Chemokine receptor modulator preparation method and use

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

[0001] The present 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

[0002] The successful operation of the host defense system is the result of several processes working together to eliminate foreign pathogens. A coordinated innate and adaptive immune response is required, and many secreted factors and cell-related factors have been identified as important mediators for coordinating and regulating these two host defense arms. Chemokines are a family of cytokines that act as chemoattractants to guide the migration of leukocytes. 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 invading pathogens. Inflammatory chemokines are released by cells in response to pathological events (e.g., proinflammatory stimuli such as IL-1 or viruses). They primarily act as chemoattractants as part of the inflammatory response and serve to guide cells of the innate and adaptive immune systems to sites of inflammation. CC chemokine receptor type 4 (CCR4) plays a role in the progression of many inflammation-related and other diseases. Identifying compounds that modulate CCR4 function is an ongoing challenge.

[0003] Cancer is a disease in which cells exhibit dysregulated replication and growth. Recent cancer models implicate the immune system in the development and progression of cancer. While significant progress has been made in understanding the biological basis of cancer, cancer remains a leading cause of death.

[0004] 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 are also produced by monocytes and dendritic cells.

[0005] CCR4 is involved in immune regulatory processes such as cell homing to specific tissues, including the homing of T lymphocytes to the skin and lungs (see, for example: 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). Modulators of CCR4 activity have been described, for example, in WO2013 / 082490.

[0006] Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is considered a key regulator of adaptive immune responses, particularly in maintaining and maintaining the components of burst T cell responses. Therefore, CTLA-4, as an immune checkpoint inhibitor, is considered a potential therapeutic target for the treatment of cancer and inflammation. CTLA-4 modulators have been described, for example, in WO2018 / 035710.

[0007] Programmed death receptor-1 (PD-1) is a transmembrane receptor protein that negatively regulates T cell function through interaction with its two natural ligands, PD-L1 and PD-L2. Like CTLA4, PD-1 is 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.

[0008] 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.

[0009] Summary of the Invention

[0010] The first aspect of the present invention relates to a compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof,

[0011] wherein Ring A is a 4-8 membered aromatic heterocycle;

[0012] X 1 or X 2 are each independently N or CH;

[0013] X 3 N or CR a ;

[0014] Y is -O- or -CH2-;

[0015] R a -H, -C1-3 Alkyl, -halogen or -CN, the -C 1-3 The alkyl group is optionally substituted with one or more halogens as allowed by the valence;

[0016] L 1 For the key, -C 1-3 Alkylene-, -C 2-4 Alkenylene- or -C 2-4 Alkynylidene-;

[0017] R 1 -C 1-3 Alkylene-OH;

[0018] R 2 -H, -CN, -C 1-5 Alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, the -C 1-5 The alkyl group is optionally substituted with one or more halogens within the allowed range of valence, and the 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl group is optionally substituted with one or more -C 1-5 Alkyl or -halogen substituted;

[0019] R 3 -C 1-5 alkyl;

[0020] R 4 are each independently halogen;

[0021] n is 0, 1, 2 or 3.

[0022] 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.

[0023] In some embodiments, Ring A is Among them E 1 、E 2 are independently CH, N, O or S, E 3 is a bond, CH or N, and E 1 、E 2 、E 3 Not CH at the same time.

[0024] In some embodiments, Ring A is

[0025] In some embodiments, E 1 S, N, O, E 2 is CH, N, O or S, E 3 is the key.

[0026] In some embodiments, E1 O, E 2 N, E 3 is a key; or E 1 N, E 2 O, E 3 is a key; or E 1 For S, E 2 N, E 3 is a key; or E 1 N, E 2 For S, E 3 is a key; or E 1 For S, E 2 CH, E 3 is the key.

[0027] In some embodiments, E 1 、E 2 、E 3 are each independently CH or N, and E 1 、E 2 、E 3 Not CH at the same time.

[0028] In some embodiments, E 1 CH, E 2 N, E 3 CH; or E 1 CH, E 2 CH, E 3 N; or E 1 N, E 2 N, E 3 CH; E 1 CH, E 2 N, E 3 N; or E 1 N, E 2 N, E 3 CH; E 1 N, E 2 N, E 3 is N.

[0029] In some embodiments, Ring A is

[0030] In some embodiments, Ring A is

[0031] In some embodiments, n is 0, 1 or 2, preferably 2.

[0032] In some embodiments, X 1 or X 2 Not N at the same time; preferably, X1 CH, X 2 N, X 3 N; or X 1 CH, X 2 N, X 3 CR a ; or X 1 N, X 2 CH, X 3 N; or X 1 CH, X 2 CH, X 3 N; or X 1 CH, X 2 CH, X 3 CR a .

[0033] In some embodiments, R a It is -H or -CN.

[0034] In some embodiments, L 1 For the key, -C 2-4 Alkenylene- or -C 2-4 Alkynylene-, preferably a bond, -C≡C- or -CH=CH-.

[0035] In some embodiments, R 1 It is -CH2CH2OH.

[0036] In some embodiments, R 2 -H, -CN, -C 1-5 Alkyl, 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl, the -C 1-5 The alkyl group is optionally substituted with one or more halogens within the allowed range of valence, and the 3-5 membered cycloalkyl group or 3-5 membered heterocycloalkyl group is optionally substituted with one or more -C 1-3 Alkyl or -halogen substituted; preferably, the -C 1-5 The 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.

[0037] In some embodiments, R 2 It 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 scope allowed by the valence, and the cyclopropyl, oxetanyl or azetidinyl is optionally substituted with one or more methyl or -F within the scope allowed by the valence.

[0038] In some embodiments, R 2-H, -CN, -CH3, -CHF2, -CF3, -C(CH3)3, cyclopropyl,

[0039] In some embodiments, R 3 -C 1-3 Alkyl group, preferably methyl group.

[0040] In some embodiments, R 4 Each is independently -F, -Cl or -Br, preferably -Cl.

[0041] In some embodiments, the compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, has the following structure:

[0042] Among them, Y, L 1 、R 2 、E 1 ~E 3 、X 1 ~X 3 As described in the compound of formula (I);

[0043] Preferably, Y is -CH2-, X 1 CH, X 2 N, X 3 is N.

[0044] In some embodiments, the compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, has the following structure:

[0045] Among them, R 3 -H or -C 1-3 alkyl;

[0046] 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 The 5- to 6-membered heteroaryl group contains 1, 2 or 3 heteroatoms.

[0047] In some embodiments, the compound represented by formula (I), formula (II) or formula (III), its stereoisomers, tautomers or pharmaceutically acceptable salts, the compound represented by formula (I), formula (II) or formula (III) is selected from the compounds represented by the following structural formulas:

[0048] In some embodiments, the compound represented by formula (I), formula (II) or formula (III), its stereoisomers, tautomers or pharmaceutically acceptable salts, the compound represented by formula (I), formula (II) or formula (III) is selected from the compounds represented by the following structural formulas:

[0049] 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:

[0050] Option 1:

[0051] Option 2: When X 3 CR a hour

[0052] Option 3: When X 3 When N

[0053] Wherein, each group is as described above.

[0054] The third aspect of the present invention provides a pharmaceutical composition comprising the compound according to 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.

[0055] 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, 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.

[0056] In some embodiments of the present invention, the CCR4-mediated disease comprises an inflammatory disease, an autoimmune disease, or cancer.

[0057] 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.

[0058] 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 compound of the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of the third aspect of the present invention. DETAILED DESCRIPTION

[0059] Unless otherwise indicated, conventional methods within the technical scope of the art, such as mass spectrometry, NMR and pharmacological methods, are used. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthesis chemistry, and pharmaceutical and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation 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 the multiple summaries and more specific documents cited and discussed in this specification.

[0060] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0061] The term "optionally" includes both "optional" and "optional". For example, "optionally substituted methyl" refers to "unsubstituted methyl" or "substituted methyl". The term "substituted" refers to the replacement of one or more hydrogen atoms on a designated atom or group with a designated group, provided that the normal valence of the designated atom is not exceeded. When the substituent is oxo or keto (i.e., =0), then two hydrogen atoms on the atom are replaced.

[0062] The term "-C 1-n "alkyl" (wherein n is an integer from 2 to n) means a non-cyclic, saturated, branched or straight-chain aliphatic hydrocarbon group having 1 to n carbon atoms, preferably an alkyl group containing 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 containing 1 to 6 carbon atoms (i.e. -C 1-6 Alkyl), more preferably an alkyl containing 1 to 3 carbon atoms. For example -C 1-3 Alkyl refers to methyl, ethyl, n-propyl and isopropyl.

[0063] The term "-C 1-n "Alkylene-" refers to a group consisting of -C 1-n The residue resulting from the removal of one hydrogen atom from an alkyl group 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., -C 1-12 Alkylene-), more preferably an alkylene group containing 1 to 6 carbon atoms (i.e. -C 1-6 Alkylene-). For example, -C 1-3Alkylene includes -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -C(CH3)2-, -CH(CH2CH3)-, -CH(CH3)-CH2-, -CH2-CH(CH3)-, etc. Alkylene can be substituted or unsubstituted, and when substituted, it can be substituted at any available point of attachment.

[0064] The term "-C 2-n "Alkenyl" refers to -C 2-n Alkyl groups contain at least one carbon-carbon double bond. Alkenyl groups (i.e., C 2-12 alkenyl), more preferably alkenyl containing 2 to 6 carbon atoms (ie, C 2- 6 alkenyl). For example, the term -C 2-3 Alkenyl groups include -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2. Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents may optionally be present in cis or trans or E or Z form, for example:

[0065] The term "-C 2-n "Alkenylene-" refers to 2-n The residue resulting from the removal of a hydrogen atom from an alkenyl group, for example, the term -C 2-3 Alkenylene- includes -CH=CH-, -CH=CH-CH2-, and -CH2-CH=CH-.

[0066] The term "-C 2-n -alkynyl" refers to -C 2-n Alkyl groups contain at least one carbon-carbon triple bond. 2-3 Alkynyl groups include -C≡CH, -C≡C-CH3, and -CH2-C≡CH.

[0067] The term "-C 2-n "Alkynylidene-" refers to 2-n The residue resulting from the removal of a hydrogen atom from an alkynyl group, e.g., -C 2-3 Alkynylene- includes -C≡C-, -C≡C-CH2-, and -CH2-C≡C-.

[0068] Refers to the presence or absence of a key, e.g. Refers to a single key or double bond

[0069] 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- to 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-isozolyl; 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.

[0070] 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, wherein the cycloalkyl ring contains 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.

[0071] 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 group is a 4-12 membered heterocyclyl group. In some embodiments of the present invention, the heterocyclyl group is a 4-6 membered heterocyclyl group, such as tetrahydrofuranyl, oxetane, or azetidine.

[0072]

[0046] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0073] The term "isotopic derivative" refers to a derivative of a compound in which one or more atoms 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), carbon isotopes (e.g. 11 C. 13 C and 14 C), isotopes of fluorine (e.g. 18 F), nitrogen isotopes (e.g. 13 N and 15 N), oxygen isotopes (e.g. 15 O. 17 O and 18O).

[0074] The terms "pharmaceutically acceptable salts" and "pharmaceutically acceptable salts" have synonymous meanings and include 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 non-toxic 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, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, 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 functional groups, which allow the compounds to be converted into either base or acid addition salts.

[0075] "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 may be included in the compositions of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solution, lactated Ringer's, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and pigments. Such formulations can be sterilized and, if desired, can be mixed with adjuvants that do not adversely react with the compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring and / or aromatic substances, and the like. Those skilled in the art will recognize that pharmaceutical excipients are useful in the present invention.

[0076] The term "treating" 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; remission; reduction or rendering the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; rendering the degenerative endpoint less debilitating; and improvement of the patient's physical or mental well-being. 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 "treating" and variations thereof may include preventing an injury, pathology, condition, or disease. In embodiments, treatment is prophylaxis. In embodiments, treatment does not include prophylaxis.

[0077] As used herein, "treatment" (and as is 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 in the extent of the disease, stabilization of the disease state (i.e., not worsening), prevention of disease transmission or spread, delay or slowing of disease progression, improvement or palliation 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.

[0078] "Treatment" includes prophylactic treatment. Treatment methods include administering to a subject 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 patient's age, 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 assays known in the art. In some instances, long-term administration may be required. For example, the composition is administered to the subject in an amount and for a duration sufficient to treat the patient.

[0079] The term "prevention" refers to reducing the occurrence of symptoms of a patient's disease. As mentioned above, prevention can be complete (no detectable symptoms) or partial prevention, so that the observed symptoms are less than the symptoms that would occur if not treated. In embodiments, prevention refers to slowing down the progression 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.

[0080] An "effective amount" is an amount that is 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 a signaling pathway, or alleviate one or more symptoms of a disease or condition) relative to the absence of the compound. An example of an "effective amount" is an amount that is sufficient to promote the treatment, prevention, or alleviation of one or more symptoms of a disease, also referred to as a "therapeutically effective amount." "Relief" of one or more symptoms (and grammatical equivalents of this phrase) means reducing the severity or frequency of the symptoms, or eliminating the symptoms. A "prophylactically effective amount" of a drug is an amount that, when administered to an individual, has the expected prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition or its symptoms. A complete prophylactic effect does not necessarily occur by administering one dose, and may only occur after administering a series of doses. Therefore, a prophylactically effective amount may be administered over the course of one or more administrations. As used herein, an "activity-reducing amount" refers to the amount of antagonist required to reduce enzyme activity relative to the absence of the 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 therapeutic goal and will be determined by one skilled in the art using known techniques (see, for example, 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 ed., 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). A therapeutically effective amount can be determined by measuring relevant physiological effects and can be adjusted based on the individual's condition, dosing regimen, diagnostic assays, and the like. 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 reduces CCR4 activity compared to a control (eg, a compound lacking the compound or a compound known to be inactive).

[0081] The term "administration" 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, to an individual. Administered by any route, including parenteral and transmucosal (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 formulations, intravenous infusions, transdermal patches, etc. "Combined administration" refers to administering a composition described herein while, just before, or just after administering 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 a 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 via topical routes and can be formulated as applicators, 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 patient ingestion. 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 further comprise 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 and are 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 for all purposes. The compositions of the present invention can 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 ed., 623-645, 1995; can be administered as a biodegradable and injectable gel formulation (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or can be administered 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, i.e., by using receptor ligands linked to the liposomes that bind to surface membrane protein receptors of the cell, thereby causing endocytosis.By using liposomes, particularly when the liposomes carry receptor ligands that are specific to target cells or preferentially involved in specific organs, the compositions of the present invention can be delivered to 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 compositions of the present invention can also be delivered as nanoparticles.

[0082] 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 variations that are readily accomplished by one of ordinary skill in the art will fall within the scope of the present invention.

[0083] Unless otherwise specified, the raw materials and reagents used in the examples of the present invention are commercially available.

[0084] The abbreviations used in the present invention have the conventional meanings in the art. For example, the following abbreviations have the following meanings:

[0085] Preparation method of compound

[0086] 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)

[0087] Step 1: Synthesis of tert-butyl (R)-3-(1-(2-hydroxyethyl)piperidin-3-yl)azetidine-1-carboxylate 1c

[0088] 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 rotary evaporation, ethyl acetate was added, and the mixture was washed with water. The aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and dried by rotary evaporation to obtain compound 1c (4.7 g). LCMS (ESI-MS) m / z: 285.1 [M+H] + .

[0089] Step 2 Synthesis of (R)-2-(3-(azetidin-3-yl)piperidin-1-yl)ethan-1-ol trifluoroacetate 1d

[0090] 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 visualized by iodine. After the reaction was complete, the mixture was dried by spin drying and then dried with toluene to obtain compound 1d (8.2 g). LCMS (ESI-MS) m / z: 185.1 [M+H] + .

[0091] Step 3 Synthesis of 4,6-dichloro-N-methoxy-N-methylnicotinamide 1f

[0092] Compound 1e (10 g, 52 mmol) was dissolved in DMF (40 mL), and methoxymethylamine (4.8 g, 79 mmol) and DIPEA (14 g, 100 mol) were added. HATU (30 g, 79 mmol) was added portionwise and stirred at room temperature for 1 hour. The reaction was complete after LCMS analysis. The mixture was extracted with ethyl acetate, and 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] + .

[0093] Step 4: Synthesis of 1g of 1-(4,6-dichloropyridin-3-yl)ethan-1-one

[0094] Compound 1f (10 g, 43 mmol) was dissolved in THF (5.0 mL), and methylmagnesium bromide (29 mL, 86 mmol) was added in an ice bath. The reaction was stirred for 1 hour. After the reaction was substantially complete, the mixture was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, and the organic phase was washed with water and then with saturated brine, and passed 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] + .

[0095] Step 5: Synthesis of (S)-1-(4,6-dichloropyridin-3-yl)ethane-1-ol 1h

[0096] Compound 1g (3.5 g, 19 mmol) was dissolved in THF (20 mL) at -78°C, and (-)-IPC2Bci (13 mL, 22 mmol) was slowly added dropwise over half an hour. The mixture was slowly warmed to room temperature and stirred for 3 hours. LCMS analysis indicated that the reaction was complete. The mixture was 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 1h (3.3 g). LCMS (ESI-MS) m / z: 192.0 [M+H] + .

[0097] Step 6 Synthesis of (R)-6-chloro-1-(1-(4,6-dichloropyridin-3-yl)ethyl)-3-iodo-1H-pyrazolo[3,4-b]pyrazine 1j

[0098] Compound 1i (4.0 g, 14 mmol) was dissolved in THF (20 mL), and compound 1h (3.3 g, 17 mmol) and PPh3 (4.3 g, 21 mmol) were added under ice-cooling. DIAD (5.6 g, 21 mmol) was added under nitrogen protection, and the mixture was stirred at room temperature overnight. LCMS was used to monitor the reaction. Upon completion, the mixture was 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 1j (3.0 g). LCMS (ESI-MS) m / z: 454.0 [M+H] + .

[0099] 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)ethan-1-ol 1k

[0100] 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-cooling. The mixture was stirred at room temperature for 2 hours and detected by LCMS. After completion of the reaction, the mixture was 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] + .

[0101] 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)ethan-1-ol 11

[0102] Compound 1k (2.2 g, 3.7 mmol) was dissolved in THF (25 mL), and Pd(PPh3)Cl2 (260 mg, 0.37 mmol), CuI (140 mg, 0.73 mmol), and triethylamine (740 mg, 7.3 mmol) were added. Under nitrogen, a solution of TMS alkyne (1.4 g, 15 mmol) in THF (5.0 mL) was added. The mixture was heated at 40°C for 15 minutes. After the reaction was complete, the mixture was filtered, dried, and passed through a flash column to obtain compound 1l (1.6 g). LCMS (ESI-MS) m / z: 572.3 [M+H]+ .

[0103] 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)ethan-1-ol 1

[0104] Compound 11 (1.1 g, 1.8 mmol) was dissolved in methanol (10 mL), and K2CO3 (770 mg, 5.6 mmol) was added. The mixture was stirred at room temperature for 10 minutes. After the reaction was complete, the mixture was filtered, dried, and passed through a flash column to obtain 600 mg of the target compound, which was then subjected to SFC separation to obtain compound 1 (430 mg). LCMS (ESI-MS) m / z: 500.2 [M+H + ).

[0105] 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).

[0106] 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)

[0107] The preparation of compound 2 was carried out according to Example 1. LCMS (ESI-MS) m / z: 500.30 [M+H] + .

[0108] 1H NMR(400MHz,DMSO-d6)δ7.98(dd,J=8.2,3.1Hz,1H),7.88(s,1H),7.59(d,J=8.1Hz,1H),6.1 7(q,J=7.0Hz,1H),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=7.0 Hz,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).

[0109] 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)

[0110] The preparation of compound 3 was carried out according to Example 1. LCMS (ESI-MS) m / z: 501.40 [M+H] + .

[0111] 1 H NMR(400MHz,DMSO-d6)δ9.03(d,J=3.0Hz,1H),7.90(s,1H),6.12(q,J=7.0H z,1H),4.57(s,1H),4.17(s,2H),3.87(s,2H),3.54(t,J=6.0Hz,2H),2.93( d,J=10.8Hz,2H),2.61–2.51(m,3H),2.15(d,J=11.8Hz,1H),1.85(d,J=7.1 Hz,5H),1.65(d,J=15.1Hz,3H),1.50(d,J=12.7Hz,1H),0.94–0.82(m,1H).

[0112] 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)

[0113] The preparation of compound 4 was carried out according to Example 1. LCMS (ESI-MS) m / z: 501 [M+H] + .

[0114] 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)

[0115] The preparation of compound 5 refers to Example 1. LCMS (ESI-MS) m / z: 501.40 [M+H] + .

[0116] 1 H NMR (400MHz, DMSO-d6) δ8.87(s,1H),7.88(s,1H),6.25(q,J=6.8Hz,1H),4.50(s,1 H),4.35(s,1H),4.12(s,2H),3.82(s,2H),3.48(t,J=6.3Hz,2H),2.75(d,J=10.5H z,2H),2.55(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).

[0117] 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)

[0118] The preparation of compound 6 was carried out according to Example 1. LCMS (ESI-MS) m / z: 502 [M+H] + .

[0119] 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)

[0120] The preparation of compound 7 was carried out according to Example 1. LCMS (ESI-MS) m / z: 500.4 [M+H] + .

[0121] 1 H NMR (400MHz, DMSO-d6) 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=10.1Hz,2H),2.55(d,J=7.7Hz,1H),2.41(t,J=6.3Hz,2H),1.98(t,J=10.9Hz,1H) ,1.81(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).

[0122] Example 8: 2-((3R)-3-(1-(1-(2-(2,5-dichloro-1λ 3 ,3λ 2 (8)

[0123] The preparation of compound 8 was carried out according to Example 1. LCMS (ESI-MS) m / z: 490 [M+H] + .

[0124] 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)

[0125] The preparation of compound 9 refers to Example 1. LCMS (ESI-MS) m / z: 490 [M+H] + .

[0126] 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)

[0127] The preparation of compound 10 refers to Example 1. LCMS (ESI-MS) m / z: 506 [M+H] + .

[0128] 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)

[0129] The preparation of compound 11 refers to Example 1. LCMS (ESI-MS) m / z: 506 [M+H] + .

[0130] 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)

[0131] The preparation of compound 12 was carried out according to Example 1. LCMS (ESI-MS) m / z: 505.3 [M+H] + .

[0132] 1 H NMR(400MHz,DMSO-d6)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).

[0133] 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)

[0134] The preparation of compound 13 was carried out according to Example 1. LCMS (ESI-MS) m / z: 514 [M+H]+ .

[0135] 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)

[0136] The preparation of compound 13 was carried out according to Example 1. LCMS (ESI-MS) m / z: 556.23 [M+H] + .

[0137] 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)

[0138] The preparation of compound 15 was carried out according to Example 1. LCMS (ESI) m / z: 540.40 [M+H] + .

[0139] 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)

[0140] The preparation of compound 16 was carried out according to Example 1. LCMS (ESI-MS) m / z: 556.23 [M+H] + .

[0141] 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)

[0142] The preparation of compound 17 was carried out according to Example 1. LCMS (ESI-MS) m / z: 569.23 [M+H] + .

[0143] 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)

[0144] The preparation of compound 18 was carried out according to Example 1. LCMS (ESI-MS) m / z: 576.23 [M+H] + .

[0145] 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)

[0146] Step 1: Synthesis of (R)-6-chloro-1-(1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazine 19a

[0147] Compound 1j (5.0 g, 11 mmol), methyl fluorosulfonyldifluoroacetate (4.2 g, 22 mmol), hexamethylphosphoric triamide (7.9 g, 44 mmol), and CuI (2.1 g, 11 mmol) were placed in a single-necked flask. DMF (50 mL) was added, the atmosphere was replaced with nitrogen, and the reaction was heated at 100°C for 4 h. The reaction solution was cooled to room temperature and poured into water. The precipitated solid was filtered and washed three times with dichloromethane. The organic phases were combined and concentrated by flash column (mobile phase PE:EA = 5:1) to obtain compound 19a (3.5 g). LCMS (ESI-MS) m / z: 396.1 [M+H] + .

[0148] 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

[0149] 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 stirred at room temperature for 24 hours. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and concentrated by flash column chromatography (DCM:MeOH = 10:1) to obtain compound 19 (660 mg). LCMS (ESI-MS) m / z: 544.28 [M+H] + .

[0150] 1 H NMR (400MHz, DMSO-d6) 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).

[0151] 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)

[0152] The preparation of compound 20 refers to Example 19. LCMS (ESI) m / z: 544.40 [M+H] + .

[0153] 1 H NMR (400MHz, DMSO-d6) 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).

[0154] 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)

[0155] 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)

[0156] In a single-necked flask, TMEDA (350 mg, 3.0 mmol) dissolved in DMF (2.0 mL) was added. Cuprous iodide (570 mg, 3.0 mmol) and potassium carbonate (830 mg, 6.0 mmol) were then added sequentially at room temperature. Stir vigorously for 20 minutes under air at room temperature. TMSCF3 (570 mg, 4.0 mmol) was added and stirred for 15 minutes under air at room temperature. The reaction mixture turned dark green. Cooled to 0°C, a mixed solution of compound 1 (1000 mg, 2.0 mmol) and TMSCF3 (570 mg, 4.0 mmol) in DMF (2.0 mL) was added. The mixture was allowed to react at 0°C for 30 minutes. The mixture was allowed to warm to room temperature and react for 6 hours. The mixture was quenched with saturated ammonium chloride (10 mL), and ethyl acetate (50 mL) was added. The organic phase was washed with water and dried over anhydrous sodium sulfate. Concentrated under reduced pressure. Purification by preparative HPLC (prepared with ammonium bicarbonate) afforded compound 21 (62 mg). LCMS (ESI) m / z: 568.40 [M+H] + .

[0157] 1 H NMR (400MHz, DMSO-d6) 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).

[0158] 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)

[0159] The preparation of compound 22 was carried out according to Example 1. LCMS (ESI) m / z: 540.40 [M+H] + .

[0160] 1 H NMR (400MHz, DMSO-d6) 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,2H),2.59–2.53(m,1H),2.35(t,J=6.4Hz,2H),1.91(t,J=10.8Hz,1H),1.84(d,J=7.2 Hz,3H),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).

[0161] 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)

[0162] Step 1 (R)-6-Bromo-1-(1-(3,5-dichloropyridin-2-yl)ethyl)-3-vinyl-1H-pyrazolo[4,3-b]pyridine (23c)

[0163] 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- The mixture was stirred overnight at room temperature for 1 hr. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was dried, filtered, and the filtrate was concentrated and purified on a silica gel column (petroleum ether:ethyl acetate = 25:1) to give compound 23c (200 mg). LCMS (ESI-MS) m / z: 397.14 [M+H] + .

[0164] 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)ethan-1-ol (23)

[0165] Compound 23c (150 mg, 0.38 mmol) and compound 1d (320 mg, 1.74 mmol) were dissolved in N,N-dimethylformamide (3.0 mL). Cesium carbonate (250 mg, 0.76 mmol) and methanesulfonic acid (4,5-bis(diphenylphosphino-9,9-dimethylxanthene)(2-amino-1,1'-biphenyl-2-yl)palladium (36 mg, 0.038 mmol) were added at room temperature, and the atmosphere was replaced with nitrogen. The mixture was heated at 100°C for 3 hours. The mixture was quenched with water, extracted with ethyl acetate, and the organic phase was dried, filtered, and the filtrate was concentrated. The crude product was purified by preparative liquid phase (ammonium bicarbonate) to give compound 23 (13 mg). LCMS (ESI-MS) m / z: 501.40 [M+H] + .

[0166] 1H NMR (400MHz, DMSO-d6) 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).

[0167] 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)

[0168] Step 1 (R)-6-bromo-1-(1-(3,5-dichloropyridin-2-yl)ethyl)-1H-pyrazolo[4,3-b]pyridine-3-carbonitrile (24a)

[0169] Compound 23a (500 mg, 1.0 mmol) was dissolved in N,N-dimethylacetamide (6.0 mL). Zinc cyanide (61 mg, 0.52 mmol), tris(dibenzylideneacetone)dipalladium (81 mg, 0.10 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (58 mg, 0.10 mmol), and tetramethylethylenediamine (35 mg, 0.30 mmol) were added at room temperature. The atmosphere was purged with nitrogen. The reaction was heated at 90°C for 1 hour. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was dried and filtered, and the filtrate was concentrated and purified on a silica gel column (petroleum ether:ethyl acetate = 25:1) to obtain compound 24a (220 mg). LCMS (ESI-MS) m / z: 396.05 [M+H] + .

[0170] 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)

[0171] Compound 24a (150 mg, 0.38 mmol) and 1d (320 mg, 1.7 mmol) were dissolved in toluene (1.2 mL). Cesium carbonate (190 mg, 0.57 mmol), (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2-aminoethylphenyl)]palladium chloride methyl tert-butyl ether complex (31 mg, 0.038 mmol), and 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (18 mg, 0.038 mmol) were added at room temperature. The atmosphere was purged with nitrogen. The mixture was heated at 100°C for 3 hours. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was dried, filtered, and the filtrate was concentrated. The crude product was purified by preparative liquid chromatography (aqueous ammonia) to give compound 24 (4.2 mg). LCMS (ESI-MS) m / z: 500.40 [M+H] + .

[0172] 1 H NMR (400MHz, DMSO-d6) 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).

[0173] 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)

[0174] Step 1: Synthesis of 6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine 25b

[0175] To a 50 mL flask, add 6-bromo-1H-pyrrolo[3,2-b]pyridine 25a (5.0 g, 25 mmol) and DMF (25 mL). Stir in an ice bath for 10 min. Add potassium hydroxide (1.1 g, 20 mmol) and iodine (2.6 g, 10 mmol). Warm the temperature to room temperature and react for 5 hours. Add saturated aqueous sodium bisulfite solution in an ice bath to quench the reaction. Extract with ethyl acetate (400 mL*3), wash with saturated aqueous sodium chloride solution (200 mL), dry over anhydrous sodium sulfate, and spin dry the solvent to obtain compound 25b (6.2 g). LCMS (ESI-MS) m / z: 323.0 [M+H] + .

[0176] Step 2 Synthesis of 6-bromo-1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]-3-iodopyrrolo[3,2-b]pyridine 25d

[0177] A 25 mL three-necked flask was charged with compound 25b (1.1 g, 3.3 mmol), dichloromethane (6.0 mL), (1R)-1-(3,5-dichloropyridin-2-yl)ethane-1-ol 25c (0.63 g, 3.3 mmol), and triphenylphosphine (1.4 g, 5.3 mmol). The mixture was stirred under nitrogen and ice-bathed for 10 min. DIAD (1.1 g, 5.6 mmol) was added and the reaction was continued with stirring for 2 hours. The mixture was then poured into water and extracted with DCM three times (20 mL x 3). The mixture was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and purified on a flash column to obtain compound 25d (1.6 g). LCMS (ESI-MS) m / z: 496.04 [M+H] + .

[0178] Step 3 Synthesis of 6-bromo-1-[(1R)-1-(3,5-dichloropyridin-2-yl)ethyl]pyrrolo[3,2-b]pyridine-3-carbonitrile 25e

[0179] A 100 mL flask was charged with 25d (1200 mg, 2.4 mmol), DMA (10 mL), Zn(CN)2 (140 mg, 1.2 mmol), Pd2(dba)3 (220 mg, 0.24 mmol), XANTPHOS (140 mg, 0.24 mmol), and 2,5-dimethyl-2,5-diazahexane (83 mg, 0.71 mmol). The mixture was stirred at 70°C for 2 h under nitrogen atmosphere. LCMS monitoring confirmed complete reaction. The reaction solution was poured into water (60 mL), saturated aqueous NaHCO3 (20 mL) and EDTA (0.50 g) were added. The product was extracted with EA three times (30 mL x 3), washed with saturated aqueous sodium chloride (60 mL), dried over anhydrous sodium sulfate, and purified on a flash column to obtain compound 25e (390 mg). LCMS (ESI-MS) m / z: 395.13 [M+H] + .

[0180] 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

[0181] A 75 mL sealed tube was added to compound 25e (220 mg, 1.2 mmol), tert-butyl alcohol (15 mL), EPhos Pd G4 (110 mg, 0.12 mmol), EPhos (65 mg, 0.12 mmol), and cesium carbonate (390 mg, 1.2 mmol). The mixture was heated at 85°C for 6 hours under nitrogen protection. LCMS monitoring confirmed that the reaction of the starting materials was complete. The mixture was filtered through celite, the solvent was dried, and the reaction was performed by HPLC to obtain compound 25 (16 mg). LCMS (ESI-MS) m / z: 250.20 [M / 2+H] + .

[0182] 1H NMR (400MHz, DMSO-d6) δ8.63(d,J=2.1Hz,1H),8.37(s,1H),8.30(d,J=2.2Hz,1H),7.82(d,J=2.3Hz,1 H),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=11.8,6.5H z,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).

[0183] Example 26: 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)

[0184] Step 1: Synthesis of 3-chloro-5-[(4-methylphenyl)dioxo-λ6-thio]pyrrolo[3,2-b]pyrazine 26b

[0185] In a single-necked flask, compound 26a (5.0 g, 33 mmol) was dissolved in DCM (50 mL). Triethylamine (23 mL, 160 mmol), p-toluenesulfonyl chloride (10 g, 39 mmol), and DMAP (0.80 g, 6.5 mmol) were added at room temperature. The reaction was allowed to proceed overnight. Water was added, the mixture was extracted with DCM, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification on a silica gel column (ethyl acetate:petroleum ether = 10-30%) afforded compound 26b (9.0 g). LCMS (ESI-MS) m / z = 308.14 [M+H] +

[0186] Step 2 Synthesis of 3-chloro-5-[(4-methylphenyl)dioxo-λ6-thio]pyrrolo[3,2-b]pyrazine-6-carbonitrile 24c

[0187] In a three-necked flask, compound 26b (1.5 g, 4.9 mmol) was dissolved in tetrahydrofuran (5.0 mL). The atmosphere was replaced with nitrogen three times at room temperature. n-Butyl lithium (2.9 mL, 7.3 mmol) was added dropwise under a dry ice ethyl acetate bath. The reaction was allowed to proceed for 1 h. A solution of 4-toluenesulfonyl cyanide (1.8 g, 9.7 mmol) in tetrahydrofuran (5.0 mL) was then added dropwise. The reaction was continued at the same temperature for 30 min and then overnight at room temperature. Saturated aqueous ammonium chloride was added, and the mixture was extracted three times with ethyl acetate. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was performed on a silica gel column (ethyl acetate:petroleum ether = 10-30%) to afford compound 26c (410 mg). LCMS (ESI-MS) m / z = 333.14 [M+H] +

[0188] Step 3 Synthesis of 3-chloro-5H-pyrrolo[3,2-b]pyrazine-6-carbonitrile 26d

[0189] To a single-necked flask, compound 26c (2.7 g, 8.2 mmol) was added sequentially and suspended in methanol (30 mL). Potassium carbonate (2.3 g, 16 mmol) was added at room temperature and allowed to react at 50°C for 1 h. The solution became clear and the solvent was evaporated under reduced pressure. Water was added, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. This afforded compound 26d (1.7 g, 9.6 mmol), which was used directly in the next step. LCMS (ESI-MS) m / z = 178.99 [M+H] +

[0190] Step 4 Synthesis of 3-chloro-7-iodo-5H-pyrrolo[3,2-b]pyrazine-6-carbonitrile 26e

[0191] To a single-necked flask, compound 26d (1.7 g, 9.6 mmol) was added sequentially, dissolved in DMF (30 mL), and potassium hydroxide (2.0 g, 35 mmol) was added portionwise. The mixture was allowed to react at room temperature for 30 min. Iodine (5.0 g, 19 mmol) in DMF (1.0 mL) was added under ice-water bath conditions. The reaction was allowed to proceed at room temperature for 20 h. Saturated aqueous sodium sulfite solution was added to quench the excess iodine. The mixture was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Compound 26e (810 mg) was obtained and used directly in the next step. LCMS (ESI-MS) m / z = 304.98 [M+H] +

[0192] Step 5 Synthesis of 3-chloro-5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-7-iodopyrrolo[3,2-b]pyrazine-6-carbonitrile 26f

[0193] To a three-necked flask, add compound 1h (190 mg, 1.0 mmol) and compound 26e (300 mg, 1.0 mmol). The atmosphere was replaced with nitrogen at room temperature. A solution of triphenylphosphine (520 mg, 2.0 mmol) in DCM (1.0 mL) was added under an ice-water bath. Simultaneously, a solution of DIAD (500 mg, 2.5 mmol) in DCM (1.0 mL) was added. The mixture was reacted at the same temperature for 2 h. Methanol was added, and the solvent was evaporated under reduced pressure. Purification on a silica gel column (ethyl acetate:petroleum ether = 10%) afforded compound 26f (380 mg). LCMS (ESI-MS) m / z = 478.07 [M+H] +

[0194] 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

[0195] In a single-necked flask, compound 26f (160 mg, 0.33 mmol) was added sequentially, dissolved in DMF (5.0 mL), followed by cuprous iodide (130 mg, 0.40 mmol), hexamethylphosphoric triamide (0.070 mL, 0.40 mmol), and methyl fluorosulfonyldifluoroacetate (260 mg, 1.3 mmol). The atmosphere was replaced with nitrogen at room temperature and the reaction was carried out at 100°C for 2 h. The mixture was cooled to room temperature, added with saturated aqueous sodium chloride solution, and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification on a silica gel column (ethyl acetate:petroleum ether = 10%) afforded compound 26g (95 mg). LCMS (ESI-MS) m / z = 420.01 [M+H] +

[0196] 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

[0197] To a single-necked flask, compound 26g (90 mg, 0.21 mmol) and compound 1d (590 mg, 0.6 mmol) were added sequentially, dissolved in acetonitrile (10 mL), and cesium carbonate (700 mg, 2.1 mmol) was added. The mixture was reacted at room temperature for 2 h. Aqueous solution was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification by preparative HPLC yielded compound 26 (48 mg). LCMS (ESI-MS) m / z = 568.40 [M+H] +

[0198] 1H NMR (400MHz, DMSO-d6) δ8.94(s,1H),7.98(d,J=1.5Hz,1H),7.80(d,J=2.2Hz,1H),6.06- 6.01(m,1H),4.59-4.33(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=7.1 Hz,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).

[0199] 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)

[0200] The preparation of compound 27 was carried out according to Example 1. LCMS (ESI-MS) m / z: 541.40 [M+H] + .

[0201] 1 H NMR(400MHz,DMSO-d6)δ9.02(d,J=2.3Hz,1H),7.86(s,1H),6.11(q,J=7.0Hz,1 H),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).

[0202] 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)hexahydropyridin-1-yl]ethan-1-ol (28)

[0203] The preparation of compound 28 refers to Example 23. LCMS (ESI-MS) m / z: 542.4 [M+H] + .

[0204] 1 H NMR (400MHz, DMSO-d6) 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 ),1.82–1.51(m,6H),1.44(t,J=12.0Hz,1H),0.89–0.76(m,3H),0.55(d,J=4.8Hz,2H).

[0205] Example 29: 5-[(1R)-1-(4,6-dichloropyridin-3-yl)ethyl]-3-{3-[(3R)-1-(2-hydroxyethyl)hexahydropyridin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyrazine-6,7-dicarbonitrile (29)

[0206] The preparation of compound 29 refers to Example 26. LCMS (ESI-MS) m / z = 525.38 [M+H] +

[0207] 1 H NMR(400MHz,DMSO-d6)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).

[0208] 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)

[0209] The preparation of compound 30 refers to Example 24. LCMS (ESI-MS) m / z: 500.40 [M+H] + .

[0210] 1 H NMR (400MHz, DMSO-d6) 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,2H),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).

[0211] 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)

[0212] The preparation of compound 31 was carried out according to Example 1. LCMS (ESI-MS) m / z: 542.40 [M+H] + .

[0213] 1H NMR (400MHz, DMSO-d6) 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=27.7Hz,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=11.8Hz,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).

[0214] 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)

[0215] The preparation of compound 32 was carried out according to Example 19. LCMS (ESI-MS) m / z: 546.35 [M+H] + .

[0216] 1 H NMR (400MHz, DMSO-d6) 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).

[0217] Example 33: 1-[1-(4,6-dichloropyridin-3-yl)ethyl]-6-{3-[(3R)-1-(2-hydroxyethyl)hexahydropyridin-3-yl]azetidin-1-yl}pyrrolo[3,2-b]pyridine-3-carbonitrile (33)

[0218] The preparation of compound 33 was carried out according to Example 25. LCMS (ESI-MS) m / z: 250.20 [1 / 2 M+H]+ .

[0219] 1 H NMR (400MHz, DMSO-d6) 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=7.2H z, 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).

[0220] 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)

[0221] The preparation of compound 34 was carried out according to Example 19. LCMS (ESI-MS) m / z: 543.39 [M+H] + .

[0222] 1 H NMR (400MHz, DMSO-d6) 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).

[0223] 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)

[0224] Step 1: Synthesis of methyl 6-chloro-4-diazinyl-1,2-diazine-3-carboxylate 35b

[0225] To a 100 mL flask, methyl 4,6-dichloro-1,2-diazolyl-3-carboxylate 35a (10 g, 48 mmol) and ethanol (100 mL) were added. Under nitrogen protection, hydrazine hydrate (7.3 mL, 120 mmol) was slowly added under ice-cooling for 5 minutes. The reaction was continued in the ice-cooling bath. After 5 minutes, a large amount of solid precipitated. The temperature was maintained for 1 hour. LCMS monitoring showed that the starting material was almost completely reacted. The filter cake was filtered, washed with water, and dried. DCM / EA (1:3, 300 mL) was added to dissolve the filter cake. The filter cake was not completely dissolved, and a large amount of suspended matter was present. Silica gel was added to mix the sample, and the sample was purified on a flash column to obtain compound 35b (4.9 g). LCMS (ESI-MS) m / z: 203.06 [M+H] + .

[0226] Step 2 Synthesis of 6-chloro-1H-pyrazolo[4,3-c][1,2]diazine 35c

[0227] Compound 35b (3.5 g, 17 mmol) and DCM (30 mL) were added to a 250 mL three-necked flask. A low-temperature thermometer was inserted and nitrogen was applied. The temperature was cooled to below -70°C in a dry ice-ethanol bath. DIBAL-H (17 mL, 26 mmol) was slowly added, maintaining the temperature below -70°C. After completion of the addition, the temperature was slowly raised to room temperature and the reaction was allowed to proceed for 4 hours. LCMS monitoring indicated that a small amount of starting material had not reacted completely. HO (2.0 mL) and ethanol (50 mL) were added, the solvent was directly evaporated, silica gel was added, and the sample was purified using a flash column to obtain compound 35c (1.4 g). LCMS (ESI-MS) m / z: 155.04 [M+H] + .

[0228] Step 3 Synthesis of 6-chloro-3-iodo-1H-pyrazolo[4,3-c][1,2]diazine 35d

[0229] To a 100 mL flask, compound 35c (1.4 g, 8.7 mmol), DMF (15 mL), and NIS (3.9 g, 17 mmol) were added. The mixture was heated at 70°C under nitrogen for 1 hour. LCMS monitoring indicated that the reaction was complete. The reaction solution was lyophilized, and ethyl acetate (50 mL) and petroleum ether (30 mL) were added to the residue. The mixture was stirred for 1 hour, filtered, and the insoluble material was spin-dried and used directly in the next step. LCMS (ESI-MS) m / z: 281.01 [M+H] + .

[0230] Step 4 Synthesis of 6-chloro-1-(5,5-dimethyl-2-oxo-5-silanyl-1-yl)-3-iodopyrazolo[4,3-][1,2]diazine 35e

[0231] To a 100 mL flask, compound 35d (2.2 g, 7.7 mmol), THF (40 mL), cesium carbonate (5.0 g, 15 mmol), and SEMCl (1.7 g, 10 mmol) were added. The mixture was stirred at room temperature overnight under nitrogen. LCMS monitoring indicated that the reaction was complete. The reaction solution was poured into water (100 mL) and extracted three times with ethyl acetate (30 mL x 3). The mixture was washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. Silica gel was added and the sample was mixed. The mixture was purified on a flash column to obtain compound 35e (1.3 g). LCMS (ESI-MS) m / z: 411.13 [M+H] + .

[0232] 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]ethan-1-ol 35f

[0233] A 15 mL sealed tube was added to compound 35e (410 mg, 1.0 mmol), DMSO (5.0 mL), compound 1d (280 mg, 1.5 mmol), and DIEA (390 mg, 3.0 mmol). The mixture was heated at 120°C for 2 hours under nitrogen protection. LCMS monitoring indicated that the reaction of the starting materials was essentially complete. The reaction solution was poured into water (50 mL) and extracted three times with ethyl acetate (10 mL x 3). The mixture was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and silica gel was added to mix the sample. The product was purified on a flash column to obtain compound 35f (140 mg). LCMS (ESI-MS) m / z: 280.18 [1 / 2 M+H] + .

[0234] 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]diazin-6-yl]azetidin-3-yl}hexahydropyridin-1-yl]ethane-1-ol

[0235] A 50 mL flask was added with compound 35f (200 mg, 0.36 mmol), THF (5.0 mL), bistriphenylphosphine palladium dichloride (50 mg, 0.072 mmol), CuI (20 mg, 0.10 mmol), TEA (0.25 mL, 1.8 mmol), and ethynylcyclopropane (71 mg, 1.1 mmol). The reaction was heated at 45°C under nitrogen for 1 h. LCMS monitoring showed that the reaction was essentially complete. The mixture was poured into water (20 mL) and extracted with ethyl acetate three times (10 mL*3). The mixture was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and purified on a flash column to obtain compound 35g (140 mg). LCMS (ESI-MS) m / z: 249.29 [1 / 2M+H + ].

[0236] 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]ethan-1-ol 35h

[0237] A 15 mL sealed tube was added with compound 35 g (110 mg, 0.22 mmol), a THF solution of TBAF (5.0 mL, 5.0 mmol), and 2,5-dimethyl-2,5-diazahexane (130 mg, 1.1 mmol). The mixture was reacted at 80 °C under nitrogen for 5 h. After LCMS monitoring, the reaction was essentially complete. The reaction was then poured into a saturated sodium chloride solution (15 mL), washed four times, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness to obtain a crude compound 35h, which was used directly in the next step.

[0238] LCMS(ESI-MS)m / z:184.17[1 / 2M+H] + .

[0239] 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]ethan-1-ol 35

[0240] A 15 mL sealed tube was added to compound 35h (60 mg, 0.16 mmol), DMF (2.0 mL), 5-(1-bromoethyl)-2,4-dichloropyridine 35i (130 mg, 0.49 mmol), and potassium carbonate (91 mg, 0.66 mmol). The mixture was stirred at room temperature overnight under nitrogen protection. LCMS monitoring showed that the starting materials were essentially reacted. The reaction solution was poured into water (30 mL) and extracted three times with EA (15 mL*3). The mixture was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was dried by spin drying. The mixture was purified by HPLC and lyophilized to obtain compound 35 (20 mg). LCMS (ESI-MS) m / z: 270.76 [1 / 2M+H] + .

[0241] 1 H NMR (400MHz, DMSO-d6) 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=5.9H z,2H),2.84–2.71(m,2H),2.59(s,1H),2.37(t,J=6.4Hz,2H),1.93(t,J=11.1Hz,1H),1.83(d, J=6.9Hz,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).

[0242] Biological activity test

[0243] Experiment 1: In vitro activity determination of the CCR4 inhibitory small molecule compound of the present invention

[0244] 1. Purpose of the experiment

[0245] In this experiment, the inhibitory effect of the compounds of the present invention on CCR4 receptor was tested by high-throughput real-time fluorescence analysis (FLIPR) method. 50 ) To evaluate the in vitro activity of the compounds.

[0246] 2. Experimental Methods

[0247] 2.1 Experimental Materials

[0248] Table 1 Experimental materials

[0249] 2.2 Experimental Procedure

[0250] (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.

[0251] (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 Direct™ No-Wash Loading Buffer into the 384-well cell culture plate.

[0252] (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.

[0253] (4) Start FLIPRTETRA

[0254] (5) Transfer 10 μL of agonist reference compound at 6×EC80 concentration to the cell plate.

[0255] (6) Reading the fluorescence signal

[0256] (7) For antagonist tests, 1 addition was used to calculate the "maximum-minimum value" from reading 1 to the maximum value allowed, and 1 addition was used to calculate the "maximum value" from reading 1 to 130 allowed.

[0257] (8) Analyze data

[0258] 2.3 Experimental Results

[0259] Table 1 In vitro activity test results of the compounds of the present invention

[0260] Conclusion: The compounds of the present invention have significant inhibitory activity on CCR4 receptor.

[0261] Experiment 2: In vivo efficacy study of the compounds of the present invention in mice

[0262] 1. Modeling and Drug Administration

[0263] 8-week-old female immunodeficient mice, with an average weight of 20 g. The experiment began after the mice had adapted for 3 days. The mice were divided into four groups: model group, FLX475 group, and Example 1 compound group, with 8 mice in each group. Humanized hepatoma cell lines Hepg2 were cultured before the experiment. After the cells reached a certain number, each mouse was subcutaneously injected with 1*10 7 After the tumors grew to a certain size, the model group, FLX475 group, and Example Compound 35 group were given 10 mL / kg by gavage and injection once daily; the model group was given an equal amount of vehicle by gavage / injection for 28 consecutive days. The specific design is shown in Table 3.

[0264] Table 3 In vivo efficacy experimental plan of compounds

[0265] 2. Experimental Animals

[0266] Female immunodeficient mice, 8 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number SCXK (Shanghai) 2017-0011, animal certificate number: 20170011003865.

[0267] 3. Experimental drugs

[0268] Example Compound 35.

[0269] 4. Drug configuration

[0270] An appropriate amount of compound was weighed, dissolved in 30% polyethylene glycol (PEG400), vortexed and sonicated, and then added to 70% saline to obtain a clear and transparent solution of 3 mg / mL.

[0271] 5. Medication

[0272] The specific administration is carried out according to the administration plan.

[0273] 6. Detection indicators and methods

[0274] The tumor size and body weight of mice were observed daily, and the tumor size was measured three times a week. The tumor was removed by dissection at the endpoint, and photographed to record the endpoint tumor size.

[0275] 7. Experimental results and conclusions

[0276] After transplantation of humanized Hepg2 tumor cells, the tumor size of mice in the model control group continued to increase over the days, while the tumor volume of Example Compound 35 began to decrease significantly after 14 days.

[0277] Experiment 3: Effects of the compounds of the present invention on hERG channel currents

[0278] 1. Purpose of the experiment

[0279] The effects of the compounds on hERG channel currents were detected by manual patch clamp technique to evaluate the cardiac safety of the compounds of the present invention.

[0280] 2. Experimental Methods

[0281] 2.1 Experimental Materials

[0282] 2.2 Experimental Procedure

[0283] 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 a temperature of 37° C. and a carbon dioxide concentration of 5%.

[0284] 2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of TrypLE TM Express solution, incubate at 37°C for approximately 0.5 min. When cells detach from the bottom of the dish, add approximately 5 mL of complete medium preheated at 37°C. Gently pipette the cell suspension to separate aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, seed the cells in a 6 cm cell culture dish at a seeding volume of 2.5 × 105 cells per dish (final volume: 5 mL).

[0285] 3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0286] 4) Patch clamp assay: cells were stained with TrypLE TM Express cells were isolated and 4 × 10 cells were plated onto coverslips in 24-well plates (final volume: 500 μL). After 18 hours, the assay was performed. The voltage stimulation protocol for whole-cell patch clamp recording of hERG potassium currents was as follows: after whole-cell seal formation, the cell membrane voltage was clamped at -80 mV. The clamping voltage was depolarized from -80 mV to -50 mV for 0.5 s (as a leak current measurement), then stepped to 30 mV for 2.5 s, and then rapidly restored to -50 mV for 4 s to stimulate the hERG channel tail current. Data were collected repeatedly every 10 s to observe the effects of drugs on hERG tail currents. A 0.5 s stimulation at -50 mV was used as a leak current measurement. Experimental data were acquired using an IPA amplifier (Sutter Instrument) and stored in Sutter Patch software.

[0287] When the hERG current recorded by the whole cell is stable, the drug is administered. After each drug concentration is applied for 5 minutes (or the current is stable), 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 to flow through the recording bath to act on the cells. At the same time, a peristaltic pump is used for liquid replacement during the recording. The current detected in the external solution without the compound for each cell 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.

[0288] 2.3 Data Analysis

[0289] First, the tail current (Peak tail current compound) after each drug concentration was normalized to the tail current of the blank solvent-treated control group (Peak tail current Control) (Normalized) (Peak tail current compound / Peak tail current Control). Then, the inhibition rate corresponding to each drug concentration was calculated (1-(Peak tail current compound / Peak tail current Control). The mean (Mean), standard deviation (SD), and standard error (SE) of the inhibition rate at each concentration were calculated. The data were expressed as Mean ± SE and saved in Excel. Y = 1 / (1 + 10^((LogIC50-X)*HillSlope). The IC of each compound was calculated using the above equation. 50 The concentration-effect curve was fitted nonlinearly, where IC 50 IC is the half inhibitory concentration. 50 Calculations and curve fitting were performed using GraphPad Prism software.

[0290] 2.4 Experimental Results

[0291] Among them, D<0.1, 0.1≤C<1.0, 1.0≤B<1.5, 1.5≤A.

[0292] 3. Experimental Conclusion

[0293] Compared with the positive reference FLX-475, the cardiac safety indicator hERG toxicity of the compound of the present invention is improved.

[0294] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and their equivalents are also within the scope of the appended claims.

Claims

1. A 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; X 1 or X 2 are each independently N or CH; X 3 N or CR a ; Y is -O- or -CH2-; R a -H, -C 1-3 Alkyl, -halogen or -CN, the -C 1-3 The alkyl group is optionally substituted with one or more halogens as allowed by the valency; L 1 For the key, -C 1-3 Alkylene-, -C 2-4 Alkenylene- or -C 2-4 Alkynylidene-; R 1 For -C 1-3 Alkylene-OH; R 2 -H, -CN, -C 1-5 alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, the -C 1-5 The alkyl group is optionally substituted with one or more halogens within the range allowed by the valence, and the 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl group is optionally substituted with one or more -C 1-5 Alkyl or -halogen substituted; R 3 For -C 1-5 alkyl; R 4 are each independently a halogen; n is 0, 1, 2 or 3.

2. The compound according to claim 1, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: Ring A is a 5-6 membered aromatic heterocyclic ring; preferably, the heteroatom of the 5-6 membered aromatic heterocyclic ring is N, O or S.

3. The compound according to claim 1 or 2, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: Ring A is Where E 1 、E 2 are independently CH, N, O or S, E 3 is a bond, CH or N, and E 1 、E 2 、E 3 Not at the same time CH; Preferably, ring A is 4. The compound according to claim 3, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: E 1 S, N, O, E 2 is CH, N, O or S, E 3 is the key; Preferably, E 1 For O, E 2 N, E 3 is a key; or E 1 N, E 2 For O, E 3 is a key; or E 1 For S, E 2 N, E 3 is a key; or E 1 N, E 2 For S, E 3 is a key; or E 1 For S, E 2 For CH, E 3 is the key.

5. The compound according to claim 3, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: E 1 、E 2 、E 3 are each independently CH or N, and E 1 、E 2 、E 3 Not at the same time CH; Preferably, E 1 For CH, E 2 N, E 3 CH; or E 1 For CH, E 2 For CH, E 3 N; or E 1 N, E 2 N, E 3 CH; E 1 For CH, E 2 N, E 3 N; or E 1 N, E 2 N, E 3 CH; E 1 N, E 2 N, E 3 is N.

6. 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 7. The compound according to any one of claims 1 to 6, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: n is 0, 1 or 2, preferably 2.

8. The compound according to any one of claims 1 to 7, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: X 1 or X 2 Not all are N; Preferably, X 1 For CH, X 2 N, X 3 N; or X 1 For CH, X 2 N, X 3 CR a ; or X 1 N, X 2 For CH, X 3 N; or X 1 For CH, X 2 For CH, X 3 N; or X 1 For CH, X 2 For CH, X 3 CR a ; More preferably, R a It is -H or -CN.

9. The compound according to any one of claims 1 to 8, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: L 1 For the key, -C 2-4 Alkenylene- or -C 2-4 Alkyne-, preferably a bond, -C≡C- or -CH=CH-; and / or R 1 It is -CH2CH2OH.

10. The compound according to any one of claims 1 to 9, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: R 2 -H, -CN, -C 1-5 alkyl, 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl, the -C 1-5 The alkyl group is optionally substituted with one or more halogens within the range allowed by the valence, and the 3-5 membered cycloalkyl or 3-5 membered heterocycloalkyl group is optionally substituted with one or more -C 1-3 Alkyl or -halogen substituted; Preferably, the -C 1-5 The 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, the cyclopropyl, oxetanyl or azetidinyl is optionally substituted with one or more methyl or -F within the range allowed by the valence; More preferably, R 2 -H, -CN, -CH3, -CHF2, -CF3, -CH(CH3)3, cyclopropyl, 11. The compound according to any one of claims 1 to 10, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: R 3 For -C 1-3 Alkyl, preferably methyl; and / or R 4 Each is independently -F, -Cl or -Br, preferably -Cl.

12. 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 Among them, Y, L 1 , R 2 、E 1 ~E 3 , X 1 ~X 3 As claimed in claim 1; Preferably, Y is -CH2-, X 1 For CH, X 2 N, X 3 is N.

13. The compound according to claim 1, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that: The compound has the following structure Among them, R 3 For -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 The 5-6 membered heteroaryl group contains 1, 2 or 3 heteroatoms.

14. 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 shown in the following structural formula:

15. 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 shown in the following structural formula:

16. A method for preparing the compound according to any one of claims 1 to 15, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, characterized in that: The method includes the steps shown in any of the following schemes 1 to 3: Option 1: Option 2: When X 3 CR a hour Option 3: When X 3 When N Wherein, each group is as described in claim 1.

17. A pharmaceutical composition, characterized in that The invention comprises a compound according to any one of claims 1 to 15, a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers and / or excipients.

18. 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.

19. A method for treating a CCR4-mediated disease, characterized in that: The method comprises administering a therapeutically effective amount of any compound according to any one of claims 1 to 15, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 17 to a patient or individual in need of such treatment.

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