WO2025098425A1 - Chemokine receptor modulator preparation method and use - Google Patents

Abstract

The present invention relates to the field of medicine, and provides a compound as shown in formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, a preparation method therefor, a pharmaceutical composition thereof, and a use thereof in preparation of a drug for preventing and/or treating CCR4-mediated diseases.

Description

Preparation method and use of chemokine receptor modulator Technical Field

The invention belongs to the field of medicine and relates to a compound for regulating the activity of a chemokine receptor, a preparation method and use thereof.

Background Art

The successful operation of the host defense system is the result of several processes working together to eliminate foreign pathogens. Coordinated innate immune responses and acquired immune responses are required, and many secreted factors and cell-related factors have been identified as important mediators for coordinating and regulating these two host defense weapons. Chemokines are a family of cytokines that guide leukocytes to move as chemical attractants. They are secreted by a variety of cells and can be functionally divided into two categories: hemostatic chemokines and inflammatory chemokines. Hemostatic chemokines are produced in certain tissues and control cells of the immune system during the immune surveillance process, such as directing lymphocytes to lymph nodes so that they can screen for pathogen invasion. Inflammatory chemokines are released by cells in response to pathological events (e.g., proinflammatory stimulants, such as IL-1 or viruses). It mainly acts as a chemical attractant as a part of the inflammatory response, and is used to guide cells of the innate immune system and the adaptive immune system to the site of inflammation. CC chemokine receptor type 4 (CCR4) plays a role in the progression of many inflammation-related and other diseases. Identifying compounds that regulate CCR4 function is a continuing challenge.

Cancer is a class of diseases in which cells exhibit dysregulated replication and growth. Recent cancer models implicate the immune system in the development and progression of cancer. While much progress has been made in understanding the biological basis of cancer, cancer remains a leading cause of death.

CCR4 was first identified by Power et al. (J. Biol. Chem. 270: 19495-19500) and is a G protein-coupled receptor that can bind to chemokines. CC chemokines are chemotactic agents for the Th2 subset of peripheral blood T cells, dendritic cells, and natural killer cells. Chemokines include CCL22 (also known as macrophage-derived chemokine, MDC). And CCL17 (also known as thymus and activation-regulated chemokine, TARC), which is also produced by monocytes and dendritic cells.

CCR4 is involved in immune regulatory processes such as homing of cells to specific tissues, including homing of T lymphocytes to the skin and lungs (see, e.g., Campbell et al., 1999, Nature, 400:776-780; Gonzalo et al., 1999, J. Immunol, 163:403-411; Lloyd et al., 2000, J. Exp. Med, 191:265-273; Kawasaki et al., 2000, J. Immunol. 166:2055-2062). Regulators of CCR4 activity have been described, e.g., in WO2013/082490.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is considered a key regulator of adaptive immune responses, particularly in maintaining burst T cell responses and components used in burst T cell responses. Therefore, CTLA-4, as an immune checkpoint inhibitor, is considered a possible therapeutic target for the treatment of cancer and inflammation. CTLA-4 modulators have been described, for example, in WO2018/035710.

Programmed death receptor-1 (PD-1) is a transmembrane receptor protein that negatively regulates the function of T cells through its interaction with its two natural ligands, PD-L1 and PD-L2. Similar to CTLA4, PD-1 is also a major regulator of the immune system and is also considered an immune checkpoint inhibitor. PD-1/PD-L1 modulators have been described, for example, in WO2018/005374.

Given the roles of cell homing and immune checkpoint pathways in cancer development and progression, there is a need to develop combination therapies that can improve cancer treatment.

Summary of the invention

The first aspect of the present invention relates to a compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof,

Wherein, Ring A is a 4-8 membered aromatic heterocycle;

^X1 or ^X2 are each independently N or CH;

^X3 is N or CR ^a ;

Y is -O- or -CH ₂ -;

^Ra is -H, _-C1-3 alkyl, -halogen or -CN, wherein the _-C1-3 alkyl is optionally substituted with one or more halogens within the range allowed by the valence;

L ¹ is a bond, -C _1-3 alkylene-, -C _2-4 alkenylene- or -C _2-4 alkynylene-;

^R1 is _{-C1-3alkylene} -OH;

^R2 is -H, -CN, _-C1-5 alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, the _-C1-5 alkyl is optionally substituted with one or more halogens within the range allowed by the valence, the 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl is optionally substituted with one or more _-C1-5 alkyl within the range allowed by the valence or -halogen;

^R3 is _-C1-5 alkyl;

R ⁴ are each independently halogen;

n is 0, 1, 2 or 3.

In some embodiments, ring A is a 5-6 membered aromatic heterocycle; preferably, the heteroatom of the 5-6 membered aromatic heterocycle is N, O or S.

In some embodiments, Ring A is wherein E ¹ and E ² are each independently CH, N, O or S, E ³ is a bond, CH or N, and E ¹ , E ² and E ³ are not CH at the same time.

In some embodiments, Ring A is

In some embodiments, ^E1 is S, N, O, ^E2 is CH, N, O or S, and ^E3 is a bond.

In some embodiments, ^E1 is O, ^E2 is N, and ^E3 is a bond; or ^E1 is N, ^E2 is O, and ^E3 is a bond; or ^E1 is S, ^E2 is N, and ^E3 is a bond; or ^E1 is N, ^E2 is S, and ^E3 is a bond; or ^E1 is S, ^E2 is CH, and ^E3 is a bond.

In some embodiments, E ¹ , E ² , and E ³ are each independently CH or N, and E ¹ , E ² , and E ³ are not CH at the same time.

In some embodiments, ^E1 is CH, ^E2 is N, and ^E3 is CH; or ^E1 is CH, ^E2 is CH, and ^E3 is N; or ^E1 is N, ^E2 is N, and ^E3 is CH; ^E1 is CH, ^E2 is N, and ^E3 is N; or ^E1 is N, ^E2 is N, and ^E3 is CH; ^E1 is N, ^E2 is N, and ^E3 is N.

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, n is 0, 1 or 2, preferably 2.

In some embodiments, ^X1 or ^X2 are not N at the same time; preferably, ^X1 is CH, ^X2 is N, and X3 is N; or ^{X1 is} CH, ^X2 is N, and ^X3 is CR ^a ; or X1 is N, ^X2 is CH, and X3 is N; or ^X1 is CH, ^X2 is CH, and X3 is N; or ^X1 is CH, ^X2 is CH, and ^X3 is N; or ^X1 is CH, ^X2 is CH, and ^X3 is CR ^a .

In some embodiments, ^Ra is -H or -CN.

In some embodiments, L ¹ is a bond, -C _2-4 alkenylene-, or -C _2-4 alkynylene-, preferably a bond, -C≡C-, or -CH=CH-.

In some embodiments, ^R1 _{is -CH2CH2OH} .

In some embodiments, ^R2 is -H, -CN, _-C1-5 alkyl, 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl, the _-C1-5 alkyl is optionally substituted with one or more halogens within the range allowed by the valence, the 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl is optionally substituted with one or more _-C1-3 alkyl or -halogens within the range allowed by the valence; preferably, the _-C1-5 alkyl is methyl or tert-butyl, the 3-5 membered cycloalkyl is cyclopropyl, and the 3-5 membered heterocycloalkyl is oxetanyl or azetidinyl.

In some embodiments, ^R2 is -H, -CN, methyl, tert-butyl, cyclopropyl, oxetanyl or azetidinyl, the methyl or tert-butyl group is optionally substituted with one or more -F groups within the range allowed by the valence, and the cyclopropyl, oxetanyl or azetidinyl group is optionally substituted with one or more methyl or -F groups within the range allowed by the valence.

In some embodiments, R ² is -H, -CN, -CH ₃ , -CHF ₂ , -CF ₃ , -C(CH ₃ ) ₃ , cyclopropyl,

In some embodiments, R ³ is -C _1-3 alkyl, preferably methyl.

In some embodiments, R ⁴ is each independently -F, -Cl or -Br, preferably -Cl.

In some embodiments, the compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, has the following structure:

Wherein, Y, L ¹ , R ² , E ¹ to E ³ , X ¹ to X ³ are as described in the compound of formula (I);

Preferably, Y is _-CH2- , ^X1 is CH, ^X2 is N, and ^X3 is N.

In some embodiments, the compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, has the following structure:

Wherein, R ³ is -H or -C _1-3 alkyl;

R ⁵ is a 5-6 membered heteroaryl group optionally substituted by one or more substituents; the substituents are each independently selected from hydrogen, halogen, -CN, hydroxyl, -C _1-3 alkyl or -C _1-3 haloalkyl, and the 5-6 membered heteroaryl group contains 1, 2 or 3 heteroatoms.

In some embodiments, the compound represented by formula (I), formula (II) or formula (III), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, the compound represented by formula (I), formula (II) or formula (III) is selected from the compounds represented by the following structural formulas:

In some embodiments, the compound represented by formula (I), formula (II) or formula (III), its stereoisomer, tautomer or its pharmaceutically acceptable salt With salt, the compound represented by formula (I), formula (II) or formula (III) is selected from the compounds represented by the following structural formula:

The second aspect of the present invention provides a method for preparing the compound described in the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, comprising the steps shown in any one of the following schemes 1 to 3:

Solution 1:

Solution 2: When X ³ is CR ^a

Solution 3: When X ³ is N

Wherein, each group is as described above.

The third aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers and/or excipients.

The fourth aspect of the present invention provides use of the compound described in the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a medicament for preventing and/or treating CCR4-mediated diseases.

In some embodiments of the invention, the CCR4-mediated disease comprises an inflammatory disease, an autoimmune disease or cancer.

In some embodiments of the present invention, the inflammatory disease and autoimmune disease are dermatitis, allergic asthma, inflammatory bowel disease or lupus erythematosus, and the cancer is liver cancer, pancreatic cancer, colon cancer, lung cancer, brain cancer or gastric cancer.

The fifth aspect of the present invention provides a method for treating CCR4-mediated diseases, comprising administering to a patient or individual in need of such treatment a therapeutically effective amount of any of the compounds described in the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described in the third aspect of the present invention.

DETAILED DESCRIPTION

Unless otherwise indicated, conventional methods within the technical scope of the art are used, such as mass spectrometry, NMR and pharmacological methods. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthesis chemistry, and drugs and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and in the treatment of patients. Conventionally, the above-mentioned techniques and methods can be implemented according to conventional methods well known in the art, based on the descriptions in multiple summaries and more specific documents cited and discussed in this specification.

Unless stated otherwise, the terms used in the specification and claims have the following meanings.

The term "optionally" includes both "optional" and "not selected". For example, "optionally substituted methyl" refers to "unsubstituted methyl" or "substituted methyl". The term "substituted" refers to the selection of one or more hydrogens on the specified atom or group by a specified group, provided that the normal valence of the specified atom is not exceeded. When the substituent is oxo or keto (i.e., =O), then 2 hydrogens on the atom are replaced.

The term "-C _1-n alkyl" (wherein n is an integer from 2 to n) refers to a non-cyclic, saturated, branched or straight-chain aliphatic hydrocarbon group having 1 to n carbon atoms, preferably an alkyl group having 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms (i.e., -C _1-12 alkyl), more preferably an alkyl group having 1 to 6 carbon atoms (i.e., -C _1-6 alkyl), and further preferably an alkyl group having 1 to 3 carbon atoms. For example, -C _1-3 alkyl refers to methyl, ethyl, n-propyl and isopropyl.

The term "-C1 _-n alkylene-" refers to a residue resulting from the removal of a hydrogen atom from a -C1 _-n alkyl group, which is a straight or branched chain group containing 1 to n carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms (i.e., _-C1-12 alkylene-), and more preferably an alkylene group containing 1 to 6 carbon atoms ₍ i.e., _-C1-6 alkylene-). For example, _-C1-3 alkylene- includes _-CH2- , _-CH2 - _CH2- , -CH( _CH3 )-, _{-CH2 -CH2} - _CH2- , -C( _CH3 ) _2- , -CH( _CH2CH3 )-, -CH( _CH3 ) _-CH2- , _-CH2 -CH( _CH3 )-, and the like. An alkylene group may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment.

The term "-C _2-n alkenyl" refers to a group containing at least one carbon-carbon double bond in a -C _2-n alkyl group. Preferably, the alkenyl group contains 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms (i.e., C _2-12 alkenyl), and more preferably contains 2 to ₆ carbon atoms (i.e., C _2-6 alkenyl). For example, the term -C _2-3 alkenyl includes -CH=CH ₂ , -CH=CH-CH ₃ , -CH ₂ -CH=CH ₂ . The alkenyl group may be substituted or unsubstituted, and when substituted, the substituent may optionally exist in cis or trans or E or Z form, for example:

The term "-C2 _-n alkenylene-" refers to a residue derived from a -C2 _-n alkenyl group by removing a hydrogen atom. For example, the term _-C2-3 alkenylene- includes -CH=CH-, -CH=CH- _CH2- , and _-CH2 -CH=CH-.

The term "-C _2-n -alkynyl" refers to a group of -C _2-n alkyl containing at least one carbon-carbon triple bond. For example, the term -C _2-3 alkynyl includes -C≡CH, -C≡C-CH ₃ , and -CH ₂ -C≡CH.

The term "-C _2-n alkynylene-" refers to a residue derived from a -C _2-n alkynyl group by removing a hydrogen atom. For example, -C _2-3 alkynylene- includes -C≡C-, -C≡C-CH ₂ -, and -CH ₂ -C≡C-.

Refers to the presence or absence of a key, e.g. Refers to a single key or double bond

The terms "aromatic heterocycle", "aromatic heterocyclic group", "heteroaromatic ring" or "heteroaromatic ring group" have the same meaning and refer to heterocyclic compounds with aromatic characteristics, including single heterocyclic aromatic groups and fused heterocyclic aromatic groups. Typical 5-6 membered heteroaryl groups include, but are not limited to, 2- or 3-thienyl; 2- or 3-furyl; 2- or 3-pyrrolyl; 2-, 4- or 5-imidazolyl; 3-, 4- or 5-pyrazolyl; 2-, 4- or 5-thiazolyl; 3-, 4- or 5-isothiazolyl; 2-, 4- or 5-oxazolyl; 3-, 4- or 5-isoxazolyl; 3- or 5-1,2,4-triazolyl; 4- or 5-1,2,3-triazolyl; tetrazolyl; 2-, 3- or 4-pyridyl; 3- or 4-pyridazinyl; 3-, 4- or 5-pyrazinyl; 2-pyrazinyl; 2-, 4- or 5-pyrimidinyl.

The term "cycloalkane" refers to a saturated monocyclic or polycyclic ring, and the term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic alkyl substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7 and 8) carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.

The term "heterocycloalkane" refers to a cycloalkane comprising at least one ring heteroatom (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom). The term "heterocycloalkyl" refers to a cycloalkyl comprising at least one ring heteroatom (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom). In some embodiments of the present invention, the heterocyclyl is a 4-12 membered heterocyclyl. In some embodiments of the present invention, the heterocyclyl is a 4-6 membered heterocyclyl, such as tetrahydrofuranyl, oxetane, or azetidine.

[0043] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

The term "isotopic derivative" refers to a derivative in which one or more atoms in a compound are replaced by an isotope thereof (an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature). Examples of isotopes include, but are not limited to, isotopes of hydrogen (e.g., ² H, ³ H), isotopes of carbon (e.g., ¹¹ C, ¹³ C, and ¹⁴ C), isotopes of fluorine (e.g., ¹⁸ F), isotopes of nitrogen (e.g., ¹³ N and ¹⁵ N), and isotopes of oxygen (e.g., ¹⁵ O, ¹⁷ O, and ¹⁸ O).

The terms "pharmaceutically acceptable salts" and "pharmaceutically acceptable salts" have the same meaning and refer to salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, etc. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

"Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of a compound to an individual and absorption by the individual and that can be included in the composition of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include: Examples include water, NaCl, physiological saline solution, lactated Ringer's, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and pigments, etc. Such preparations can be sterilized and, if necessary, can be mixed with auxiliary agents that do not adversely react with the compounds of the invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring and/or aromatic substances, etc. Those skilled in the art will recognize that pharmaceutical excipients are useful in the present invention.

The term "treatment" refers to any marker of successful treatment or improvement of an injury, disease, pathology, or condition, including any objective or subjective parameter, such as alleviation of symptoms; relief; reduction or making an injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the degenerative endpoint less debilitating; improving the patient's physical or mental health. Treatment or improvement of symptoms can be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and/or psychiatric evaluation. The term "treatment" and variations thereof may include prevention of an injury, pathology, condition, or disease. In embodiments, treatment is prevention. In embodiments, treatment does not include prevention.

"Treatment" as used herein (and as known in the art) also broadly includes any method that can achieve a beneficial or desired result (including a clinical result) in an individual's condition. Favorable or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms or symptoms, reduction of the extent of the disease, stabilization of the disease state (i.e., no worsening), prevention of disease transmission or spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence and remission, whether partial or complete and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, improvement or prevention of a disease. Treatment can prevent the disease from occurring; inhibit the spread of the disease; alleviate the symptoms of the disease, completely or partially remove the underlying cause of the disease, shorten the duration of the disease, or a combination of these events.

"Treatment" includes preventive treatment. The treatment method includes administering to an individual a therapeutically effective amount of a compound described herein. The administration step may include a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of the compound, the activity of the composition used for treatment, and combinations thereof. It should also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage can be obtained and become apparent through standard diagnostic analyses known in the art. In some instances, long-term administration may be required. For example, the composition is administered to an individual in an amount and duration sufficient to treat the patient.

The term "prevention" refers to reducing the occurrence of disease symptoms in patients. As mentioned above, prevention can be complete (no detectable symptoms) or partial prevention, so that the observed symptoms are less than the symptoms that may occur when not treated. In embodiments, prevention refers to slowing down the progress of a disease, illness or condition, or inhibiting its progression to a harmful or other undesirable state. "Patient" or "individual in need" refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administering a pharmaceutical composition provided herein. Non-limiting examples include people, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer and other non-mammals. In some embodiments, the patient is a person.

An "effective amount" is an amount sufficient to achieve the specified purpose of the compound (e.g., to achieve the effect of its administration, treat a disease, reduce enzyme activity, increase enzyme activity, reduce signaling pathways, or alleviate one or more symptoms of a disease or condition) relative to the absence of the compound. An "effective amount" example is an amount sufficient to promote the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Relief" of one or more symptoms (and grammatical equivalents of the phrase) means reducing the severity or frequency of the symptoms, or eliminating the symptoms. A "prophylactic effective amount" of a drug is an amount that has an expected preventive effect when the drug is administered to an individual, such as preventing or delaying the onset (or recurrence) of damage, disease, pathology, or condition, or reducing the likelihood of onset (or recurrence) of damage, disease, pathology, or condition or its symptoms. A complete preventive effect does not necessarily occur by administering one dose, and may only occur after a series of doses are administered. Therefore, a prophylactic effective amount may be administered during one or more administrations. As used herein, an "activity reduction amount" refers to the amount of antagonist required to reduce enzyme activity relative to the absence of an antagonist. As used herein, a "function-disrupting amount" refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amount will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). The therapeutically effective amount can be determined by measuring relevant physiological effects and can be adjusted in conjunction with individual dosing regimens and diagnostic analyses of the condition. For example, measuring serum levels of a CCR4 inhibitor (or, for example, a metabolite thereof) at a specific time after administration can indicate whether a therapeutically effective amount has been administered. A "CCR4 inhibitor" is a compound (eg, a compound described herein) that decreases CCR4 activity compared to a control (eg, a compound lacking the compound or known to be inactive).

The term "administering" means oral administration, suppository administration, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or implanting a sustained release device such as a micro-osmotic pump into a subject. Administration by any route, including parenteral and intramuscular administration, is contemplated. Membranes (e.g., oral, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal). Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other modes of delivery include, but are not limited to, the use of liposome preparations, intravenous infusions, transdermal patches, etc. "Combined administration" refers to the administration of the compositions described herein at the same time, just before or just after the administration of one or more additional therapies (e.g., anticancer agents, chemotherapy or treatment of neurodegenerative diseases). The compounds of the present invention can be administered alone or can be administered to the patient in combination. When necessary, the preparation can also be combined with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered transdermally by a local route and can be formulated into smear sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, coatings, powders and aerosols. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, and the like suitable for ingestion by a patient. Solid form formulations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form formulations include solutions, suspensions, and emulsions, such as aqueous solutions or water/propylene glycol solutions. The compositions of the present invention may additionally contain components that provide sustained release and/or comfort. Such components include high molecular weight anionic mucomimetic polymers, gelling polysaccharides, and finely divided drug carrier matrices, as discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention may also be delivered as microspheres for slow release in vivo. For example, the microspheres can be administered by intradermal injection of drug-containing microspheres that are slowly released subcutaneously (see Rao, J. Biomater Sci. Polym. 7th edition, 623-645, 1995; can be administered as a biodegradable and injectable gel formulation (see, e.g., Gao Pharm. Res. 12: 857-863, 1995); or as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49: 669-674, 1997). In another embodiment, the formulation of the composition of the present invention can be delivered by using liposomes that fuse with cell membranes or are internalized by cells, that is, by using receptor ligands linked to liposomes that bind to surface membrane protein receptors of cells, thereby causing endocytosis. By using liposomes, especially carrying In the case of receptor ligands specifically targeting target cells or preferentially involving specific organs, the composition of the present invention can be delivered to the target cells in vivo. (See, for example, Al-Muhammed, J. Microencapsul. 13: 293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6: 698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The composition of the present invention can also be delivered as nanoparticles.

The present invention is further described by way of the following examples, which are illustrative and do not limit the present invention in any way. Any modifications or changes that are easily realized by a person of ordinary skill in the art to the present invention will fall within the scope of the present invention.

Unless otherwise specified, the raw materials or reagents used in the examples of the present invention are commercially available.

The abbreviations used in the present invention have the conventional meanings in the art, for example, the meanings of the following abbreviations are as follows:

Preparation method of compound

Example 1: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (1)

The first step is the synthesis of tert-butyl (R)-3-(1-(2-hydroxyethyl)piperidin-3-yl)azetidine-1-carboxylate 1c

Compound 1a (4.0 g, 17 mmol) was dissolved in acetonitrile (40 mL), and compound 1b (4.3 g, 25 mmol) and cesium carbonate (8.1 g, 25 mmol) were added and stirred at room temperature overnight. After the reaction was completed, the mixture was dried by spin drying, ethyl acetate was added, washed with water, and the aqueous phase was extracted with ethyl acetate twice. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and dried by spin drying to obtain compound 1c (4.7 g). LCMS (ESI-MS) m/z: 285.1 [M+H] ⁺ .

Step 2 Synthesis of (R)-2-(3-(azetidin-3-yl)piperidin-1-yl)ethane-1-ol trifluoroacetate 1d

Compound 1c (4.7 g, 17 mmol) was dissolved in DCM (20 mL), TFA (10 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was detected by TLC and developed by iodine flask. After the reaction was completed, the mixture was dried by spin drying and toluene was used to dry the mixture to obtain compound 1d (8.2 g). LCMS (ESI-MS) m/z: 185.1 [M+H] ⁺ .

Step 3 Synthesis of 4,6-dichloro-N-methoxy-N-methylnicotinamide 1f

Compound 1e (10 g, 52 mmol) was dissolved in DMF (40 mL), methoxymethylamine (4.8 g, 79 mmol), DIPEA (14 g, 100 mol) were added, HATU (30 g, 79 mmol) was added in batches, and stirred at room temperature for 1 hour. The reaction was completed by LCMS detection, and the mixture was extracted with ethyl acetate. The organic phase was passed through a flash column (petroleum ether/ethyl acetate = 0-20%) to obtain compound 1f (10 g). LCMS (ESI-MS) m/z: 235.0 [M+H] ⁺ .

Step 4 Synthesis of 1-(4,6-dichloropyridin-3-yl)ethane-1-one 1g

Compound 1f (10 g, 43 mmol) was dissolved in THF (5.0 mL), methylmagnesium bromide (29 mL, 86 mmol) was added in an ice bath, and the reaction was stirred for 1 hour. After the reaction was basically completed, the mixture was quenched with a saturated aqueous solution of ammonium chloride, extracted with ethyl acetate, and the organic phase was washed with water and then washed with saturated brine through a Flash column (petroleum ether/ethyl acetate = 0-10%) to obtain compound 1g (7.0 g). LCMS (ESI-MS) m/z: 190.0 [M+H] ⁺ .

Step 5 Synthesis of (S)-1-(4,6-dichloropyridin-3-yl)ethane-1-ol 1h

Compound 1g (3.5g, 19mmol) was dissolved in THF (20mL) at -78°C, (-)-IPC ₂ Bci (13mL, 22mmol) was slowly added dropwise within half an hour, and the temperature was slowly raised to room temperature and stirred for 3 hours. After LCMS detection, the reaction was completed, quenched with water, extracted with ethyl acetate, the organic phase was washed with water, and saturated The mixture was washed with brine, dried over anhydrous sodium sulfate and passed through a Flash column to obtain compound 1h (3.3 g). LCMS (ESI-MS) m/z: 192.0 [M+H] ⁺ .

Step 6 Synthesis of (R)-6-chloro-1-(1-(4,6-dichloropyridin-3-yl)ethyl)-3-iodo-1H-pyrazolo[3,4-b]pyrazine 1j

Compound 1i (4.0 g, 14 mmol) was dissolved in THF (20 mL), and compound 1h (3.3 g, 17 mmol) and PPh ₃ (4.3 g, 21 mmol) were added under ice bath, and DIAD (5.6 g, 21 mmol) was added under nitrogen protection, and stirred at room temperature overnight. After LCMS detection, the reaction was completed, quenched with water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried with anhydrous sodium sulfate, and passed through a Flash column to obtain compound 1j (3.0 g). LCMS (ESI-MS) m/z: 454.0 [M+H] ⁺ .

Step 7 Synthesis of 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-iodo-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethane-1-ol 1k

Compound 1j (2.5 g, 5.5 mmol) was dissolved in acetonitrile (30 mL), and compound 1d (59%, 3.9 g, 8.3 mmol) and cesium carbonate (9.0 g, 28 mmol) were added under ice bath, and stirred at room temperature for 2 hours. After LCMS detection, the reaction was completed, quenched with water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and passed through a Flash column to obtain compound 1k (2.2 g). LCMS (ESI-MS) m/z: 602.2 [M+H] ⁺ .

Step 8 Synthesis of 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-((trimethylsilyl)ethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethane-1-ol 11

Compound 1k (2.2 g, 3.7 mmol) was dissolved in THF (25 mL), and Pd(PPh ₃ )Cl ₂ (260 mg, 0.37 mmol), CuI (140 mg, 0.73 mmol), and triethylamine (740 mg, 7.3 mmol) were added. A THF (5.0 mL) solution of TMS alkyne (1.4 g, 15 mmol) was added under nitrogen protection, and the mixture was heated at 40°C for 15 minutes. After the reaction was completed, the mixture was filtered and dried by passing through a Flash column to obtain compound 1l (1.6 g). LCMS (ESI-MS) m/z: 572.3 [M+H] ⁺ .

Step 9 Synthesis of 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethane-1-ol 1

Compound 11 (1.1 g, 1.8 mmol) was dissolved in methanol (10 mL), K ₂ CO ₃ (770 mg, 5.6 mmol) was added, and stirred at room temperature for ten minutes. After the reaction was completed, the mixture was filtered, dried, and passed through a flash column to obtain 600 mg of the target compound, which was then sent to SFC for separation to finally obtain compound 1 (430 mg). LCMS (ESI-MS) m/z: 500.2 [M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d6) δ8.57(s,1H),7.89(s,1H),7.81(s,1H),6.20(m,1H),4.53(s,1H),4.33(m,1H),4.16(m,2H),3.87( m,2H),3.48(m,2H),2.75(m,2H),2.56(m,2H),2.36(m,2H),1.89(m,1H),1.85(d,J＝8.0Hz,3H),1.64(m,4H),1.43(m,1H).

Example 2: 2-((3R)-3-(1-(1-(2-(2,6-dichloropyridin-3-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (2)

The preparation of compound 2 refers to Example 1. LCMS (ESI-MS) m/z: 500.30 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ7.98(dd,J＝8.2,3.1Hz,1H),7.88(s,1H),7.59(d,J＝8.1Hz,1H),6.17(q,J＝7.0Hz,1 H),4.53(s,1H),4.16(d,J＝9.3Hz,2H),3.87(s,3H),3.47(t,J＝6.3Hz,2H),2.80–2.68( m,2H),2.54(s,1H),2.35(t,J＝6.3Hz,2H),1.92(t,J＝11.1Hz,1H),1.80(d,J＝7.0Hz,3H ),1.66(td,J＝30.0,27.3,13.5Hz,4H),1.43(t,J＝11.8Hz,1H),0.82(d,J＝12.0Hz,1H).

Example 3: 2-((3R)-3-(1-(1-(2-(2,4-dichloropyrimidin-5-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (3)

The preparation of compound 3 refers to Example 1. LCMS (ESI-MS) m/z: 501.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ9.03(d,J＝3.0Hz,1H),7.90(s,1H),6.12(q,J＝7.0Hz,1H),4.57(s,1H),4.17(s,2H),3.87(s,2H),3.54(t,J＝6.0Hz,2H),2.93(d,J＝10.8H z,2H),2.61–2.51(m,3H),2.15(d,J＝11.8Hz,1H),1.85(d,J＝7.1Hz,5H),1.65(d,J＝15.1Hz,3H),1.50(d,J＝12.7Hz,1H),0.94–0.82(m,1H).

Example 4: 2-((3R)-3-(1-(1-(4-(4,6-dichloropyridazin-3-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (4)

The preparation of compound 4 was carried out according to Example 1. LCMS (ESI-MS) m/z: 501 [M+H] ⁺ .

Example 5: 2-((3R)-3-(1-(1-(3-(3,5-dichloropyrazin-2-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (5)

The preparation of compound 5 refers to Example 1. LCMS (ESI-MS) m/z: 501.40 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ )δ8.87(s,1H),7.88(s,1H),6.25(q,J＝6.8Hz,1H),4.50(s,1H),4.35(s,1H) ,4.12(s,2H),3.82(s,2H),3.48(t,J＝6.3Hz,2H),2.75(d,J＝10.5Hz,2H),2.5 5(d,J＝7.0Hz,1H),2.36(t,J＝6.3Hz,2H),1.94(t,J＝11.2Hz,1H),1.81(d,J＝6 .8Hz,3H),1.75–1.55(m,4H),1.42(d,J＝12.1Hz,1H),0.83(d,J＝11.3Hz,1H).

Example 6: 2-((3R)-3-(1-(1-(3-(3,5-dichloro-1,2,4-triazin-6-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl]azetidin-3-yl)piperidin-1-yl)ethan-1-ol (6)

The preparation of compound 6 refers to Example 1. LCMS (ESI-MS) m/z: 502 [M+H] ⁺ .

Example 7: 2-((3R)-3-(1-(1-(3-(3,5-dichloropyridin-2-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (7)

The preparation of compound 7 was carried out according to Example 1. LCMS (ESI-MS) m/z: 500.4 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.61(d,J＝2.2Hz,1H),8.23(d,J＝2.2Hz,1H),7.88(s,1H),6.27(q,J＝6.8Hz,1H),4 .49(s,1H),4.15(s,2H),3.85(s,2H),3.65(s,1H),3.50(t,J＝6.2Hz,2H),2.79(d,J＝1 0.1Hz,2H),2.55(d,J＝7.7Hz,1H),2.41(t,J＝6.3Hz,2H),1.98(t,J＝10.9Hz,1H),1.8 1(d,J＝6.9Hz,3H),1.77–1.52(m,4H),1.46(t,J＝12.2Hz,1H),0.84(d,J＝11.8Hz,1H).

Example 8: 2-((3R)-3-(1-(1-(2-(2,5-dichloro-1λ ³ ,3λ ² -oxazol-4-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (8)

The preparation of compound 8 refers to Example 1. LCMS (ESI-MS) m/z: 490 [M+H] ⁺ .

Example 9: 2-((3R)-3-(1-(1-(2-(2,4-dichlorooxazol-5-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (9)

The preparation of compound 9 refers to Example 1. LCMS (ESI-MS) m/z: 490 [M+H] ⁺ .

Example 10: 2-((3R)-3-(1-(1-(2-(2,4-dichlorothiazol-5-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (10)

The preparation of compound 10 refers to Example 1. LCMS (ESI-MS) m/z: 506 [M+H] ⁺ .

Example 11: 2-((3R)-3-(1-(1-(2-(2,5-dichlorothiazol-4-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (11)

The preparation of compound 11 refers to Example 1. LCMS (ESI-MS) m/z: 506 [M+H] ⁺ .

Example 12: 2-((3R)-3-(1-(1-(2-(2,5-dichlorothien-3-yl)ethyl)-3-ethynyl-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (12)

The preparation of compound 12 refers to Example 1. LCMS (ESI-MS) m/z: 505.3 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.01(s,1H),7.23(s,1H),6.04-6.01(m,1H),5.32(s,1H),4.72(s,1H),4.19(s2H),3.90-3.88(m,2H),3.57-3.55(m,2H),3.01 (s,2H),2.65(s,2H),2.58-2.54(m,2H),1.89(s,1H),1.80-1.79(d,3H),1.74-1.71(m,2H),1.54-1.52(m,2H),0.94-0.91(m,1H).

Example 13: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(prop-1-yn-1-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (13)

The preparation of compound 13 refers to Example 1. LCMS (ESI-MS) m/z: 514 [M+H] ⁺ .

Example 14: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(3,3-dimethylbut-1-yn-1-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (14)

The preparation of compound 13 refers to Example 1. LCMS (ESI-MS) m/z: 556.23 [M+H] ⁺ .

Example 15: 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]pyrazolo[4,3-b]pyrazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethan-1-ol (15)

The preparation of compound 15 refers to Example 1. LCMS (ESI) m/z: 540.40 [M+H] ⁺ .

Example 16: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(oxetan-3-ylethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (16)

The preparation of compound 16 refers to Example 1. LCMS (ESI-MS) m/z: 556.23 [M+H] ⁺ .

Example 17: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-((1-methylazetidin-3-yl)ethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (17)

The preparation of compound 17 refers to Example 1. LCMS (ESI-MS) m/z: 569.23 [M+H] ⁺ .

Example 18: 2-((3R)-3-(1-(1-((R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-((2,2-difluorocyclopropyl)ethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (18)

The preparation of compound 18 refers to Example 1. LCMS (ESI-MS) m/z: 576.23 [M+H] ⁺ .

Example 19: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (19)

Step 1 Synthesis of (R)-6-chloro-1-(1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazine 19a

Compound 1j (5.0 g, 11 mmol), methyl fluorosulfonyl difluoroacetate (4.2 g, 22 mmol), hexamethylphosphoric triamide (7.9 g, 44 mmol), CuI (2.1 g, 11 mmol) were added to a single-mouth bottle, and DMF (50 mL) was added to replace nitrogen. The reaction mixture was heated at 100 ° C for 4 h. The reaction solution was cooled to room temperature and poured into water to precipitate solids. The solids were filtered and washed with dichloromethane three times. The organic phases were combined and concentrated through a flash column (mobile phase PE: EA = 5: 1) to obtain compound 19a (3.5 g). LCMS (ESI-MS) m/z: 396.1 [M + H] ⁺ .

Step 2 Synthesis of 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol 19

Compound 19a (1.0 g, 2.5 mmol) and compound 1d (0.46 g, 2.5 mmol) were dissolved in acetonitrile (40 mL), cesium carbonate (4.1 g, 13 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Water was added to quench the mixture, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and concentrated through a flash column (DCM: MeOH = 10:1) to obtain compound 19 (660 mg). LCMS (ESI-MS) m/z: 544.28 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.58(s,1H),8.01(s,1H),7.84(s,1H),6.26(q,J＝7.0Hz,1H),4.19(s,2H),3.90(s,2H),3.53(s,2H),2.98–2.74( m,2H),2.56(d,J＝8.1Hz,2H),1.89(d,J＝7.1Hz,4H),1.80(s,1H),1.69(d,J＝13.4Hz,3H),1.53(s,2H),0.88(s,2H).

Example 20: ((R)-3-(1-(1-(R)-1-(3,5-dichloropyridin-2-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (20)

The preparation of Compound 20 refers to Example 19. LCMS (ESI) m/z: 544.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.61(s,1H),8.27(s,1H),7.99(s,1H),6.33(q,J＝6.8Hz,1H),4.38(t,J＝5.6Hz,1H) ,4.16(s,2H),3.86(s,2H),3.49(t,J＝5.8Hz,2H),2.75(d,J＝10.6Hz,2H),2.55(dt,J＝ 9.0,4.8Hz,1H),2.35(t,J＝6.4Hz,2H),1.95–1.88(m,1H),1.85(d,J＝6.8Hz,3H),1.65 (tt,J＝17.8,9.2Hz,4H),1.42(dd,J＝25.0,13.4Hz,1H),0.81(dd,J＝23.2,10.8Hz,1H).

Example 21: 2-[(3R)-3-(1-{1-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-(3,3,3-trifluoroprop-1-ynyl)pyrazolo[4,3-b]pyrazin-6-yl}azetidin-3-yl)hexahydropyridin-1-yl]ethan-1-ol (21)

Step 1 Synthesis of 2-[(3R)-3-(1-{1-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-(3,3,3-trifluoroprop-1-ynyl)pyrazolo[4,3-b]pyrazin-6-yl}azetidin-3-yl)hexahydropyridin-1-yl]ethan-1-ol (21)

Take a single-mouth bottle, add TMEDA (350mg, 3.0mmol) dissolved in DMF (2.0mL), add cuprous iodide (570mg, 3.0mmol) and potassium carbonate (830mg, 6.0mmol) in turn at room temperature. Stir vigorously for 20min under normal temperature air atmosphere. Add TMSCF ₃ (570mg, 4.0mmol), and stir for 15min under normal temperature air atmosphere. The reaction turns dark green. Cool to 0℃, add a mixed solution of compound 1 (1000mg, 2.0mmol) and TMSCF ₃ (570mg, 4.0mmol) in DMF (2.0mL). React at 0℃ for 30min. Warm to room temperature and react for 6h. Quench with saturated ammonium chloride (10mL), add ethyl acetate (50ml), wash the organic phase with water, and dry over anhydrous sodium sulfate. Concentrate under reduced pressure. Purify by preparative HPLC (prepared by ammonium bicarbonate system) to obtain compound 21 (62mg). LCMS(ESI)m/z:568.40[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.60(s,1H),7.98(s,1H),7.83(s,1H),6.26(q,J＝7.0Hz,1H),4.34(t,J＝5.4Hz,1H),4.18(s,2H),3.89(s,2H),3.46(q,J＝6.0Hz,2H), 2.74-2.72(m,2H),2.56-2.54(m,1H),2.34(t,J=6.4Hz,2H),1.93-1.87(m,4H),1.73-1.56(m,4H),1.42-1.39(m,1H),0.82-0.80(m,1H).

Example 22: 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]pyrazolo[4,3-b]pyrazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethan-1-ol (22)

The preparation of Compound 22 refers to Example 1. LCMS (ESI) m/z: 540.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.57(s,1H),7.85(d,J＝0.8Hz,1H),7.83(d,J＝0.8Hz,1H),6.18(q,J＝7.0Hz,1H),4.36 (t,J＝5.4Hz,1H),4.21–4.12(m,2H),3.87(s,2H),3.48(q,J＝6.0Hz,2H),2.78–2.72(m,2 H),2.59–2.53(m,1H),2.35(t,J＝6.4Hz,2H),1.91(t,J＝10.8Hz,1H),1.84(d,J＝7.2Hz,3 H),1.78–1.57(m,6H),1.47–1.38(m,1H),0.95–0.90(m,2H),0.79(dd,J=5.4,2.4Hz,2H).

Example 23: 2-((R)-3-(1-(1-(R)-1-(3,5-dichloropyridin-2-yl)ethyl)-3-vinyl-1H-pyrazolo[4,3-b]pyridin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (23)

Step 1 (R)-6-Bromo-1-(1-(3,5-dichloropyridin-2-yl)ethyl)-3-vinyl-1H-pyrazolo[4,3-b]pyridine (23c)

Compound (R)-6-bromo-1-(1-(3,5-dichloropyridin-2-yl)ethyl)-3-iodo-1H-pyrazolo[4,3-b]pyridine 23a (500 mg, 1.0 mmol) was dissolved in 1,4-dioxane/water (5.0 mL/1.0 mL), and cesium carbonate (650 mg, 2.0 mmol) and methanesulfonic acid [(4-(N,N-dimethylamino)phenyl]di-tert-butylphosphine (2-amino-1,1-biphenyl-2-yl) palladium (250 mg, 0.35 mmol) were added at room temperature, and nitrogen was replaced. Ethylene was added in an ice bath. Boronic acid pinacol ester 23b (170 mg, 1.1 mmol) was stirred at room temperature overnight. The mixture was quenched with water, extracted with ethyl acetate, and the organic phase was dried, filtered, and the filtrate was concentrated and purified by silica gel column (petroleum ether: ethyl acetate = 25:1) to give compound 23c (200 mg). LCMS (ESI-MS) m/z: 397.14 [M+H] ⁺ .

Step 4: 2-((R)-3-(1-(1-(R)-1-(3,5-dichloropyridin-2-yl)ethyl)-3-vinyl-1H-pyrazolo[4,3-b]pyridin-6-yl)azetidin-3-yl)piperidin-1-yl)ethane-1-ol (23)

Compound 23c (150 mg, 0.38 mmol) and compound 1d (320 mg, 1.74 mmol) were dissolved in N,N-dimethylformamide (3.0 mL), and cesium carbonate (250 mg, 0.76 mmol) and methanesulfonic acid (4,5-bis(diphenylphosphine)-9,9-dimethyloxanthene) (2-amino-1,1'-biphenyl-2-yl) palladium (36 mg, 0.038 mmol) were added at room temperature, and nitrogen was replaced. The mixture was heated at 100°C for 3 hours. The mixture was quenched with water, extracted with ethyl acetate, the organic phase was dried, filtered, and the filtrate was concentrated. The crude product was purified by preparative liquid phase (ammonium bicarbonate) to obtain compound 23 (13 mg). LCMS (ESI-MS) m/z: 501.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.68(d,J＝2.2Hz,1H),8.23(d,J＝2.2Hz,1H),7.92(d,J＝2.4Hz,1H),6.83–6.76(m,2H),6. 58(dd,J＝17.8,2.6Hz,1H),6.25(d,J＝6.8Hz,1H),5.49(dd,J＝11.2,2.6Hz,1H),4.05–3.98( m,2H),3.67(td,J＝8.8,8.4,4.0Hz,2H),3.57(s,2H),2.93(s,2H),2.62–2.53(m,2H),1.91– 1.83(m,1H),1.80(d,J＝6.8Hz,3H),1.78–1.61(m,4H),1.60–1.44(m,2H),1.02–0.81(m,2H).

Example 24: 1-(((R)-1-(3,5-dichloropyridin-2-yl)ethyl)-6-(3-(((R)-1-(2-hydroxyethyl)piperidin-3-yl)azetidin-1-yl)-1H-pyrazolo[4,3-b]pyridine-3-carbonitrile (24)

First step (R)-6-bromo-1-(1-(3,5-dichloropyridin-2-yl)ethyl)-1H-pyrazolo[4,3-b]pyridine-3-carbonitrile (24a)

Compound 23a (500 mg, 1.0 mmol) was dissolved in N,N-dimethylacetamide (6.0 mL), and zinc cyanide (61 mg, 0.52 mmol), tris(dibenzylideneacetone)dipalladium (81 mg, 0.10 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (58 mg, 0.10 mmol) and tetramethylethylenediamine (35 mg, 0.30 mmol) were added at room temperature, and nitrogen was replaced. The reaction was heated at 90°C for 1 hour. Water was added to quench, and the organic phase was extracted with ethyl acetate. The organic phase was dried and filtered, and the filtrate was concentrated and purified by silica gel column (petroleum ether: ethyl acetate = 25:1) to obtain compound 24a (220 mg). LCMS (ESI-MS) m/z: 396.05 [M+H] ⁺ .

Step 2 1-(((R)-1-(3,5-dichloropyridin-2-yl)ethyl)-6-(3-(((R)-1-(2-hydroxyethyl)piperidin-3-yl)azetidin-1-yl)-1H-pyrazolo[4,3-b]pyridine-3-carbonitrile (24)

Compound 24a (150 mg, 0.38 mmol) and 1d (320 mg, 1.7 mmol) were dissolved in toluene (1.2 mL), and cesium carbonate (190 mg, 0.57 mmol), (2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl) [2-(2-aminoethylphenyl)] palladium chloride methyl tert-butyl ether complex (31 mg, 0.038 mmol) and 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl (18 mg, 0.038 mmol) were added at room temperature, and nitrogen was replaced. The mixture was heated at 100°C for 3 hours. The mixture was quenched with water, extracted with ethyl acetate, the organic phase was dried, filtered, and the filtrate was concentrated. The crude product was purified by preparative liquid phase (ammonia water) to obtain compound 24 (4.2 mg). LCMS (ESI-MS) m/z: 500.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.68(d,J＝2.2Hz,1H),8.30(d,J＝2.2Hz,1H),8.09(d,J＝2.2Hz,1H),6.94(t,J＝2.0Hz ,1H),6.45(d,J＝6.8Hz,1H),4.37(s,1H),4.08–4.03(m,2H),3.73(d,J＝8.4Hz,2H),3.49 (t,J＝6.0Hz,2H),2.80–2.74(m,2H),2.57(d,J＝7.8Hz,1H),2.39–2.34(m,2H),1. 85(d,J＝6.8Hz,3H),1.72–1.62(m,4H),1.48–1.40(m,2H),0.86(d,J＝5.4Hz,1H).

Example 25: 1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]-6-{3-[(3R)-1-[(2-hydroxyethyl)hexahydropyridin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyridine-3-carbonitrile (25)

Step 1 Synthesis of 6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine 25b

Take a 50mL flask, add 6-bromo-1H-pyrrolo[3,2-b]pyridine 25a (5.0g, 25mmol), DMF (25mL), stir in an ice bath for 10min, add potassium hydroxide (1.1g, 20mmol), iodine (2.6g, 10mmol), warm to room temperature for 5 hours, add saturated sodium bisulfite aqueous solution in an ice bath to quench the reaction, extract with ethyl acetate (400mL*3), wash with saturated sodium chloride aqueous solution (200mL), dry with anhydrous sodium sulfate, spin dry the solvent to obtain compound 25b (6.2g). LCMS (ESI-MS) m/z: 323.0 [M+H] ⁺ .

Step 2 Synthesis of 6-bromo-1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]-3-iodopyrrolo[3,2-b]pyridine 25d

Take a 25mL three-necked flask, add compound 25b (1.1g, 3.3mmol), dichloromethane (6.0mL), (1R)-1-(3,5-dichloropyridin-2-yl)ethane-1-ol 25c (0.63g, 3.3mmol), triphenylphosphine (1.4g, 5.3mmol), protect with nitrogen, stir and react in an ice bath for 10min, add DIAD (1.1g, 5.6mmol), continue to stir and react for 2 hours, pour into water, extract with DCM three times (20mL*3), wash with saturated sodium chloride aqueous solution (30mL), dry with anhydrous sodium sulfate, purify with Flash column to obtain compound 25d (1.6g). LCMS (ESI-MS) m/z: 496.04 [M+H] ⁺ .

Step 3 Synthesis of 6-bromo-1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]pyrrolo[3,2-b]pyridine-3-carbonitrile 25e

Take a 100mL flask, add 25d (1200mg, 2.4mmol), DMA (10mL), Zn(CN) ₂ (140mg, 1.2mmol), Pd ₂ (dba) ₃ (220mg, 0.24mmol), XANTPHOS (140mg, 0.24mmol), 2,5-dimethyl-2,5-diazahexane (83mg, 0.71mmol), protect with nitrogen, stir and react at 70℃ for 2h, monitor by LCMS, the reaction of raw materials is complete, pour the reaction solution into water (60mL), add saturated NaHCO ₃ aqueous solution (20mL), add EDTA (0.50g), extract with EA three times (30mL*3), wash with saturated sodium chloride aqueous solution (60mL), dry with anhydrous sodium sulfate, and purify with Flash column to obtain compound 25e (390mg). LCMS(ESI-MS)m/z:395.13[M+H] ⁺ .

Step 4 Synthesis of 1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]-6-{3-[(3R)-1-[(2-hydroxyethyl)hexahydropyridin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyridine-3-carbonitrile 25

Take 75mL sealed tube, add compound 25e (220mg, 1.2mmol), tert-butyl alcohol (15mL), EPhos Pd G4 (110mg, 0.12mmol), EPhos (65mg, 0.12mmol), cesium carbonate (390mg, 1.2mmol), protect with nitrogen, heat at 85℃ for 6 hours, monitor by LCMS, the raw material reacts completely, filter with diatomaceous earth, spin dry the solvent, prepare by HPLC, and obtain compound 25 (16mg). LCMS (ESI-MS) m/z: 250.20 [M/2+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.63(d,J＝2.1Hz,1H),8.37(s,1H),8.30(d,J＝2.2Hz,1H),7.82(d,J＝2.3Hz,1H),6.80(d,J＝2. 4Hz,1H),6.21(q,J＝6.8Hz,1H),4.36(s,1H),3.93(dt,J＝13.3,7.6Hz,2H),3.56(q,J＝7.1Hz,2H) ,3.48(t,J＝6.4Hz,2H),2.75(t,J＝6.9Hz,2H),2.36(t,J＝6.3Hz,2H),1.91(dt,J＝11.8,6.5Hz,1H ),1.83(d,J＝6.8Hz,3H),1.76–1.54(m,5H),1.42(d,J＝12.3Hz,1H),0.84(dd,J＝17.2,7.6Hz,1H).

Example 26: 5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-{3-[(3R)-1-(2-hydroxyethyl)piperidin-3-yl]azetidin-1-yl}-7-(trifluoromethyl)pyrrolo[3,2-b]pyrazine-6-carbonitrile (26)

Step 1 Synthesis of 3-chloro-5-[(4-methylphenyl)dioxo-λ6-thio]pyrrolo[3,2-b]pyrazine 26b

Take a single-mouth bottle, dissolve compound 26a (5.0 g, 33 mmol) in DCM (50 mL), add triethylamine (23 mL, 160 mmol), p-toluenesulfonyl chloride (10 g, 39 mmol), DMAP (0.80 g, 6.5 mmol) at room temperature. React overnight at room temperature. Add water, extract with DCM, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify on a silica gel column (ethyl acetate: petroleum ether = 10-30%) to obtain compound 26b (9.0 g). LCMS (ESI-MS) m/z = 308.14 [M+H] ⁺

Step 2 Synthesis of 3-chloro-5-[(4-methylphenyl)dioxo-λ6-thio]pyrrolo[3,2-b]pyrazine-6-carbonitrile 24c

Take a three-necked flask, dissolve compound 26b (1.5 g, 4.9 mmol) in tetrahydrofuran (5.0 mL), replace with nitrogen three times at room temperature, add n-butyl lithium (2.9 mL, 7.3 mmol) dropwise under dry ice ethyl acetate bath, react for 1 h, add 4-toluenesulfonyl cyanide (1.8 g, 9.7 mmol) in tetrahydrofuran (5.0 mL) dropwise. React at the same temperature for 30 min, and react overnight at room temperature. Add saturated ammonium chloride aqueous solution, extract with ethyl acetate three times, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify on a silica gel column (ethyl acetate: petroleum ether = 10-30%) to obtain compound 26c (410 mg). LCMS (ESI-MS) m/z = 333.14 [M+H] ⁺

Step 3 Synthesis of 3-chloro-5H-pyrrolo[3,2-b]pyrazine-6-carbonitrile 26d

Take a single-mouth bottle, add compound 26c (2.7 g, 8.2 mmol) in turn, suspend in methanol (30 mL), add potassium carbonate (2.3 g, 16 mmol) at room temperature, and react at 50°C for 1 h. After it becomes transparent and clear, evaporate the solvent under reduced pressure. Add water, extract with ethyl acetate, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Compound 26d (1.7 g, 9.6 mmol) is obtained and used directly in the next step. LCMS (ESI-MS) m/z = 178.99 [M + H] ⁺

Step 4 Synthesis of 3-chloro-7-iodo-5H-pyrrolo[3,2-b]pyrazine-6-carbonitrile 26e

Take a single-mouth bottle, add compound 26d (1.7 g, 9.6 mmol) in turn, dissolve in DMF (30 mL), add potassium hydroxide (2.0 g, 35 mmol) in batches, and react at room temperature for 30 min. Add iodine (5.0 g, 19 mmol) in DMF (1.0 mL) under ice-water bath conditions. React at room temperature for 20 h. Add saturated sodium sulfite aqueous solution to quench excess iodine, extract three times with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure. Compound 26e (810 mg) is obtained and used directly in the next step. LCMS (ESI-MS) m/z = 304.98 [M + H] ⁺

Step 5 Synthesis of 3-chloro-5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-7-iodopyrrolo[3,2-b]pyrazine-6-carbonitrile 26f

Take a three-necked flask, add compound 1h (190 mg, 1.0 mmol), compound 26e (300 mg, 1.0 mmol), replace with nitrogen at room temperature, add triphenylphosphine (520 mg, 2.0 mmol) in DCM (1.0 mL) solution under ice-water bath, and add DIAD (500 mg, 2.5 mmol) in DCM (1.0 mL) solution at the same temperature for 2 h. Add methanol and evaporate the solvent under reduced pressure. Purify on a silica gel column (ethyl acetate: petroleum ether = 10%) to obtain 26f (380 mg). LCMS (ESI-MS) m/z = 478.07 [M+H] ⁺

Step 6: Synthesis of 26 g of 3-chloro-5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-7-(trifluoromethyl)pyrrolo[3,2-b]pyrazine-6-carbonitrile

Take a single-mouth bottle, add compound 26f (160 mg, 0.33 mmol) in sequence, dissolve in DMF (5.0 mL), add cuprous iodide (130 mg, 0.40 mmol), hexamethylphosphoric triamide (0.070 mL, 0.40 mmol), methyl fluorosulfonyl difluoroacetate (260 mg, 1.3 mmol), replace with nitrogen at room temperature, react at 100 ° C for 2 h. Cool to room temperature, add saturated sodium chloride aqueous solution, extract three times with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure. Purify on a silica gel column (ethyl acetate: petroleum ether = 10%) to obtain compound 26g (95 mg). LCMS (ESI-MS) m/z = 420.01 [M + H] ⁺

Step 7 Synthesis of 5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-{3-[(3R)-1-(2-hydroxyethyl)hexahydropyridin-3-yl]azetidin-1-yl}-7-(trifluoromethyl)pyrrolo[3,2-b]pyrazine-6-carbonitrile 26

Take a single-mouth bottle, add compound 26g (90 mg, 0.21 mmol), compound 1d (590 mg, 0.6 mmol) in sequence, dissolve in acetonitrile (10 mL), add cesium carbonate (700 mg, 2.1 mmol), and react at room temperature for 2 h. Add aqueous solution, extract three times with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure. Purify by preparative HPLC to obtain compound 26 (48 mg). LCMS (ESI-MS) m/z = 568.40 [M + H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ )δ8.94(s,1H),7.98(d,J＝1.5Hz,1H),7.80(d,J＝2.2Hz,1H),6.06-6.01(m,1H),4.5 9-4.33(m,1H),4.20-3.95(m,2H),3.87(t,J＝7.4Hz,1H),3.69(s,1H),3.49-3.44(m ,2H),2.80-2.70(m,2H),2.60-2.50(m,1H),2.40-2.30(m,2H),2.02(d,J＝7.1Hz,3H ),1.90(t,J＝11.1Hz,1H),1.75–1.54(m,4H),1.50-1.35(m,1H),0.90-0.75(m,1H).

Example 27: 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-[1-(2,4-dichloropyrimidin-5-yl)ethyl]pyrazolo[4,3-b]pyrazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethan-1-ol (27)

The preparation of compound 27 was carried out according to Example 1. LCMS (ESI-MS) m/z: 541.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ9.02(d,J＝2.3Hz,1H),7.86(s,1H),6.11(q,J＝7.0Hz,1H),4.41(s,1H), 4.17(s,2H),3.88(s,2H),3.51(s,2H),2.80(s,2H),2.52(s,2H),2.41(s, 1H),1.99(s,1H),1.85(d,J＝7.0Hz,3H),1.78–1.56(m,4H),1.46(s,1H),0 .93(dq,J＝6.5,3.9,3.5Hz,2H),0.86(s,1H),0.78(dq,J＝7.0,4.0Hz,2H).

Example 28: 2-[(3R)-3-(1-{3-[(1E)-2-cyclopropylvinyl]-1-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]pyrazolo[4,3-b]pyrazin-6-yl}azetidin-3-yl)piperidin-1-yl]ethan-1-ol (28)

The preparation of compound 28 refers to Example 23. LCMS (ESI-MS) m/z: 542.4 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.56(d,J＝2.4Hz,1H),7.81(d,J＝5.3Hz,2H),6.59(d,J＝4.4Hz,2H),6.17(q,J＝ 7.1Hz,1H),4.40(s,1H),4.14(t,J＝8.4Hz,2H),3.84(q,J＝6.6Hz,2H),3.49(t,J＝ 6.4Hz,2H),2.76(d,J＝10.6Hz,2H),2.36(t,J＝6.2Hz,2H),1.96–1.82(m,4H),1.8 2–1.51(m,6H),1.44(t,J＝12.0Hz,1H),0.89–0.76(m,3H),0.55(d,J＝4.8Hz,2H).

Example 29: 5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-{3-[(3R)-1-(2-hydroxyethyl)piperidin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyrazine-6,7-dicarbonitrile (29)

The preparation of compound 29 refers to Example 26. LCMS (ESI-MS) m/z = 525.38 [M + H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ )δ8.92(s,1H),8.01(s,1H),7.82(s,1H),6.08-6.02(m,1H),4.34(t,J＝5.4Hz,1H) ,4.20-4.00(m,2H),3.92-3.84(m,1H),3.80-3.60(m,1H),3.47(q,J＝6.1Hz,2H),2 .78–2.69(m,2H),2.52-2.60(m,1H),2.34(dd,J＝7.2,5.6Hz,2H),2.01(d,J＝7.0Hz ,3H),1.96-1.86(m,1H),1.75-1.55(m,4H),1.47-1.37(m,1H),0.90-0.75(m,1H).

Example 30: 1-(1-(4,6-dichloropyridin-3-yl)ethyl)-6-(3-((R)-1-(2-hydroxyethyl)piperidin-3-yl)azetidin-1-yl)-1H-pyrazolo[4,3-b]pyridine-3-carbonitrile (30)

The preparation of compound 30 refers to Example 24. LCMS (ESI-MS) m/z: 500.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.59(d,J＝1.7Hz,1H),8.09(d,J＝2.2Hz,1H),7.85(s,1H),7.04(d,J＝2.3Hz,1H),6.36(q,J＝ 6.8,6.3Hz,1H),4.35(t,J＝4.8Hz,1H),4.06(p,J＝7.4Hz,2H),3.72(s,2H),3.47(q,J＝5.9Hz,2 H),2.75(d,J＝10.5Hz,2H),2.56(s,1H),2.34(t,J＝6.3Hz,2H),1.97(dd,J＝16.6,10.0Hz,1H), 1.88(d,J＝6.8Hz,3H), 1.64(dt,J＝25.6,12.8Hz,4H), 1.42(d,J＝9.4Hz,1H), 0.87–0.80(m,1H).

Example 31: 2-((R)-2-(1-(3-(cyclopropylethynyl)-1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azetidin-3-yl)morpholinyl)ethan-1-ol (31)

The preparation of compound 31 refers to Example 1. LCMS (ESI-MS) m/z: 542.40 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.54(d,J＝1.3Hz,1H),7.83(d,J＝12.5Hz,2H),6.17(q,J＝7.0Hz,1H),4.12(s,2H),3.97(d,J＝27.7 Hz,2H),3.78(d,J＝11.6Hz,1H),3.59(td,J＝7.4,7.0,3.7Hz,1H),3.55–3.45(m,3H),2.76(d,J＝11.8H z,2H),2.69(d,J＝11.3Hz,1H),2.37(t,J＝6.2Hz,3H),2.03(dd,J＝11.6,8.4Hz,1H),1.83(d,J＝7.1Hz, 3H),1.72(td,J＝10.7,3.1Hz,1H),1.64–1.57(m,1H),0.90(dt,J＝8.1,3.2Hz,2H),0.81–0.72(m,2H).

Example 32: 2-[2-(1-{1-[1-(4,6-dichloropyridin-3-yl)ethyl]-3-(trifluoromethyl)pyrazolo[4,3-b]pyrazin-6-yl}azetidin-3-yl)-1,4-oxazin-4-yl]ethan-1-ol (32)

The preparation of compound 32 refers to Example 19. LCMS (ESI-MS) m/z: 546.35 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.56(d,J＝1.8Hz,1H),8.01(s,1H),7.83(s,1H),6.26(q,J＝7.0Hz,1H),4.27–3.91(m,4H),3.78 (d,J＝11.0Hz,1H),3.63–3.47(m,5H),2.89–2.63(m,3H),2.37(t,J＝6.2Hz,2H),2.14–1.61(m,5H).

Example 33: 1-[1-(4,6-dichloropyridin-3-yl)ethyl]-6-{3-[(3R)-1-(2-hydroxyethyl)piperidin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyridine-3-carbonitrile (33)

The preparation of compound 33 refers to Example 25. LCMS (ESI-MS) m/z: 250.20 [1/2M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.53(d,J＝2.8Hz,1H),8.36(d,J＝2.8Hz,1H),7.95–7.79(m,2H),6.90(s,1H),6.09( d,J＝6.9Hz,1H),4.38(s,1H),3.95(dq,J＝8.0,4.0Hz,2H),3.59(q,J＝7.2Hz,2H),3.49 (t,J＝6.3Hz,2H),2.76(d,J＝10.1Hz,2H),2.36(t,J＝6.4Hz,2H),1.90(d,J＝7.2Hz,4H) ,1.65(dq,J＝23.5,12.2,11.4Hz,5H),1.44(t,J＝12.3Hz,1H),0.83(d,J＝11.9Hz,1H).

Example 34: 2-((3R)-3-(1-(1-(4-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[4,3-b]pyridin-6-yl)azetidin-3-yl)piperidin-1-yl)ethan-1-ol (34)

The preparation of compound 34 refers to Example 19. LCMS (ESI-MS) m/z: 543.39 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.55(d,J＝1.8Hz,1H),8.06(d,J＝2.2Hz,1H),7.85(d,J＝1.6Hz,1H),6.97(d,J＝2.4Hz,1H),6. 32(q,J＝6.8Hz,1H),4.37(s,1H),4.06(h,J＝4.6,3.8Hz,2H),3.73(d,J＝8.6Hz,2H),3.49(t,J＝8 .2Hz,2H),2.81–2.74(m,2H),2.61–2.55(m,1H),2.38(t,J＝6.2Hz,2H),1.97(d,J＝10.4Hz,1H), 1.90(d,J＝6.8Hz,3H),1.75–1.60(m,4H),1.44(d,J＝12.4Hz,1H),0.86(dd,J＝22.4,10.6Hz,1H).

Example 35: 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-[1-(4,6-dichloropyridin-3-yl)ethyl]pyrazolo[4,3-c][1,2]diazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethan-1-ol (35)

The first step is the synthesis of methyl 6-chloro-4-diazine-1,2-diazine-3-carboxylate 35b

Take a 100mL flask, add 4,6-dichloro-1,2-diazo-3-carboxylic acid methyl ester 35a (10g, 48mmol), ethanol (100mL), nitrogen protection, slowly add hydrazine hydrate (7.3mL, 120mmol) under ice bath, add completely in 5min, keep ice bath reaction, a large amount of solid precipitates after 5min, keep temperature reaction for 1 hour, LCMS monitoring, the raw material is basically reacted, filter, wash the filter cake with water, spin dry the filter cake, add DCM/EA (1:3, 300mL) to dissolve the filter cake, the filter cake is not completely dissolved, there is a large amount of suspended matter, add silica gel to mix the sample, and purify with Flash column to obtain compound 35b (4.9g). LCMS (ESI-MS) m/z: 203.06 [M + H] ⁺ .

Step 2 Synthesis of 6-chloro-1H-pyrazolo[4,3-c][1,2]diazine 35c

Take a 250mL three-necked flask, add compound 35b (3.5g, 17mmol), DCM (30mL), insert a low-temperature thermometer, protect with nitrogen, cool to below -70℃ in a dry ice-ethanol bath, slowly add DIBAL-H (17mL, 26mmol), keep the temperature below -70℃, after the addition, slowly warm to room temperature and react for 4 hours. LCMS monitoring shows that a small amount of raw materials have not reacted completely, add H ₂ O (2.0mL), ethanol (50mL), directly spin dry the solvent, add silica gel to mix the sample, and purify with a Flash column to obtain compound 35c (1.4g). LCMS (ESI-MS) m/z: 155.04 [M+H] ⁺ .

Step 3 Synthesis of 6-chloro-3-iodo-1H-pyrazolo[4,3-c][1,2]diazine 35d

Take a 100mL flask, add compound 35c (1.4g, 8.7mmol), DMF (15mL), NIS (3.9g, 17mmol), protect with nitrogen, heat at 70℃ for 1 hour, monitor by LCMS, the reaction is complete, freeze-dry the reaction solution, add ethyl acetate (50mL) and petroleum ether (30mL) to the residue, stir and slurry for 1 hour, filter, spin dry the insoluble matter, and use it directly in the next step. LCMS (ESI-MS) m/z: 281.01 [M+H] ⁺ .

Step 4 Synthesis of 6-chloro-1-(5,5-dimethyl-2-oxo-5-silanyl-1-yl)-3-iodopyrazolo[4,3-][1,2]oxadiazine 35e

Take a 100mL flask, add compound 35d (2.2g, 7.7mmol), THF (40mL), cesium carbonate (5.0g, 15mmol), SEMCl (1.7g, 10mmol), protect with nitrogen, stir at room temperature overnight, monitor by LCMS, the raw materials react completely, pour the reaction solution into water (100mL), extract with ethyl acetate three times (30mL*3), wash with saturated sodium chloride aqueous solution (100mL), dry with anhydrous sodium sulfate, filter, spin dry the solvent, add silica gel to mix, and purify with Flash column to obtain compound 35e (1.3g). LCMS (ESI-MS) m/z: 411.13 [M+H] ⁺ .

Step 5 Synthesis of 2-[(3R)-3-{1-[1-(5,5-dimethyl-2-oxo-5-silanyl-1-yl)-3-iodopyrazolo[4,3-][1,2]diazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethane-1-ol 35f

Take 15 mL sealed tube, add compound 35e (410 mg, 1.0 mmol), DMSO (5.0 mL), compound 1d (280 mg, 1.5 mmol), DIEA (390 mg, 3.0 mmol), protect with nitrogen, heat at 120 ° C for 2 hours, monitor by LCMS, the raw materials are basically reacted, pour the reaction solution into water (50 mL), extract with ethyl acetate three times (10 mL * 3), wash with saturated sodium chloride aqueous solution (20 mL), dry with anhydrous sodium sulfate The product was dried, filtered, and silica gel was added to mix the sample. The product was purified by Flash column to obtain compound 35f (140 mg). LCMS (ESI-MS) m/z: 280.18 [1/2M+H] ⁺ .

Step 6 Synthesis of 35 g of 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-(5,5-dimethyl-2-oxo-5-silanyl-1-yl)pyrazolo[4,3-][1,2]diazine-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethane-1-ol

Take a 50mL flask, add compound 35f (200mg, 0.36mmol), THF (5.0mL), bistriphenylphosphine palladium dichloride (50mg, 0.072mmol), CuI (20mg, 0.10mmol), TEA (0.25mL, 1.8mmol), ethynylcyclopropane (71mg, 1.1mmol), heat at 45℃ under nitrogen for 1h, monitor by LCMS, the reaction is basically complete, pour into water (20mL), extract with ethyl acetate three times (10mL*3), wash with saturated sodium chloride solution (20mL), dry with anhydrous sodium sulfate, filter, and purify with Flash column to obtain compound 35g (140mg). LCMS (ESI-MS) m/z: 249.29 [1/2M+H ⁺ ].

Step 7 Synthesis of 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1H-pyrazolo[4,3-][1,2]diazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethane-1-ol 35h

Take 15 mL sealed tube, add compound 35g (110 mg, 0.22 mmol), TBAF THF solution (5.0 mL, 5.0 mmol), 2,5-dimethyl-2,5-diazahexane (130 mg, 1.1 mmol), react at 80 ° C under nitrogen for 5 h, monitor by LCMS, and the reaction is basically complete. Pour into saturated sodium chloride solution and wash four times (15 mL), dry over anhydrous sodium sulfate, spin dry the solvent to obtain a crude compound 35h, which is directly used in the next step.

LCMS(ESI-MS)m/z:184.17[1/2M+H] ⁺ .

Step 8 Synthesis of 2-[(3R)-3-{1-[3-(cyclopropylethynyl)-1-[1-(4,6-dichloropyridin-3-yl)ethyl]pyrazolo[4,3-c][1,2]diazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethane-1-ol 35

Take 15mL sealed tube, add compound 35h (60mg, 0.16mmol), DMF (2.0mL), 5-(1-bromoethyl)-2,4-dichloropyridine 35i (130mg, 0.49mmol), potassium carbonate (91mg, 0.66mmol), protect with nitrogen, stir at room temperature and react overnight. LCMS monitoring shows that the raw materials are basically reacted. Pour the reaction solution into water (30mL), extract with EA three times (15mL*3), wash with saturated sodium chloride aqueous solution (20mL), dry with anhydrous sodium sulfate, filter, spin dry the solvent, purify by HPLC, and freeze-dry to obtain compound 35 (20mg). LCMS (ESI-MS) m/z: 270.76 [1/2M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ )δ8.60(s,1H),7.83(d,J＝2.0Hz,1H),6.80(d,J＝1.8Hz,1H),6.16(q,J＝6.8Hz,1H),4.37 (t,J＝5.4Hz,1H),4.14(q,J＝7.4Hz,2H),3.82(t,J＝7.2Hz,2H),3.49(q,J＝5.9Hz,2H),2.8 4–2.71(m,2H),2.59(s,1H),2.37(t,J＝6.4Hz,2H),1.93(t,J＝11.1Hz,1H),1.83(d,J＝6.9 Hz,3H),1.79–1.55(m,5H),1.44(d,J=12.2Hz,1H),1.00–0.92(m,2H),0.91–0.78(m,3H).

Biological activity test

Experiment 1: In vitro activity determination of the CCR4 inhibitor small molecule compound of the present invention

1. Purpose of the experiment

In this experiment, the inhibitory effect of the compound of the present invention on CCR4 receptor was tested by high-throughput real-time fluorescence analysis (FLIPR) method, and the in vitro activity of the compound was evaluated according to the half inhibitory concentration (IC ₅₀ ).

2. Experimental Methods

2.1 Experimental Materials

Table 1 Experimental materials

2.2 Experimental Procedure

(1) Use Echo to serially dilute the test compound 3-fold to 10 in 100% DMSO. Then transfer 900 nL of the compound to the target plate. Add 30 μL of assay buffer to the compound plate.

(2) Remove the cell plate from the incubator, discard the culture medium, and add 20 μL of assay buffer. Use a pipette to gently dispense 20 μL of 2X Fluo-4 DirectTM No-Wash Loading Buffer into the 384-well cell culture plate.

(3) Then transfer 10 μL of reference and compound from the compound plate to the cell plate. The final volume in the cell plate is 50 μL.

(4) Start FLIPRTETRA

(5) Transfer 10 μL of 6×EC80 concentration of agonist reference compound to the cell plate.

(6) Reading the fluorescence signal

(7) For antagonist tests, 1 was added to calculate the “maximum-minimum” value from reading 1 to the maximum value allowed, and 1 was added to calculate the “maximum” value from reading 1 to 130 allowed.

(8) Analyze data

2.3 Experimental Results

Table 1 Results of in vitro activity experiments of the compounds of the present invention

Conclusion: The compounds of the present invention have significant inhibitory activity on CCR4 receptor.

Experiment 2: In vivo efficacy test of the compounds of the present invention in mice

1. Modeling and drug administration

Female immunodeficient mice aged 8 weeks, with an average weight of 20 g. The experiment began after the mice adapted for 3 days. The experiment was divided into four groups, model group, FLX475 group and Example 1 compound group, with 8 mice in each group. Humanized hepatoma cell Hepg2 was cultured before the experiment. After culturing to a certain number, 1*10 ⁷ cells were injected subcutaneously to each mouse. After the tumor grew to a certain size, the model group, FLX475 group and Example compound 35 group were gavaged and injected at 10 mL/kg once a day; the model group was gavaged/injected with an equal amount of solvent for 28 consecutive days. The specific design is shown in Table 3.

Table 3 In vivo efficacy experimental plan of compounds

2. Experimental Animals

Female immunodeficient mice, 8 weeks old. All mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number SCXK (Shanghai) 2017-0011, animal certificate number: 20170011003865.

3. Experimental drugs

Example Compound 35.

4. Drug configuration

An appropriate amount of compound was weighed, dissolved in 30% polyethylene glycol (PEG400), vortexed and sonicated, and then added with 70% saline to obtain a clear and transparent solution of 3 mg/mL.

5. Drug administration

The specific administration is carried out according to the administration plan.

6. Detection indicators and methods

The tumor size and body weight of mice were observed daily, and the tumor size was measured three times a week. The endpoint was dissected, the tumor was removed, and photos were taken to record the endpoint tumor size.

7. Experimental results and conclusions

After humanized Hepg2 tumor cell transplantation, the tumor size of mice in the model control group continued to increase with the number of days, while the tumor volume of Example Compound 35 began to decrease significantly after the 14th day.

Experiment 3: Effects of the compounds of the present invention on hERG channel current

1. Purpose of the experiment

The effects of the compounds on hERG channel current were detected by manual patch clamp technique to evaluate the cardiac safety of the compounds of the present invention.

2. Experimental Methods

2.1 Experimental Materials

2.2 Experimental Procedure

1) HEK-293 cells stably expressing hERG potassium channel were cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg/mL G418 at 37°C and 5% carbon dioxide concentration.

2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of TrypLE ^TM Express solution and incubate at 37°C for about 0.5 min. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and collect the cells by centrifugation at 1000 rpm for 5 min. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish, with 2.5×105 cells inoculated in each cell culture dish (final volume: 5 mL).

3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

4) Patch clamp detection. Before the experiment, cells were separated with TrypLE ^TM Express, 4×103 cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, the test was performed. The voltage stimulation scheme for recording hERG potassium current by whole-cell patch clamp is as follows: after the whole-cell seal is formed, the cell membrane voltage is clamped at -80 mV. The clamping voltage is depolarized from -80 mV to -50 mV for 0.5 s (as a leakage current detection), then stepped to 30 mV for 2.5 s, and then quickly restored to -50 mV for 4 s to stimulate the tail current of the hERG channel. Data was collected repeatedly every 10 s to observe the effect of the drug on the hERG tail current. A 0.5 s stimulation of -50 mV was used as a leakage current detection. The experimental data were collected by an IPA amplifier (Sutter Instrument) and stored in the Sutter Patch software.

When the hERG current recorded by the whole cell is stable, the drug administration begins. After each drug concentration acts for 5 minutes (or the current stabilizes), the next concentration is detected. One or more concentrations are detected for each test compound. The coverslip with cells is placed in the recording bath in an inverted microscope. The test compound working solution and the external solution without the compound are administered by gravity perfusion from low concentration to high concentration through the recording bath to act on the cells. At the same time, a peristaltic pump is used for liquid replacement during recording. The current detected by each cell in the external solution without the compound serves as its own control group. At least two cells are used for each concentration and the test is repeated twice independently. All electrophysiological experiments are performed at room temperature.

2.3 Data Analysis

First, the tail current (Peak tail currentcompound) after each drug concentration and the tail current (Peak tail currentControl) of the blank solvent treatment group were normalized (Peak tail currentcompound/Peak tail currentControl), and then the inhibition rate corresponding to each drug concentration was calculated (1-(Peak tail currentcompound/Peak tail currentControl). The mean (Mean), standard deviation (SD) and standard error (SE) of the inhibition rate were calculated, and the data were expressed as Mean±SE and saved in Excel. Y=1/(1+10^((LogIC50-X)*HillSlope), the _IC50 value of each compound was calculated using the above equation, and the concentration effect curve was nonlinearly fitted, where _IC50 was the half-inhibitory concentration. The calculation of _IC50 and curve fitting were completed using GraphPad Prism software.

2.4 Experimental Results

Among them, D<0.1, 0.1≤C＜1.0, 1.0≤B＜1.5, 1.5≤A.

3. Experimental conclusion

Compared with the positive reference FLX-475, the cardiac safety indicator hERG toxicity of the compound of the present invention is improved.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the claims attached hereto.

Claims (19)

The compound represented by formula (I), its stereoisomer, tautomer or a pharmaceutically acceptable salt thereof,
It is characterized in that ring A is a 4-8 membered aromatic heterocycle; ^X1 or ^X2 are each independently N or CH; ^X3 is N or CR ^a ; Y is -O- or -CH ₂ -; ^Ra is -H, _-C1-3 alkyl, -halogen or -CN, wherein the _-C1-3 alkyl is optionally substituted with one or more halogens within the range allowed by the valence; L ¹ is a bond, -C _1-3 alkylene-, -C _2-4 alkenylene- or -C _2-4 alkynylene-; ^R1 is _{-C1-3alkylene} -OH; ^R2 is -H, -CN, _-C1-5 alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, the _-C1-5 alkyl is optionally substituted with one or more halogens within the range allowed by the valence, the 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl is optionally substituted with one or more _-C1-5 alkyl within the range allowed by the valence or -halogen; ^R3 is _-C1-5 alkyl; R ⁴ are each independently halogen; n is 0, 1, 2 or 3. The compound, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 1, characterized in that ring A is a 5-6 membered aromatic heterocycle; preferably, the heteroatom of the 5-6 membered aromatic heterocycle is N, O or S. The compound according to claim 1 or 2, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that ring A is wherein E ¹ and E ² are each independently CH, N, O or S, E ³ is a bond, CH or N, and E ¹ , E ² and E ³ are not CH at the same time; Preferably, ring A is The compound, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 3, characterized in that ^E1 is S, N, O, ^E2 is CH, N, O or S, ^{and E3} is a bond; Preferably, ^E1 is O, ^E2 is N, and ^E3 is a bond; or ^E1 is N, ^E2 is O, and ^E3 is a bond; or ^E1 is S, ^E2 is N, and ^E3 is a bond; or ^E1 is N, ^E2 is S, and ^E3 is a bond; or ^E1 is S, ^E2 is CH, and ^E3 is a bond. The compound, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 3, characterized in that E ¹ , E ² , and E ³ are each independently CH or N, and E ¹ , E ² , and E ³ are not CH at the same time; Preferably, ^E1 is CH, ^E2 is N, and ^E3 is CH; or ^E1 is CH, ^E2 is CH, and ^E3 is N; or ^E1 is N, ^E2 is N, and ^E3 is CH; ^E1 is CH, ^E2 is N, and ^E3 is N; or ^E1 is N, ^E2 is N, and ^E3 is CH; ^E1 is N, ^E2 is N, and ^E3 is N. The compound according to any one of claims 1 to 3, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that ring A is Preferably, ring A is The compound, its stereoisomer, tautomer or pharmaceutically acceptable salt according to any one of claims 1 to 6, characterized in that n is 0, 1 or 2, preferably 2. The compound according to any one of claims 1 to 7, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that ^X1 or ^X2 are not N at the same time; Preferably, ^X1 is CH, ^X2 is N, and ^X3 is N; or ^X1 is CH, ^X2 is N, and ^X3 is CR ^a ; or ^X1 is N, ^X2 is CH, and ^X3 is N; or ^X1 is CH, ^X2 is CH, and ^X3 is N; or ^X1 is CH, X2 is CH, and ^X3 is ^{CR a} ; More preferably, ^Ra is -H or -CN. The compound according to any one of claims 1 to 8, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in ^{that L1} is a bond, _-C2-4 alkenylene- or _-C2-4 alkynylene-, preferably a bond, -C≡C- or -CH=CH-; and/or ^R1 _{is -CH2CH2OH} . The compound according to any one of claims 1 to 9, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that R ² is -H, -CN, -C _1-5 alkyl, 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl, the -C _1-5 alkyl is optionally substituted with one or more halogens within the range allowed by the valence, the 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl is optionally substituted with one or more -C _1-3 alkyl or -halogen within the range allowed by the valence; Preferably, the -C _1-5 alkyl group is methyl or tert-butyl, the 3-5 membered cycloalkyl group is cyclopropyl, and the 3-5 membered heterocycloalkyl group is oxetanyl or azetidinyl; More preferably, R ² is -H, -CN, methyl, tert-butyl, cyclopropyl, oxetanyl or azetidinyl, the methyl or tert-butyl group is optionally substituted with one or more -F within the range allowed by the valence, and the cyclopropyl, oxetanyl or azetidinyl group is optionally substituted with one or more methyl or -F within the range allowed by the valence; More preferably, R ² is -H, -CN, -CH ₃ , -CHF ₂ , -CF ₃ , -CH(CH ₃ ) ₃ , cyclopropyl, The compound according to any one of claims 1 to 10, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that R ³ is -C _1-3 alkyl, preferably methyl; and/or R ⁴ is each independently -F, -Cl or -Br, preferably -Cl. The compound according to any one of claims 1 to 11, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that the compound has the following structure
Wherein, Y, L ¹ , R ² , E ¹ to E ³ , X ¹ to X ³ are as described in claim 1; Preferably, Y is _-CH2- , ^X1 is CH, ^X2 is N, and ^X3 is N. The compound according to claim 1, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that the compound has the following structure
Wherein, R ³ is -C _1-3 alkyl; R ⁵ is a 5-6 membered heteroaryl group optionally substituted by one or more substituents; the substituents are each independently selected from hydrogen, halogen, -CN, hydroxyl, -C _1-3 alkyl or -C _1-3 haloalkyl, and the 5-6 membered heteroaryl group contains 1, 2 or 3 heteroatoms. The compound according to any one of claims 1 to 13, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that the compound is selected from the compounds represented by the following structural formula:



The compound according to any one of claims 1 to 13, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that the compound is selected from the compounds represented by the following structural formula:


The method for preparing the compound according to any one of claims 1 to 15, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that it comprises the steps shown in any one of the following schemes 1 to 3: Option 1:
Solution 2: When X ³ is CR ^a
Solution 3: When X ³ is N
Wherein, each group is as described in claim 1. A pharmaceutical composition, characterized in that it comprises the compound according to any one of claims 1 to 15, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers and/or excipients. Use of the compound according to any one of claims 1 to 15, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for preventing and/or treating a CCR4-mediated disease; preferably, the CCR4-mediated disease comprises an inflammatory disease, an autoimmune disease or cancer; more preferably, the inflammatory disease and the autoimmune disease are dermatitis, allergic asthma, inflammatory bowel disease or lupus erythematosus, and the cancer is liver cancer, pancreatic cancer, colon cancer, lung cancer, brain cancer or gastric cancer. A method for treating a CCR4-mediated disease, comprising administering to a patient or individual in need of such treatment a therapeutically effective amount of any compound according to any one of claims 1 to 15, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to claim 17.