCl ₃ ) δ9.95 (s, 1H), 9.64 (s, 1H), 8.54 (d, J = 5.2Hz, 1H), 8.05 (s, 1H), 7.81 (d, J = 11.1Hz, 1H), 7.68 (s, 1H), 7.15 (d, J = 5.2Hz, 1H), 6.79 (s, 1H) , 6.48 (s, 2H), 5.75--5.67 (m, 1H), 4.84--4.72 (m, 1H), 3.91 (s, 3H), 3.03--2.93 (m, 2H), 2.72 (s, 3H), 2.51 --2.42 (m, 2H), 2.38 (s, 6H), 1.68 (d, J = 7.0Hz, 6H). ¹ H NMR (500MHz, DMSO-d ₆ ): δ9.97 (s, 1H), 8.89 ( s, 1H), 8.46 (s, 1H), 8.25–8.08 (m, 2H), 7.89 (d, J = 12.8 Hz, 1H), 7.63--7.44 (m, 1H), 6.99 (s, 1H), 6.68 --6.36 (m, 1H), 6.23 (d, J = 16.5Hz, 1H), 5.72 (d, J = 8.5Hz, 1H), 4.87--4.72 (m, 1H), 3.84 (s, 3H), 3.03-- 2.88 (m, 2H), 2.66 (s, 3H), 2.61-2.51 (m, 2H), 2.33 (s, 6H), 1.56 (d, J = 5.8Hz, 6H).

Example 5 N- (2-((2- (dimethylamino) ethyl) (methyl) amino) -5-((4- (4-fluoro-1-isopropyl-2-methyl- 1H-benzo [d] imid Preparation of azole-6-yl-5-d) pyrimidin-2-yl) amino) -4- (methoxy-d ₃ ) phenyl) acrylamide (Compound I-5).

Use the following route for synthesis:

Step 1 Synthesis of Compound 14

At room temperature, pTSA · H ₂ O (342 mg 1.80 mmol) was added to compound 13 (450 mg, 1.50 mmol) and 4-fluoro-2- (methoxy-d ₃ ) -5-nitroaniline (312 mg, 1.65 mmol In a solution of 2-pentanol (10 mL), the reaction was performed at 115 ° C. for 10 hrs, cooled to room temperature, the solid was filtered, and the solid was washed with CH ₃ CN (3 mL). Add the solid to 10mL of water, adjust the pH to alkaline with concentrated ammonia water, and stir for 2.5hrs. The solid was filtered, washed with H ₂ O (30mL). 600 mg earthy gray solid dried under vacuum at 70 ° C. Yield: 87.33%. LC-MS (APCI) = 458.0 (M + 1) ⁺ .

Step 2 Synthesis of Compound 15

K ₂ CO ₃ (365 mg, 2.62 mmol) was added to compound 14 (600 mg, 1.31 mmol) and N ¹ , N ¹ , N ² -trimethylethane-1,2-diamine (161 mg, 1.58) at room temperature. In a CH ₃ CN (15 mL) solution, the reaction was heated to reflux and stirred for 5 hrs. The reaction solution was removed under reduced pressure to obtain a red solid. The solid was extracted with H ₂ O (30 mL) and DCM (30 mL x 3). The organic layer was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 600 mg of a brown-red solid. Yield: 84.82%. LC-MS (APCI) = 540.0 (M + 1) ⁺ .

Step 3 Synthesis of Compound 16

Pd / C (5 g) was added to a solution of compound 15 (600 mg, 1.11 mmol) in methanol (30 mL) at room temperature, and the air was replaced with hydrogen three times. The reaction was allowed to react at room temperature overnight. Filter through celite, wash the filter cake with MeOH, and concentrate the filtrate under reduced pressure to obtain 450 mg of a yellow solid. Yield: 79.65%. LC-MS (APCI) = 510.2 (M + 1) ⁺ .

Step 4 Synthesis of Compound I-5

Under nitrogen protection, compound 15 (450 mg, 0.88 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and DIPEA (0.35 mL, 1.76 mmol) was added dropwise. After the dropwise addition was completed, the reaction system was cooled to -10 ° C. Acryloyl chloride (1.42 mL, 0.88 mmol, 0.618 mmol / mL) was slowly added at this temperature, and stirred for 1.0 hrs. Water was added, and dichloromethane (50 mL x 3) was extracted. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 10: 1) 300 mg was obtained with a yield of 60.4%. HPLC: 96.28%. LC-MS (APCI): m / z = 564.3 (M + 1) ⁺ , ¹ H NMR (300MHz, DMSO-d ₆ ) δ 10.06 (s, 1H), 8.94 (s, 1H), 8.46 (d, J = 3.5Hz, 1H), 8.20 (s, 2H), 7.90 (d, J = 11Hz, 1H), 7.54 (s, 1H), 6.97 (s, 1H), 6.51 (s, 1H), 6.22 ( d, J = 16.8 Hz, 1H), 5.71 (d, J = 11.2 Hz, 1H), 4.87–4.69 (m, 1H), 2.98–2.83 (m, 2H), 2.65 (s, 3H), 2.58 (s , 3H), 2.46--2.34 (m, 2H), 2.26 (s, 6H), 1.53 (d, J = 5.5Hz, 6H).

Example 6 N- (5-((4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [d] imidazol-6-yl) pyrimidin-2-yl) amino)- 4-methoxy Preparation of 2- (methyl (2- (methyl (methyl-d ₃ ) amino) ethyl) amino) phenyl) acrylamide (Compound I-6).

Use the following route for synthesis:

Step 1 Synthesis of Compound 18

K ₂ CO ₃ (1.52 g, 11.0 mmol) was added to compound 17 (2.50 g, 5.50 mmol) and N ¹ , N ¹ , N ² -trimethylethane-1,2-diamine (600 mg) at room temperature. , 6.60 mmol) in a solution of CH ₃ CN (40 mL), the reaction was heated to reflux and stirred overnight, and the reaction solution was removed under reduced pressure to obtain a red solid. The solid was slurried with H ₂ O (100 mL) and stirred for 3 hrs. The solid was filtered, and the solid was washed with cold ethanol (3 mL x 2) and dried under vacuum at 55 ° C to obtain a red solid (2.5 g). Yield: 87.01%. LC-MS (APCI): m / z = 523.3 (M + 1) ⁺ .

Step 2 Synthesis of Compound 19

K ₂ CO ₃ (276 mg, 2.00 mmol) was added to a solution of compound 18 (523 mg, 1.0 mmol) and TsO-CD ₃ (208 mg, 1.10 mmol) in CH ₃ CN (40 mL) at room temperature, and the reaction was warmed to 50 ° C. After stirring overnight, the reaction solution was removed under reduced pressure to obtain a red solid. The solid was slurried with H ₂ O (100 mL) and stirred for 3 hrs. The solid was filtered, and the solid was washed with cold ethanol (2 mL x 2) and dried under vacuum at 55 ° C. to obtain a red solid (200 mg). Yield: 37.01%. LC-MS (APCI): m / z = 540.3 (M + 1) ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.72 (s, 1H), 8.52 (d, J = 5.3Hz, 1H) , 8.32 (s, 1H), 8.22 (s, 1H), 7.83 (d, J = 12.2 Hz, 1H), 7.61 (d, J = 5.3 Hz, 1H), 6.85 (s, 1H), 4.89--4.79 ( m, 1H), 3.96 (s, 3H), 3.30--3.26 (m, 2H), 2.84 (s, 3H), 2.62 (s, 3H), 2.61--2.55 (m, 2H), 2.25 (s, 3H) 1.58 (d, J = 6.9 Hz, 6H).

Step 3 Synthesis of Compound 20

Pd / C (50 mg) was added to a solution of compound 19 (200 mg) in methanol (30 mL) at room temperature, and the air was replaced with hydrogen three times. The reaction was allowed to react at room temperature overnight. The celite was filtered, the filter cake was washed with dichloromethane, the filtrate was concentrated under reduced pressure, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 12) to obtain 120 mg with a yield of 65%. LC-MS (APCI): m / z = 510.2 (M + 1) ⁺ .

Step 4 Synthesis of Compound I-6

Under nitrogen protection, compound 20 (120 mg, 0.23 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and DIPEA (0.08 mL, 0.5 mmol) was added dropwise. After the dropwise addition was completed, the reaction system was cooled to -20 ° C. Acryloyl chloride (0.37 mL, 0.23 mmol, 0.618 mmol / mL) was slowly added at this temperature, and stirred for 1.0 hrs. Water was added, and dichloromethane (30 mL x 3) was extracted. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 9%) 75 mg was obtained in a yield of%. HPLC: 96.55%. LC-MS (APCI): m / z = 564.3 (M + 1) ⁺ .

Example 7 N- (2-((2- (bis (methyl-d ₂₎ amino) ethyl) (methyl) amino) -5-((4- (4-fluoro-1-isopropyl- 2-methyl-1H-benzo [d] Preparation of imidazole-6-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (Compound 1-7).

Use the following route for synthesis:

Step 1 Synthesis of Compound 21

K ₂ CO ₃ (920 mg, 6.60 mmol) was added to compound 17 (1.50 g, 3.30 mmol) and tert-butyl (2- (methylamino) ethyl) carbamate (700 mg, 3.96 mmol) at room temperature. In CH ₃ CN (40 mL) solution, the reaction was heated to reflux and stirred overnight. The reaction solution was removed under reduced pressure to obtain a red solid. The solid was slurried with H ₂ O (100 mL) and stirred for 3 hrs. The solid was filtered, and the solid was washed with cold ethanol (3 mL x 2) and dried under vacuum at 55 ° C. to obtain a red solid (2.5 g). Yield: 87.01%. LC-MS (APCI): m / z = 609.2 (M + 1) ⁺ .

Step 2 Synthesis of Compound 22

Trifluoroacetic acid (10 mL) was added to a solution of compound 21 (1.70 g, 2.79 mmol) in dichloromethane (20 mL), and the reaction was stirred in a greenhouse for 1 hr. The reaction solution was concentrated under reduced pressure to remove the reaction solution, and the pH was adjusted to be alkaline with a saturated sodium bicarbonate solution. Water (30 mL) was added and stirred overnight. The solid was filtered, the solid was washed with water, and then washed with cold ethanol (3 mL x 2), and dried under vacuum at 55 ° C to obtain 1.1 g of a red solid, yield: 77.44%. LC-MS (APCI): m / z = 509.2 (M + 1) ⁺ .

Step 3 Synthesis of Compound 23

In an ice bath, deuterated formaldehyde (1 mL, 20% w) was added to a mixture of deuterated chloroform (15 mL) and deuterated methanol (5 mL) of compound 22 (600 mg, 1.18 mmol), and the reaction was continued stirring in the ice bath For 30 min, sodium triacetylborohydride (525 mg, 2.48 mmol) was added and the reaction was allowed to proceed at room temperature overnight. The reaction was quenched with a saturated sodium bicarbonate solution, and the methanol was removed under reduced pressure. The aqueous layer was extracted with dichloromethane (50 mL × 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column separation (eluent: dichloromethane / methanol (v / v) = 8%) gave 220 mg with a yield of 34.49%. LC-MS (APCI): m / z = 541.2 (M + 1) +.

Step 4 Synthesis of Compound 24

Pd / C (50 mg) was added to a solution of compound 23 (220 mg, 0.40 mmol) in methanol (30 mL) at room temperature, and the air was replaced with hydrogen three times. The reaction was performed at room temperature for 1.5 hrs. Filter through diatomaceous earth, wash the filter cake with dichloromethane, and concentrate the filtrate under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 14%) to obtain 180 mg with a yield of 86.62%. . LC-MS (APCI): m / z = 511.2 (M + 1) ⁺ .

Step 5 Synthesis of Compound I-7

Under nitrogen protection, compound 24 (180 mg, 0.35 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and DIPEA (0.11 mL, 0.71 mmol) was added dropwise. After the dropwise addition was completed, the reaction system was cooled to -20 ° C. Acryloyl chloride (0.57 mL, 0.35 mmol, 0.618 mmol / mL) was slowly added at this temperature, and stirred for 1.0 hrs. Water was added, and dichloromethane (30 mL x 3) was extracted. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 9%) yielded 145 mg with a yield of 72.85%. HPLC: 95.36%. LC-MS (APCI): m / z = 565.4 (M + 1) ⁺ .

Example 8 N- (2-((2- (bis (methyl-d ₃ ) amino) ethyl) (methyl) amino) -5-((4- (4-fluoro-1-isopropyl- Preparation of 2-methyl-1H-benzo [d] imidazol-6-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (Compound I-8).

Use the following route for synthesis:

Step 1 Synthesis of Compound 25

In an ice bath, deuterated formaldehyde (1 mL, 20% w) was added to a mixture of deuterated chloroform (15 mL) and deuterated methanol (10 mL) of compound 22 (400 mg, 0.78 mmol). The reaction was continued to stir in the ice bath 30min. Added to sodium cyanoborodeuteride (104 mg, 1.57 mmol) and reacted at room temperature overnight. The reaction was quenched with a saturated sodium bicarbonate solution, and the methanol was removed under reduced pressure. The aqueous layer was extracted with dichloromethane (50 mL x 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column separation (eluent: dichloromethane / methanol (v / v) = 8%) gave 180 mg with a yield of 42.17%. LC-MS (APCI): m / z = 543.2 (M + 1) ⁺ .

Step 2 Synthesis of Compound 26

Pd / C (50 mg) was added to a solution of compound 25 (180 mg, 0.33 mmol) in methanol (30 mL) at room temperature, hydrogen was used to replace the air three times, and the reaction was performed at room temperature for 1.5 hrs. Filtered with celite, washed the filter cake with dichloromethane, and concentrated the filtrate under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 14%) to obtain 140 mg with a yield of 82.33%. . LC-MS (APCI): m / z = 513.2 (M + 1) ⁺ .

Step 3 Synthesis of Compound I-8

Under the protection of nitrogen, compound 26 (140 mg, 0.26 mmol) was dissolved in 30 mL of anhydrous dichloromethane, and DIPEA (0.10 mL, 0.54 mmol) was added dropwise. After the dropwise addition was completed, the reaction system was cooled to -20 ° C. Acryloyl chloride (0.42 mL, 0.26 mmol, 0.618 mmol / mL) was slowly added at this temperature, and stirred for 1.0 hrs. Water was added, and dichloromethane (30 mL x 3) was extracted. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 9%) 50 mg was obtained with a yield of 33.50%. HPLC: 99.24%. LC-MS (APCI): m / z = 567.5 (M + 1) ⁺ ; ¹ H NMR (300MHz, DMSO-d ₆ ): δ 10.04 (s, 1H), 8.91 (s, 1H), 8.49 (d , J = 5.2 Hz, 1H), 8.22 (d, J = 1.5 Hz, 2H), 7.92 (d, J = 12.5 Hz, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.02 (s, 1H ), 6.68--6.45 (m, 1H), 6.26 (dd, J = 17.0, 1.9 Hz, 1H), 5.88--5.67 (m, 1H), 4.83 (dt, J = 14.0, 6.9 Hz, 1H), 3.86 ( s, 3H), 3.05--2.91 (m, 2H), 2.69 (s, 3H), 2.62 (s, 3H), 2.52--2.50 (m, 2H), 1.58 (d, J = 6.9Hz, 6H).

Biological activity test.

(1) Kinase inhibition

Reagents and consumables:

WT EGFR (Carna, Catalog No. 08-115), EGFR [L858R] (Carna, Catalog No. 08-502), EGFR [L858R / T790M] (Carna, Catalog No. 08-510), ATP (Sigma, Catalog No. A7699- 1G), DMSO (Sigma, catalog number D2650), 96-well plate (Corning, catalog number 3365), 384-well plate (Greiner, catalog number 784076), HTRF Kinase TK kit (Cisbio, catalog number 62TK0PEJ), erlotine (Selleckchem, catalog number S7787), EGFR [d746-750] (Life Technologies, catalog number PV6178), 5x kinase buffer A (Life Technologies, catalog number PV3186), kinase tracer 199 (Life Technologies, catalog number PV5830) ),

Eu-anti-GST antibody (Life Technologies, catalog number PV5594).

Specific experimental methods:

Compound formulation: The test compound was dissolved in DMSO to prepare a 20 mM mother liquor. Then, a 3-fold dilution in DMSO was performed ten times. When adding medicine, dilute it with buffer 10 times.

Detection of WT EGFR and EGFR [L858R / T790M] kinase: In 5x kinase buffer A, mix WT EGFR or EGFR [L858R / T790M] kinase with a compound of different concentration prepared in a pre-diluted form for 10 minutes, and duplicate the wells at each concentration . Add the corresponding substrate and ATP, and react at room temperature for 20 minutes (wherein a negative positive control is set: a negative is a blank control and a positive is erlotinib). After the reaction, add detection reagents (reagents in the HTRF Kinase TK kit), and incubate for 30 minutes at room temperature. Then use an Evnvision microplate reader to measure the enzyme activity in the presence of each concentration of the compound of the invention, and calculate the compound concentration of different concentrations. Inhibition activity of enzyme activity, then according to the four-parameter equation, Graphpad 5.0 software was used to fit the inhibitory activity of enzyme activity at different concentrations of compounds to calculate the IC ₅₀ value.

The compound of the present invention was tested in the above kinase inhibition experiment, and it was found that the compound of the present invention has a potent activity on EGFR [L858R / T790M] and an excellent selectivity over WT EGFR compared to the non-deuterated compound T. The results for representative example compounds are summarized in Table 1 below.

Table 1:

Zh	EGFR (WT)	L858R / T790M
Compound T	1 ～ 5	<1

Compound I-1	1 ～ 5	<1
Compound I-2	1 ～ 5	<1
Compound I-3	1 ～ 5	<1
Compound I-4	1 ～ 5	<1
Compound I-5	1 ～ 5	<1
Compound I-6	1 ～ 5	<1
Compound I-7	1 ～ 5	<1
Compound I-8	1 ～ 5	<1

(2) Cytotoxicity test

The anti-proliferative activity of the compound of the present invention on three tumor cells cultured in vitro was detected by the MTS method. The experimental results show that the compound of the present invention has an inhibitory effect on the in vitro proliferation of cancer cells cultured in vitro; wherein the inhibitory effect on the proliferation of lung cancer cells in vitro is stronger than the inhibitory effect on the proliferation of skin cancer cells in vitro.

Cell line:

Skin cancer cells A431 (purchased from the American Standard Biological Collection (ATCC)); lung cancer cells NCI-H1975 (purchased from the American Standard Biological Collection (ATCC)) and HCC827 (purchased from the American Standard Biological Collection (ATCC)) ); All were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin.

Reagents and consumables:

RPMI-1640 (GIBCO, catalog number A10491-01); fetal bovine serum (GIBCO, catalog number 10099141); 0.25% trypsin-EDTA (GIBCO, catalog number 25200); penicillin-streptomycin, liquid (GIBCO, catalog number 15140-122); DMSO (Sigma, catalog number D2650); MTS test kit (Promega, catalog number G3581), 96-well plate (Corning, catalog number 3365).

Specific experimental methods:

Compound preparation: The test compound was dissolved in DMSO to prepare a 20 mM mother liquor, and stored at -20 ° C. When using, dilute with DMSO and other gradients 3 times, 10 times. At the time of dosing, it was diluted 4 times with cell culture medium RPMI-1640.

MTS cell viability test: 0.25% trypsin-EDTA digested cells in logarithmic growth phase, inoculated 150 μl in a 96-well plate according to the optimized density, 24 hours after adding the medium to dilute the compound 4 times, 50 μl / well Concentration: 100, 33.3, 11.1, 3.70, 1.23, 0.412, 0.137, 0.0457, 0.0152, 0.00508 μM). As a control, the same volume of 0.5% DMSO was added. After the cells were cultured for 72 hours, MTS was used to detect cell viability.

Specific operation: adherent cells, discard the culture medium, and add a mixed solution containing 20 μL MTS and 100 μL culture medium to each well. Put it in the incubator and continue to incubate for 1-4 hours to detect OD490, and use the OD650 value as a reference. Using GraphPad Prism software to make dose-response curves and calculate IC _50.

The compound of the present invention was tested in the above cytotoxicity experiment, and it was found that the compound of the present invention has strong activity on lung cancer cells NCI-H1975 and HCC827 and an excellent choice better than skin cancer cell A431 compared with the non-deuterated compound T. Sex. The results of representative example compounds are summarized in Table 2 below.

Table 2:

(3) Evaluation of metabolic stability

Microsomal experiments: human liver microsomes: 0.5mg / mL, Xenotech; rat liver microsomes: 0.5mg / mL, Xenotech; coenzyme (NADPH / NADH): 1mM, Sigma Life Science; magnesium chloride: 5mM, 100mM phosphate buffered Agent (pH 7.4).

Preparation of the stock solution: A certain amount of powder of the example compound and the reference compound was precisely weighed, and dissolved to 5 mM with DMSO, respectively.

Preparation of phosphate buffer solution (100 mM, pH 7.4): Take the pre-mixed 150 mL of 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution and mix with 0.5 M dipotassium phosphate solution The pH value of the solution was 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer solution (100 mM), which contained 100 mM potassium phosphate, 3.3 mM magnesium chloride, and had a pH of 7.4.

Prepare NADPH regeneration system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U / mL G-6-PD, 3.3mM magnesium chloride), and place on wet ice before use.

Preparation of stop solution: an acetonitrile solution containing 50ng / mL propranolol hydrochloride and 200ng / mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer solution (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg / mL. Take 25057.5 μL phosphate buffer solution (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL SD rat liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg / mL.

Sample incubation: Dilute the stock solution of the corresponding compound to 0.25 mM with 70% acetonitrile in water, and use it as a working solution. 398 μL of human liver microsomes or rat liver microsomes dilutions were added to 96-well incubation plates (N = 2), 2 μL of 0.25 mM working solution were added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate, and placed on ice as a stop plate. Place the 96-well incubation plate and NADPH regeneration system in a 37 ° C water bath, shake at 100 rpm, and pre-incubate for 5 minutes. Take 80 μL of the incubation solution from each well of the incubation plate and add it to the termination plate, mix well, and add 20 μL of the NADPH regeneration system solution as a 0min sample. Add 80 μL of NADPH regeneration system solution to each well of the incubation plate, start the reaction, and start timing. The reaction concentration of the corresponding compound was 1 μM, and the protein concentration was 0.5 mg / mL. When the reaction was 10, 30, and 90 minutes, 100 μL of each reaction solution was taken, added to the stop plate, and vortexed for 3 minutes to stop the reaction. The stop plate was centrifuged at 5000 × g for 10 min at 4 ° C. Take 100 μL of the supernatant into a 96-well plate pre-added with 100 μL of distilled water, mix well, and use LC-MS / MS for sample analysis.

Data analysis: The peak areas of the corresponding compounds and internal standard were detected by LC-MS / MS system, and the ratio of compound to internal standard peak area was calculated. The slope was measured by plotting the natural logarithm of the percentage of the remaining compound against time, and calculated t _1/2 and CL _int according to the following formula, where V / M is equal to 1 / protein concentration.

t _1/2 (min); CL _int (μL / min / mg).

The compounds of the present invention and their non-deuterated compounds were tested and compared simultaneously to evaluate their metabolic stability in human and rat liver microsomes. Undeuterated compound T was used as a reference. In human and rat liver microsome experiments, the compounds of the present invention can significantly improve metabolic stability by comparison with the undeuterated compound T. The results for representative example compounds are summarized in Table 3 below.

table 3:

(4) Pharmacokinetic experiments in rats

Six male Sprague-Dawley rats, 7-8 weeks old, weighing about 210 g, were divided into 2 groups of 3 rats each by intravenous or oral administration of a single dose of the compound (oral 10 mg / kg), and their pharmacokinetic differences were compared. .

Rats were raised on a standard diet and given water. Fasting began 16 hours before the test. The drug was dissolved with PEG400 and dimethyl sulfoxide. Orbital blood was collected at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

Rats were anesthetized briefly after inhaling ether, and 300 μL of blood samples were collected in test tubes from the orbit. The test tube contained 30 μL of a 1% heparin salt solution. Before use, test tubes were dried at 60 ° C overnight. After blood samples were collected at the last time point, rats were sacrificed after ether anesthesia.

Immediately after the blood sample is collected, gently invert the test tube at least 5 times to ensure that it is fully mixed and placed on ice. The blood sample was centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate the plasma from the red blood cells. Pipette 100 μL of plasma into a clean plastic centrifuge tube with the name and time point of the compound. Plasma was stored at -80 ° C before analysis. LC-MS / MS was used to determine the concentration of a compound of the invention in plasma. Pharmacokinetic parameters were calculated based on the blood drug concentration of each animal at different time points.

Experiments show that the compounds of the present invention have better pharmacokinetic properties in animals, and therefore have better pharmacodynamics and therapeutic effects.

The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without deviating from the concept of the present invention, several simple deductions or replacements can be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims (16)

1. A compound of formula (I), or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof:

   

   among them,

   Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , Y ₈ and Y ₉ are each independently selected from hydrogen, deuterium, halogen, or trifluoromethyl;

   R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from hydrogen or deuterium;

   X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D;

   The additional condition is that the above compound contains at least one deuterium atom.
2. The compound according to claim 1, or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof, wherein,

   Y ₁ , Y ₂ , R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from hydrogen or deuterium;

   X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are each independently selected from CH ₃ , CD ₃ , CHD ₂ or CH ₂ D;

   Y ₃ , Y ₄ , Y ₅ , Y ₆ , Y ₇ , Y ₈ and Y ₉ are hydrogen;

   The additional condition is that the above compound contains at least one deuterium atom.
3. The compound according to any one of claims 1-2, wherein Y ₁ is hydrogen.
4. The compound according to any one of claims 1-3, wherein R ₂ , R ₃ , R ₄ and R ₅ are hydrogen.
5. The compound according to any one of claims 1-4, wherein X ₅ is CH ₃ .
6. The compound according to any one of claims 1 to 5, wherein Y ₂ is hydrogen.
7. The compound according to any one of claims 1-6, wherein R ₁ is hydrogen.
8. The compound according to any one of claims 1 to 7, wherein X ₁ and X ₂ are CD ₃ .
9. The compound according to any one of claims 1 to 8, wherein X ₃ is CD ₃ .
10. The compound according to any one of claims 1-9, wherein X ₆ and X ₇ are each independently selected from CD ₃ , CD ₂ H, or CH ₃ .
11. The compound according to any one of claims 1 to 10, wherein X ₄ is CD ₃ .
12. The compound according to any one of claims 1-10, wherein the compound can be selected from any one of the following structures:

    

    
13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula (I) according to any one of claims 1-12, or a pharmaceutically acceptable salt, prodrug, hydrate or Solvent compounds, crystalline forms, stereoisomers, or isotopic variations.
14. The compound of formula (I) or a pharmaceutically acceptable salt, prodrug, hydrate or solvent compound, crystalline form, stereoisomer or isotope variant thereof according to any one of claims 1-15, or claim Use of the pharmaceutical composition according to any one of the claims 13 to 13 for preparing a medicament for treating a disease mediated by EGFR.
15. The use according to claim 14, wherein the EGFR-mediated disease is selected from cancer, cell proliferative disease, inflammation, infection, immune disease, organ transplant, viral disease, cardiovascular disease or metabolic disease.
16. The use according to claim 14, the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cancer, stomach Intestinal stromal tumor, leukemia, histiocytic lymphoma, and nasopharyngeal carcinoma.