Method for preparing chemokine receptor modulator and use thereof

Abstract

Provided are a class of compounds as shown in formula III or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing same. Further provided are the use of the compounds or pharmaceutically acceptable salt thereof, and the pharmaceutical composition containing same in the preparation of a drug for preventing and / or treating CCR4-mediated diseases, wherein the CCR4-mediated diseases comprise inflammatory diseases, autoimmune diseases or cancers.

Description

Preparation methods and uses of chemokine receptor modulators Technical Field

[0001] This invention belongs to the field of medicine and relates to compounds for regulating the activity of chemokine receptors and their uses. Background Technology

[0002] The successful operation of the host defense system is the result of several processes working together to eliminate foreign pathogens. Coordinated innate and adaptive immune responses are required, and many secreted and cell-associated factors have been identified as important mediators coordinating and regulating these two host defense weapons. Chemokines are a family of cytokines that act as chemical attractants to guide the movement 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 the cells of the immune system during immune surveillance processes, such as directing lymphocytes to lymph nodes to screen for pathogen invasion. Inflammatory chemokines are released by cells in response to pathological events (e.g., pro-inflammatory stimuli such as IL-1 or viruses). They primarily function as chemical attractants as part of the inflammatory response and are used to guide cells of both 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 regulate CCR4 function remains an ongoing challenge.

[0003] Cancer is a class of diseases in which cells exhibit disordered replication and growth. Recent cancer models have shown that the immune system, including cellular homing and immune checkpoints, is involved in cancer development and progression. Although significant progress has been made in understanding the biological basis of cancer, it remains a leading cause of death.

[0004] CCR4, first identified by Power et al. (J. Biol. Chem. 270:19495-19500), is a G protein-coupled receptor that binds to chemokines. CCR4 chemokines are chemokines of the Th2 subset of peripheral blood T cells, dendritic cells, and natural killer (NK) cells. Chemokines include CCL22 (also known as macrophage-derived chemokine (MDC)) and CCL17 (also known as thymic and activation-regulated chemokine (TARC)), which are also produced by monocytes and dendritic cells.

[0005] CCR4 is involved in immunomodulatory processes such as cell homing to specific tissues, including the homing of T lymphocytes to the skin and lungs (see 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, for example, in WO2013 / 082490.

[0006] Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is considered a key regulator of adaptive immune responses, playing a central role, particularly in maintaining and utilizing components of invasive T-cell responses. Therefore, CTLA-4, as an immune checkpoint inhibitor, is considered a potential therapeutic target for 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 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 considered an immune checkpoint inhibitor. PD-1 / PD-L1 modulators have been described, for example, in WO2018 / 005374.

[0008] Given the role 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

[0009] This invention provides a class of compounds and compositions for inhibiting CCR4 chemokine receptor type 4, and pharmaceutical compositions comprising the same. It also provides methods for treating or preventing diseases, conditions, or symptoms, such as by modulating (e.g., inhibiting) CCR4-mediated diseases, conditions, or symptoms; and provides combination therapies of CCR4 antagonists and one or more checkpoint inhibitors in cancer treatment.

[0010] The first aspect of this invention provides compounds of formula III, pharmaceutically acceptable salts or isotope derivatives thereof.

[0011] in: Indicates a single bond or a double bond;

[0012] Ring A is a 6-membered heteroaryl ring, in which Z 1 For C, Z3 Z 4 Each can be independently represented as C or N, Z 2 Z 6 Each can be independently represented as N or CH, Z 5 For N or CR z Z 2 ~Z 6 There are 1, 2, or 3 N;

[0013] R z For H or C, R connected to it 1 and Z 4 Form ring C;

[0014] R 3 -H or -CH3;

[0015] R 6 or R 7 Each can be independently -H, halogen, -CN, hydroxyl, or -C. 1-3 Alkyl or -C 1-3 Halogenated alkyl groups;

[0016] X is NR 2 or CR 2a R 2b ;

[0017] R 2 -H, -C 1-3 Alkyl, or R 2 and the N and R connected to it 1 Z 3 Z 4 Form ring B;

[0018] R 2a R 2b Each is independently -H, -C 1-3 Alkyl groups or C, R groups connected to them as bonds 1 Z 3 Z 4 Form ring B;

[0019] R 1 -H, -OC 1-3 Alkyl, -C 1-3 Alkyl, -P(O)(C 1-3 alkyl)2, or R 1 and Z 4 Z 5 Forming rings C and / or R 1 and X, Z 3 Z 4 Form ring B;

[0020] Rings B and C are each independently 5- or 6-membered rings, and each ring B and C are independently and optionally bounded by one or more -R groups within the range allowed by the valence. 8 replace,

[0021] R 8 Selected from =O, halogen, -C 1-3 Alkyl, -C 1-3 Halogenated alkyl groups, -OC 1-3 Alkyl, -OC 1-3 Halogenated alkyl groups, -CN, -C 2-4 Alkyne group, 5-8 membered heteroaryl group, or 3-8 membered cycloalkyl group;

[0022] The 5-8 nucleotide heteroaryl group is optionally surrounded by one or more -C groups within the range allowed by the valence. 1-3 Alkyl substitution;

[0023] The -C 2-4 The alkynyl group is optionally replaced by a 4-8-membered heterocyclic alkyl group or a 5-8-membered heteroaryl group, wherein the 5-8-membered heteroaryl group is optionally replaced by one or more -C groups within the range permitted by the valence. 1-3 Alkyl or -C 1-3 Halogenated alkyl substitution.

[0024] In some embodiments of the present invention, Z 2 ~Z 6 There is one N and Z 5 Let N be the number of elements in the array.

[0025] In some embodiments of the present invention, Z 1 Z 3 Z 4 For C, Z 2 Z 6 For CH, Z 5 Let N be the number of elements in the array.

[0026] In some embodiments of the present invention, Z 2 ~Z 6 There are two N's, for example, Z. 4 Z 6 For N, or Z 2 Z 5 For N, or Z 3 Z 5 For N, or Z 2 Z 6 For N, or Z 5 Z 6 Let N be the number of elements in the array.

[0027] In some embodiments of the present invention, Z 1 Z 3 For C, Z 5 For CRz Z 2 For CH, Z 4 Z 6 Let N be the number of elements in the array.

[0028] In some embodiments of the present invention, Z 1 Z 3 Z 4 For C, Z 5 For CR z Z 2 Z 6 Let N be the number of elements in the array.

[0029] In some embodiments of the present invention, Z 1 Z 3 For C, Z 4 Z 6 For CH, Z 2 Z 5 Let N be the number of elements in the array.

[0030] In some embodiments of the present invention, Z 1 Z 3 Z 4 For C, Z 6 For CH, Z 2 Z 5 Let N be the number of elements in the array.

[0031] In some embodiments of the present invention, Z 1 Z 4 For C, Z 2 Z 6 For CH, Z 3 Z 5 Let N be the number of elements in the array.

[0032] In some embodiments of the present invention, Z 1 Z 3 Z 4 For C, Z 5 For CR z Z 2 Z 6 Let N be the number of elements in the array.

[0033] In some embodiments of the present invention, Z 1 Z 3 Z 4 For C, Z 2 For CH, Z 5 Z 6 Let N be the number of elements in the array.

[0034] In some embodiments of the present invention, Z 2 ~Z 6 There are 3 N's.

[0035] In some embodiments of the present invention, Z 1 Z 3 For C, Z 5 For CR z Z 2 Z 4 Z 6 Let N be the number of elements in the array.

[0036] In some embodiments of the present invention, X is NR. 2 R 2 -H or -CH3 or R 2 and the N and R connected to it 1 Z 3 Z 4 Ring B is formed.

[0037] In some embodiments of the present invention, X is NR. 2 R 2 It can be -H or -CH3.

[0038] In some embodiments of the present invention, X is CR 2a R 2b R 2a R 2b The key is C and R connected to it. 1 Z 3 Z 4 Ring B is formed.

[0039] In some embodiments of the present invention, R 1 It can be -H, -OMe, -Me, -Et, or -P(O)Me2.

[0040] In some embodiments of the present invention, ring A is selected from the following structures: Preferably, ring A has the following connection method:

[0041] In some embodiments of the present invention, R 1 and X, Z 3 Z 4 A ring B is formed, wherein ring B is a 5-membered heterocycle containing 1 or 2 N atoms; preferably, ring B is a 5-membered heterocycle containing 2 N atoms.

[0042] In some embodiments of the present invention, ring B is selected from the following structures:

[0043] In some embodiments of the present invention, ring A and ring B are selected from the following structures:

[0044] In some embodiments of the present invention, R 1 and Z 4 Z 5 A ring C is formed, which is a 5- or 6-membered ring containing 0, 1, or 2 atoms chosen from N, O, or S atoms.

[0045] In some embodiments of the present invention, ring C is selected from the following structures:

[0046] In some embodiments of the present invention, the ring A and C are selected from the following structures:

[0047] In some embodiments of the present invention, R 1 and Z 4 Z 5 Forming a ring C and connecting with X and Z 3 Z 4 Ring B is formed, wherein ring B is a 5-membered heterocyclic alkane containing one nitrogen atom, and ring C is a 6-membered cyclic alkane.

[0048] In some embodiments of the present invention, ring B and ring C are selected from the following structures:

[0049] In some embodiments of the present invention, the fused ring formed by rings A, B, and C has the following structure:

[0050] In some embodiments of the present invention, R 6 or R 7 Each is independently -Cl or -F; preferably R 6 and R 7 It is -Cl.

[0051] In some embodiments of the present invention, R 8 Selected from =O, -F, -Cl, -Me, -Et, -halomethyl, -OMe, -OEt, -O-halomethyl, -CN, -C≡CH, 5-membered heteroaryl, or 3-4-membered cycloalkyl; wherein the 5-membered heteroaryl is optionally substituted with one or more methyl groups within the range allowed by the valence; wherein the -C≡CH is optionally substituted with one or more 4-5-membered heterocycloalkyl or 5-membered heteroaryl, wherein the 5-membered heteroaryl is optionally substituted with one or more -C groups within the range allowed by the valence. 1-3 Halogenated alkyl substitution.

[0052] In some embodiments of the present invention, R 8 Selected from =O, -F, -Cl, -CF3, -OMe, -Me, cyclopropyl, -CN, -OCF3,

[0053] In some embodiments of the present invention, the compound represented by formula III or its pharmaceutically acceptable salt or isotope derivative has a structure as shown in formulas III-a, III-b, III-c, III-d, III-e, III-f, III-g, III-h, III-i, III-j, III-k, and III-l:

[0054] Among them, R 1 R 6 R 7 R 8 The groups are as described in Formula III.

[0055] In some embodiments of the present invention, the compound represented by formula III or its pharmaceutically acceptable salt or isotope derivative has a structure as shown in formulas IV-a, IV-b, IV-c, IV-d, IV-e, IV-f, IV-g, IV-h, and IV-i:

[0056] Among them, R 2 R 6 R 7 R 8 The groups are as described in Formula III.

[0057] In some embodiments of the present invention, the compounds of the present invention or their pharmaceutically acceptable salts or isotope derivatives are selected from the following compounds or their pharmaceutically acceptable salts or isotope derivatives:

[0058] A second aspect of the present invention provides a pharmaceutical composition comprising the compounds of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or pharmaceutically acceptable salts or isotopic derivatives thereof, and optionally one or more pharmaceutically acceptable carriers and / or excipients.

[0059] The third aspect of the present invention provides the use of the compounds of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or their pharmaceutically acceptable salts or isotope derivatives, or the pharmaceutical compositions of the present invention in the preparation of medicaments for the prevention and / or treatment of CCR4-mediated diseases.

[0060] The fourth aspect of the present invention provides the use of the compounds of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or their pharmaceutically acceptable salts or isotope derivatives, or the use of the pharmaceutical compositions of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases, including inflammatory diseases, autoimmune diseases or cancer.

[0061] 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 stomach cancer.

[0062] The fifth aspect of the present invention provides a method for treating CCR4-mediated diseases, comprising administering to a patient requiring such treatment a therapeutically effective amount of any compound of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or a pharmaceutically acceptable salt or isotope derivative thereof, or a pharmaceutical composition according to the present invention.

[0063] The sixth aspect of the present invention provides the use of the compounds of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or their pharmaceutically acceptable salts or isotope derivatives, or the pharmaceutical compositions of the present invention for the prevention and / or treatment of diseases, including inflammatory diseases, autoimmune diseases or cancer.

[0064] The seventh aspect of the present invention provides the use of the compounds of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or their pharmaceutically acceptable salts or isotope derivatives, or pharmaceutical compositions according to the present invention, for the prevention and / or treatment of CCR4-mediated diseases.

[0065] The eighth aspect of the present invention provides a method for inhibiting CCR4, the method comprising contacting CCR4 with a compound of the present invention (including the compounds of the examples or formulas III, III-a to III-l, IV-a to IV-i) or a pharmaceutically acceptable salt or isotope derivative thereof, or a pharmaceutical composition of the present invention. Detailed Implementation

[0066] Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, and pharmacological methods, are employed. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. Reactions and purifications can be carried out using the manufacturer's instructions for use of kits, or in accordance with methods known in the art or the descriptions of this invention. The techniques and methods described herein are generally carried out according to conventional methods well known in the art, based on the descriptions in several summary and more specific documents cited and discussed in this specification.

[0067] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0068] The term "substitution" refers to the selective substitution of one or more hydrogen atoms on a specified atom or group by a specified group, provided that the substitution does not exceed the normal valence state of the specified atom. When the substituent is an oxo or ketone group (i.e., =O), the two hydrogen atoms on the atom are replaced by O. When the substituent is a double bond (i.e., =), it can also be considered that the two hydrogen atoms on the atom are replaced by C.

[0069] The term "optional" includes both selection and non-selection. The term "optional substitution" includes both substitution and non-substitution.

[0070] The term "-C" 1-n "alkyl" (where n is an integer from 2 to n) represents a non-cyclic, saturated, branched, or straight-chain aliphatic hydrocarbon group having 1 to n carbon atoms, preferably -C 1-12 Alkyl, more preferably -C 1-6 Alkyl groups, more preferably -C 1-3 Alkyl groups (referring to methyl, ethyl, n-propyl, and isopropyl).

[0071] The term "-C" 1-n "alkylene-" refers to the part from -C 1-n The residue resulting from removing a hydrogen atom from an alkyl group is a straight-chain or branched group containing 1 to n carbon atoms, preferably -C. 1-12 alkylene-, more preferably -C 1-6 Alkylene-, further preferably -C 1-3 Alkylenes (e.g., -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -C(CH3)2-, -CH(CH2CH3)-, -CH(CH3)-CH2-, -CH2-CH(CH3)-, etc.). Alkylenes can be substituted or unsubstituted, and when substituted, they can be substituted at any usable linker.

[0072] The term "-C" 2-n "-alkynyl group" refers to -C 2-n The alkyl group contains at least one carbon-carbon triple bond. For example, the term -C 2-3 Alkyne groups include -C≡CH, -C≡C-CH3, and -CH2-C≡CH.

[0073] An alkoxy group is an alkyl group that is attached to the rest of the molecule via an oxygen linker (-O-).

[0074] Unless otherwise specified, the term "haloalkyl" refers to an alkyl group in which one or more H atoms are replaced by a halogen (such as a fluorine, chlorine, bromine, or iodine atom), for example, C10. 1-3 Haloalkyl refers to C 1-3 One or more H atoms in an alkyl group are replaced by halogens.

[0075] C replaced by one or more fluorine1-3 Alkyl groups refer to methyl, ethyl, n-propyl, or isopropyl groups substituted with one or more fluorine molecules. For example, methyl groups substituted with one or more fluorine molecules include CF3, CHF2, and CH2F; ethyl groups substituted with one or more fluorine molecules include CH2CF3, CH2CHF2, CH2CH2F, CHFCF3, CHFCHF2, CHFCH2F, CF2CF3, CF2CHF2, and CF2CH2F.

[0076] This refers to whether a key exists or not, for example... single key or double bond

[0077] Unless otherwise stated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a monocyclic or fused plurality (preferably 1 to 3) rings connected directly or by covalent bonds.

[0078] When both ring B and ring C are present, ring B and ring C fuse together.

[0079] The terms “aromatic heterocyclic,” “aromatic heterocyclic group,” “heteroaromatic ring,” or “heteroaromatic group” have the same meaning: they refer to heterocyclic compounds with aromatic characteristics, including mono-heterocyclic aryl and fused heterocyclic aryl.

[0080] The term "cycloalkane" refers to a saturated monocyclic or polycyclic ring, and the term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic alkane 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, and even more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl. The term "heterocyclic alkane" refers to a cycloalkane containing at least one cyclic heteroatom (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom). The term "heterocyclic alkyl" refers to a cycloalkyl group containing at least one cyclic heteroatom (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom). In some embodiments of the invention, the heterocyclic group is a 4-12 membered heterocyclic group. In some embodiments of the invention, the heterocyclic group is a 4-6 membered heterocyclic group, such as tetrahydrofuranyl, oxetane, or azirone.

[0081] Unless otherwise specified, the terms “halogenated” or “halogen”, either on their own or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0082] Some compounds of the present invention have asymmetric carbon atoms (optical or chiral centers) or double bonds; based on absolute stereochemistry, enantiomers, racemates, diastereomers, tautomers, geometric isomers, and stereoisomers of amino acids and individual isomers can be defined as (R)- or (S)-, or (D)- or (L)-, and are all covered within the scope of the present invention.

[0083] The "compounds" of this invention, or the "compounds shown" of this invention, include pharmaceutically acceptable salts thereof, stereoisomers, tautomers, optical isomers, meso compounds, enantiomers, diastereomers, racemates, isotopic derivatives, or mixtures thereof. The compounds of this invention may also exist as hydrates or solvates.

[0084] The term "isotope derivative" refers to a derivative of a compound in which one or more atoms are replaced by their isotopes (atoms with the same atomic number but different atomic masses or mass numbers from the dominant atomic mass or mass number found 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), isotopes of oxygen (e.g. 15 O、 17 O and 18 O). The compounds of the present invention include isotopic derivatives of the compounds of the present invention, for example, compounds in which one or more atomic hydrogen atoms are replaced by deuterium or tritium isotopic derivatives.

[0085] In addition to the salt form, the present invention also provides compounds in prodrug form. The prodrugs of these compounds are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. The prodrugs of the compounds described herein can be converted in vivo after administration. Furthermore, the prodrugs can be converted into the compounds of the present invention in an in vitro environment by chemical or biochemical methods, for example, upon contact with suitable enzymes or chemical reagents.

[0086] Some compounds of this invention may exist in polycrystalline or amorphous forms. Generally, all physical forms are equivalent to the uses covered by this invention and are intended to fall within the scope of this invention.

[0087] The terms "pharmaceutical-grade salt" and "pharmaceutically acceptable salt" have the same meaning, including salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds in neutral form with a sufficient amount of the desired base (pure 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 the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds in neutral form with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Certain specific compounds of the present invention contain both basic and acidic functional groups, which allows the compounds to be converted into base or acid addition salts.

[0088] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate the administration and absorption of a compound to an individual and can be included in the compositions of the present invention without causing significant adverse toxicity to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solution, 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, hydroxymethyl cellulose, polyvinylpyrrolidone, and pigments. Such formulations are sterilizable and, if desired, can be mixed with adjuvants that do not react adversely with the compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring and / or aromatic substances, etc.

[0089] The term "treatment" refers to any indication of successfully treating or improving an injury, disease, pathology, or symptom, including any objective or subjective parameter such as symptom relief; remission; reduction or making the injury, pathology, or symptom more tolerable for the patient; slowing the rate of degeneration or decline; reducing the degree of weakness at the degenerative endpoint; or improving the patient's physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term "treatment" and variations thereof may include prevention of injury, pathology, symptom, or disease. In this implementation, treatment is prevention. In this implementation, treatment does not include prevention.

[0090] As used herein, “treatment” (and as is known in the art) also broadly includes any method that can achieve a favorable or desired outcome (including clinical outcomes) in an individual’s condition. Favorable or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, a reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), prevention of the spread or diffusion of the disease, delay or slowing the progression of the disease, improvement or mitigation of the disease state, reduction of disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. In other words, as used herein, “treatment” includes any cure, improvement, or prevention of a disease. Treatment can prevent the occurrence of the disease; inhibit the spread of the disease; alleviate the symptoms of the disease; completely or partially eliminate the root cause of the disease; shorten the duration of the disease; or a combination of these events.

[0091] As used herein, “treatment” includes preventative treatment. Treatment methods include administering a therapeutically effective amount of the compound described herein to an individual. Administration may include a single dose or a series of doses. The duration of treatment depends on various factors, such as the severity of symptoms, the patient's age, the concentration of the compound, the activity of the composition used for treatment, and their combination. It should also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease as a particular treatment or prevention regimen progresses. Dosage changes can be obtained and become apparent through standard diagnostic analyses known in the art. In some instances, prolonged administration may be required. For example, administering the composition to an individual in an amount and for a duration sufficient to treat the patient. The term “prevention” refers to reducing the occurrence of disease symptoms in a patient. As mentioned above, prevention can be complete (no detectable symptoms) or partial prevention, resulting in fewer observed symptoms than would be possible without treatment. In embodiments, prevention refers to slowing the progression of a disease, symptom, or condition, or inhibiting its progression to a harmful or other undesirable state.

[0092] "Patient" or "individual in need" means a living organism that suffers from or is susceptible to a disease or symptom that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0093] "Effective amount" is the amount of a compound sufficient to achieve its intended purpose (e.g., to achieve the effect of administration, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce signaling pathways, or to alleviate one or more symptoms of a disease or condition) relative to the absence of such an effective amount. An example of an "effective amount" is an amount sufficient to promote the treatment, prevention, or relief of one or more symptoms of a disease, also referred to as a "therapeutic effective amount." "Relief" (and its grammatical equivalent) of one or more symptoms means a reduction in the severity or frequency of the symptoms, or the elimination of the symptoms. A "preventive effective amount" of a drug is the amount of the drug that, when administered to an individual, would have the intended preventive 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 such an injury, disease, pathology, or condition or its symptoms. A complete preventive effect does not necessarily occur with the administration of a single dose and may only occur after a series of doses. Therefore, a preventive effective amount can be administered over a single or multiple administrations. As used herein, "activity reduction amount" refers to the amount of antagonist required to reduce enzyme activity relative to the absence of an antagonist. As used herein, “functionally disruptive dose” refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of an antagonist. The exact dose will depend on the therapeutic purpose and will be determined by someone skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 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). Therapeuticly effective doses can be determined by measuring the relevant physiological effects and can be adjusted in conjunction with individual symptom management, dosing regimens, and diagnostic analyses. For example, measuring serum levels of a CCR4 inhibitor (or, for example, its metabolites) at a specific time after administration can indicate whether a therapeutically effective dose has been administered.

[0094] In this document, the term "administration" means oral administration, administration as a suppository, local contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration, or implantation of a sustained-release device, such as a micro-osmotic pump, into an individual. Administration is via any route, including parenteral and transmucosal (e.g., oral, sublingual, palate, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intravenous, and intracranial administration. Other delivery modalities include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. "Combined administration" means administration of the composition described herein simultaneously, immediately before, or immediately after administration of one or more additional therapies (e.g., anticancer agents, chemotherapy, or treatment for neurodegenerative diseases). The compounds of the present invention can be administered alone or in combination to a patient. Combined administration is intended to include administration of one or more compounds or agents simultaneously or sequentially, alone or in combination. Therefore, when necessary, the formulation 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 a local route and can be formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, coatings, powders, and aerosols. Oral formulations include tablets, pills, powders, sugar-coated pills, capsules, liquids, lozenges, capsules, gels, syrups, slurries, suspensions, etc., suitable for patient ingestion. Solid forms include powders, tablets, pills, capsules, capsules, suppositories, and dispersible granules. Liquid forms 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 fragmented drug carrier matrices. These components 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 via intradermal injection as drug-containing microspheres that release slowly under the skin (see Rao, J. Biomater Sci. Polym. 7th ed., 623-645, 1995); as biodegradable and injectable gel formulations (see, for example, Gao Pharm. Res. 12:857-863, 1995); or as microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).In another embodiment, the formulation of the composition of the present invention can be delivered using liposomes fused to or endocytosed by cell membranes, i.e., by employing receptor ligands linked to the liposomes, which bind to cell surface membrane protein receptors, thereby inducing endocytosis. By using liposomes, particularly when the liposomes carry receptor ligands specifically targeting target cells or preferentially involving specific organs, the composition of the present invention can be delivered concentratedly 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.

[0095] "CCR4 inhibitor" refers to a compound (such as the compound described herein) that reduces CCR4 activity compared to a control (such as a compound that does not contain the compound or is known to be inactive).

[0096] The invention is further illustrated by the following examples, which are illustrative and do not limit the invention in any way. Any modifications or alterations to the invention that are readily achievable by those skilled in the art will fall within the scope of the invention.

[0097] Unless otherwise specified, all raw materials or reagents used in the embodiments of this invention are commercially available.

[0098] The abbreviations used in this invention have their conventional meanings in the art. For example, the meanings of the following abbreviations are as follows:

[0099] Preparation methods of compounds

[0100] Example 1: 2-(3-(1-(5-((1-(2,4-dichlorophenyl)ethyl)amino)-6-methoxypyridin-3-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (1)

[0101] Step 1: Preparation of compound 1b

[0102] Compound 1a (3.0 g, 12.9 mmol) was dissolved in EtOH (30 mL) and water (10 mL). Fe (2.88 g, 51.5 mmol) and ammonium chloride (2.75 g, 51.5 mmol) were added sequentially, and the mixture was stirred in an oil bath at 80 °C for 8 h. The reaction was monitored by LC-MS until complete. The reaction solution was directly filtered, and the filtrate was extracted with EA (30 mL). The organic phase was collected, washed with saturated brine, and separated. The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain compound 1b (2.50 g). LC-MS (ESI-MS) m / z: 203.0 [M+H] + .

[0103] Step 2: Preparation of compound 1d

[0104] Compound 1b (1.0 g, 4.9 mmol) and compound 1c (1.25 g, 4.92 mmol) were dissolved in DMF (10 mL), and NaH (473 mg, 19.7 mmol) was slowly added at 0 °C. The mixture was stirred overnight at room temperature. After the reaction was complete, water (5 mL) was added to quench the reaction mixture. The mixture was washed with ethyl acetate and extracted. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and flash purified (PE:EA = 0–7%) to give compound 1d (1.40 g). LCMS (ESI-MS) m / z: 374.96 [M+H] + .

[0105] Step 3: Preparation of Compound 1

[0106] Compound 1d (100 mg, 0.31 mmol) and compound 1e (95 mg, 0.31 mmol) were dissolved in DMF (3 mL), and Xantphos PdG3 (40 mg, 0.04 mmol) and cesium carbonate (200 mg, 0.62 mmol) were added sequentially. The mixture was reacted under nitrogen protection at 100 °C in an oil bath with stirring for 16 h. The reaction was confirmed by LCMS. The reaction solution was filtered, and the filtrate was purified by HPLC to obtain compound 1 (11 mg). LCMS (ESI-MS) m / z: 479.3 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ7.58-7.57(d,1H),7.50-7.48(s,1H),7.38-7.35(m,1 H),6.50-6.49(m,1H),5.75-5.74(d,1H),5.53(s,1H),4.73-4.70(m,1H),3.78 (m,3H),3.91-3.88(m,2H),3.66-3.59(m,4H),3.51(s,2H),3.21(s,2H),2.36 (s,2H),2.01-2.97(m,2H),1.52-1.50(m,2H),1.45-1.43(d,4H),1.22(m,2H).

[0107] Example 2: 2-((3R)-3-(1-(4-((1-(2,4-dichlorophenyl)ethyl)amino)imidazo[1,2-a][1,3,5]triazin-2-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (2)

[0108] Step 1: Preparation of compound 2c

[0109] Compound 2a (3.3 g, 20 mmol) and 1-(2,4-dichlorophenyl)ethyl-1-amine 2b (3.8 g, 20 mmol) were dissolved in ACN (70 mL), stirred overnight at room temperature, and then concentrated directly. The resulting solution was flash purified (PE:EA = 0–18%) to give compound 2c (2.7 g). LCMS (ESI-MS) m / z: 318.1 [M+H] +

[0110] Step 2: Preparation of compound 2d

[0111] Compound 2c (955 mg, 3.0 mmol) and compound 1e (1.0 g, 3.3 mmol) were dissolved in DMF (20 mL), and cesium carbonate (4.77 g, 15.0 mmol) was added. The mixture was reacted overnight at room temperature, then stirred at 50 °C for 1 h. After filtration, the filtrate was washed with water (60 mL), extracted with EA, dried over anhydrous sodium sulfate, filtered, concentrated, and flash purified (DCM: MeOH = 0–20%) to give compound 2d (670 mg). LCMS (ESI-MS) m / z: 466.3 [M+H] +

[0112] Step 3: Preparation of Compound 2

[0113] Compound 2d (590 mg, 1.27 mmol) and chloroacetaldehyde hydrate 2e (996 g, 5.08 mmol) were dissolved in DMSO (5 mL) and reacted in an oil bath at 120 °C with stirring for 3 h. The reaction was monitored by LCMS until complete. The mixture was washed with water (5 mL), extracted with ethyl acetate, separated, and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and flash purified (DCM:MeOH = 0–30%) to give compound 2 (70 mg). LCMS (ESI-MS) m / z: 490.3 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.68-8.66(d,1H),7.72-7.71(m,1H),7.59-7.58(d,1H), 7.49-7.46(m,1H),7.40-7.38(m,1H),7.10-7.09(d,1H),5.49(s,1H),5.39(s,1H ),3.91(s,2H),3.63(s,2H),3.46-3.45(m,2H),2.74-2.71(m,2H),2.35-2.31(m, 4H),1.91-1.86(m,1H),1.58-1.55(m,4H),1.51-1.49(d,3H),1.41-1.37(m,1H).

[0114] Example 3: 2-[(3R)-3-[1-(5-{[(1R)-1-(2,4-dichlorophenyl)ethyl]amino}imidazo[3,2-a]pyrimidin-7-yl)azacyclobutane-3-yl]hexahydropyridin-1-yl]ethane-1-ol (3)

[0115] Step 1: Preparation of compound 3c

[0116] Compound 3a (500 mg, 2.66 mmol), compound 3b (505 mg, 2.66 mmol), and DIEA (1.03 g, 7.98 mmol) were dissolved in ethanol (10 mL) and reacted in an oil bath at 80 °C for 3 h. After the reaction was complete, the solution was concentrated under reduced pressure and flash purified (PE / EA, 0–30%) to give compound 3c (670 mg). LCMS (ESI-MS) m / z: 341.16 [M+H] + .

[0117] Step 2: Preparation of Compound 3

[0118] Compound 3c (320 mg, 0.94 mmol), compound 1e (863 mg, 4.7 mmol), and potassium carbonate (518 mg, 3.76 mmol) were dissolved in anhydrous DMF (5 mL) and reacted in an oil bath at 120 °C for 7 h under an inert atmosphere. After the reaction was complete, water was added, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and flash purified (DMC / MeOH, 0–15%). Compound 3 (102 mg) was prepared by liquid chromatography. LCMS (ESI-MS) m / z: 489.44 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.79–7.62(m,3H),7.52–7.39(m,2H),7.15(d,J=1.6Hz,1H),4 .97(p,J=6.7Hz,1H),4.66(s,1H),4.33(t,J=5.4Hz,1H),3.84(dt,J=32.0,8.1Hz,2H) ,3.64–3.55(m,1H),3.45(q,J=6.1Hz,3H),2.76–2.65(m,2H),2.43–2.24(m,3H),1.86 (t,J=11.1Hz,1H),1.66–1.46(m,7H),1.39(d,J=12.5Hz,1H),0.75(d,J=11.4Hz,1H).

[0119] Example 4: 2-((3R)-3-(1-(4-((1-(2,4-dichlorophenyl)ethyl)amino)-7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)azacyclobutane-3-yl)piperidin-1-yl)ethane-1-ol (4)

[0120] Step 1: Preparation of compound 4b

[0121] Compound 4a (4.0 g, 23.7 mmol) was dissolved in DMF (50 mL), and the mixture was placed in an ice bath at 0 °C. NaH (1.2 g, 30 mmol, 60%) was added to replace the nitrogen atmosphere, and the mixture was stirred for half an hour. SEMCl (4.4 g, 26 mmol) was then slowly added dropwise. After the addition was complete, the reaction mixture was allowed to rise naturally to room temperature, stirred for 2 hours, quenched with ice water, extracted with EA, dried and concentrated the organic phase, and purified by normal column chromatography (SiO2, PE:EA = 1:0 to 3:1) to give compound 4b (5.30 g). LCMS (ESI-MS) m / z: 299.2 [M+H] + .

[0122] Step 2: Preparation of compound 4c

[0123] Compound 4b (5.77 g, 19 mmol) was dissolved in DMF (50 mL). NaH (1.16 g, 29 mmol, 60%) was added under an ice bath at 0 °C to replace nitrogen. The mixture was stirred at this temperature for half an hour, and intermediate 1c (5.39 g, 21.2 mmol) was slowly added dropwise. After the addition was complete, the reaction solution was allowed to warm naturally to room temperature and stirred at room temperature for 2 hours. The reaction was then quenched with ice water, extracted with ethyl acetate, dried, concentrated, and purified by normal column chromatography (SiO2, PE:EA = 1:0 to 10:1) to give compound 4c (6.40 g). LCMS (ESI-MS) m / z: 471.3 [M+H] + .

[0124] Step 3: Preparation of compound 4d

[0125] Compound 4c (500 mg, 1.06 mmol) and compound 1e (258 mg, 1.40 mmol) were dissolved in DMSO (1.0 mL) and CH3CN (1.0 mL). K2CO3 (440 mg, 3.18 mmol) and DIEA (410 mg, 3.17 mmol) were added, purging nitrogen. The reaction mixture was heated in a microwave oven at 90 °C for 12 hours. After the reaction was completed, the mixture was filtered, the filter cake was washed with ethyl acetate, the organic phase was dried, concentrated, and passed through a normal column (SiO2, DCM:MeOH = 1:0 to 10:1) to obtain compound 4d (290 mg). LCMS (ESI-MS) m / z: 619.5 [M+H] + .

[0126] Step 4: Preparation of Compound 4

[0127] Compound 4d (230 mg, 0.37 mmol) was dissolved in DCM (1 mL), and TFA (1 mL) was added. The mixture was stirred at 20 °C for 1 hour. After the reaction was complete, the solution was concentrated to obtain 230 mg of crude product. 30 mg of this crude product was used to prepare pre-HPLC purified compound 4 (8.5 mg). LCMS (ESI-MS) m / z: 519.4 [M+H] + . 1H NMR(400MHz, DMSO-d6)δ8.19(s,1H),7.77(d,J=6.8Hz,1H),7.53(s,1H),7.41(t,J=8.0Hz,1H),7.32–7.34(m, 1H),6.81–6.84(m,1H),6.53(s,1H),5.48–5.54(m,1H),5.27(s,2H),3.80–3.85(m,2H),3.75–3.77(m,1H),3. 49–3.58(m,4H),3.26–3.35(m,2H),2.78–2.84(m,2H),2.67–2.83(m,1H),2.46–2.49(m,1H),2.26–2.32(m,1H ),2.03(t,J=11.6Hz,1H),1.70(t,J=10.4Hz,1H),1.57–1.61(m,2H),1.43(d,J=7.2Hz,3H),0.75–0.81(m,1H).

[0128] Example 5: 2-[(3R)-3-[1-(4-{[1-(2,4-dichlorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-2-yl)azacyclobutane-3-yl]hexahydropyridin-1-yl]ethanol (5)

[0129] Preparation of compound 5

[0130] Compound 4 crude product (230 mg, 0.44 mmol) was dissolved in methanol (2 mL), and K2CO3 (304 mg, 2.2 mmol) was added. The mixture was stirred at 20 °C for 1 hour at room temperature. The reaction was detected by LCMS. After the reaction was completed, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by pre-HPLC to obtain compound 5 (36.5 mg). LCMS (ESI-MS) m / z: 489.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.90(s,1H),7.72(d,J=6.8Hz,1H),7.53(d,J=2.0Hz,1H),7.42–7.46(m,1H),7.35( d,J=8.4Hz,1H),6.71(s,1H),6.46(s,1H),5.53(q,J=7.2Hz,1H),3.81(t,J=8.0Hz,1H),3.73(s,1H),3.53(q ,J=5.6Hz,3H),3.29–3.34(m,2H),2.86–2.90(m,1H),2.67–2.83(m,2H),2.49–2.51(m,2H),2.26–2.32(m,1H ),2.10(t,J=12.0Hz,1H),1.80(t,J=10.4Hz,1H),1.61(s,3H),1.43(d,J=7.2Hz,3H),0.81(t,J=11.2Hz,1H).

[0131] Example 6: 2-[(3R)-3-[1-(4-{[1-(2,4-dichlorophenyl)ethyl]amino}-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)azacyclobutane-3-yl]hexahydropyridin-1-yl]ethanol (6)

[0132] Step 1: Preparation of compound 6b

[0133] Compound 6a (10 g, 66.2 mmol) was dissolved in trifluoroacetic acid (100 mL), and triethylsilane (23 g, 199 mmol) was added at room temperature. The mixture was stirred overnight at room temperature, concentrated, and evaporated to dryness to give compound 6b (16.8 g), which was directly added to the next step. LCMS (ESI-MS) m / z: 154.0 [M+H] + .

[0134] Step 2: Preparation of compound 6c

[0135] Compound 6b (16.8 g) was dissolved in POCl3 (50 mL), and DIPEA (25 g, 199 mmol) was added. The reaction mixture was heated to 90 °C and stirred for 4 hours. After cooling, the solution was evaporated to dryness and purified by silica gel column chromatography to obtain compound 6c (7 g). LCMS (ESI-MS) m / z: 190.0 [M+H] + .

[0136] Step 3: Preparation of compound 6d

[0137] Compound 6c (7 g, 36.8 mmol) was dissolved in DCM (60 mL), and (Boc)₂O (8 g, 36.8 mmol), triethylamine (7.4 g, 73.6 mmol), and DMAP (449 mg, 3.68 mmol) were added. The mixture was stirred overnight at room temperature. After the reaction was complete, compound 6d (7.5 g) was purified by column chromatography. LCMS (ESI-MS) m / z: 290.0 [M+H] + .

[0138] Step 4: Preparation of compound 6e

[0139] Compound 6d (7.5 g, 25.9 mmol) was dissolved in dioxane (25 mL), and ammonia (10 mL) was added. The mixture was stirred at 60 °C for 8 hours. After cooling, the solution was purified by silica gel column chromatography to give compound 6e (3 g). LCMS (ESI-MS) m / z: 271.0 [M+H] + .

[0140] Step 5: Preparation of compound 6f

[0141] Compound 6e (1 g, 3.7 mmol) and compound 1c (940 mg, 3.7 mmol) were dissolved in DMF (10 mL), and cesium carbonate (2.4 g, 7.4 mmol) was added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was poured into water (50 mL), extracted three times with dichloromethane, dried over the organic phase, filtered, concentrated, and purified by silica gel column chromatography to give compound 6f (540 mg). LCMS (ESI-MS) m / z: 443.0 [M+H] + .

[0142] Step 6: Preparation of 6g of compound

[0143] Compound 6f (540 mg, 1.22 mmol) and compound 1e (224 mg, 1.22 mmol) were dissolved in DMF (5 mL), and cesium carbonate (795 mg, 2.44 mmol) was added. The mixture was stirred at 100 °C for 8 hours. After the reaction was complete as determined by LCMS, the mixture was cooled to room temperature, poured into water (30 mL), extracted three times with dichloromethane, dried over the organic phase, filtered, concentrated, and purified by silica gel column chromatography to obtain compound 6 g (75 mg). LCMS (ESI-MS) m / z: 591.4 [M+H] + .

[0144] Step 7: Preparation of Compound 6

[0145] Compound 6 g (65 mg, 0.11 mmol) was added to ethyl acetate hydrochloride solution (2 mL), and the reaction mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by LC-MS. The reaction mixture was then evaporated to dryness at low temperature, and purified by reverse reaction to give compound 6 (21 mg). LC-MS (ESI-MS) m / z: 491.4 [M+H] + . 1 HNMR(400MHz, Methanol-d4)δ8.45(s,1H),7.56–7.27(m,2H),7.21(dd,J=8.3, 2.1Hz,1H),5.29(dd,J=11.5,6.7Hz,1H),4.04–3.86(m,2H),3.86–3.64(m,5H) ,3.03–2.69(m,4H),2.56(q,J=5.7Hz,2H),2.43–2.26(m,1H),2.06(t,J=11.6H z,1H),1.66(d,J=40.8Hz,5H),1.39(dd,J=7.0,1.7Hz,3H),1.37-1.17(m,1H).

[0146] Example 7: 2-((3R)-3-(1-(6-((1-(2,4-dichlorophenyl)ethyl)(methyl)amino)pyrazin-2-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol-1-ol (7)

[0147] Step 1: Preparation of compound 7b

[0148] Compound 2b (500 mg, 2.63 mmol) was dissolved in DMSO (5 mL), 2,6-dichloropyrazine 7a (391.9 mg, 2.63 mmol), and cesium fluoride (1.2 g, 7.89 mmol). The mixture was heated to 75 °C and stirred for 1 hour. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated brine, concentrated under reduced pressure, and purified by silica gel column chromatography (0–30% EA / PE) to give compound 7b (600 mg). LCMS m / z (ESI): 302.2 [M+H] + .

[0149] Step 2: Preparation of compound 7c

[0150] Compound 7b (500 mg, 1.65 mmol) was dissolved in DMF (10 mL). Under nitrogen protection, sodium hydride (133 mg, 3.3 mmol) was added in an ice bath, and the mixture was stirred for 30 minutes. Iodimethane (305 mg, 2.15 mmol) was then added in an ice bath, and the mixture was stirred at room temperature for 2 hours. The mixture was then quenched with water, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (0–15% EA / PE) to give compound 7c (290 mg). LCMS m / z (ESI): 316.11 [M+H] + .

[0151] Step 3: Preparation of Compound 7

[0152] Compound 7c (150 mg, 0.47 mmol) was dissolved in DMF (4 mL), followed by the addition of (R)-2-(3-(azacyclobutan-3-yl)piperidin-1-)ethanol-1-ol 1e (114 mg, 0.62 mmol) and cesium carbonate (772 mg, 2.37 mmol). The mixture was stirred in a microwave at 130 °C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature and purified by prep-HPLC to give compound 7 (20 mg). LCMS m / z (ESI): 464.40 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.56(d,J=2.0Hz,1H),7.45–7.37(m,2H),7.32(d,J=1.3Hz,1H),7.02(s,1H),5.76(dd ,J=7.0,3.4Hz,1H),4.31(s,1H),3.91(dt,J=13.3,8.1Hz,2H),3.60(ddd,J=8.5,6.1,2.8Hz,1H),3.53(td,J=8 .4,6.0Hz,1H),3.45(dt,J=8.3,4.0Hz,2H),2.82(d,J=3.4Hz,3H),2.73(d,J=10.9Hz,3H),2.43(s,1H),2.33( t,J=6.5Hz,2H),1.89(t,J=10.9Hz,1H),1.58(d,J=16.4Hz,4H),1.46(d,J=7.0Hz,3H),1.40(d,J=12.2Hz,1H).

[0153] Example 8: 3-((R)-1-(2,4-dichlorophenyl)ethyl)-5-(3-(((R)-1-(2-hydroxyethyl)piperidin-3-yl)azacyclobutane-1-yl)-1-methyl-1,3-dihydro-2H-imidazo[4,5-b]pyrazin-2-one (8)

[0154] Step 1: Preparation of compound 8b

[0155] Compound 8a (500 mg, 1.87 mmol), compound 3b (356 mg, 1.87 mmol), DIEA (242 mg, 1.87 mmol), and NMP (3 mL) were added to a sealed tube and reacted at 180 °C for 5 hours. LC-MS showed the reaction was complete. The reaction was quenched with water, extracted with EA, washed three times with sodium chloride aqueous solution, concentrated the organic phase, and purified by flash to give compound 8b (450 mg). LC-MS (ESI-MS) m / z: 375.14 [M+H] + .

[0156] Step 2: Preparation of compound 8c

[0157] Compound 8b (450 mg, 1.197 mmol) and DIEA (463.96 mg, 3.59 mmol) were added to a three-necked flask, followed by the addition of dichloromethane (10 mL) to displace nitrogen. The mixture was cooled to 0°C in an ice-salt bath with stirring. A DCM solution of triphosgene (710 mg, 2.39 mmol) was added dropwise. After the addition was complete, the mixture was brought to room temperature. The reaction proceeded for 16 hours until complete. The mixture was neutralized with triethylamine (1 mL), quenched with water, extracted with DCM, washed once with sodium chloride aqueous solution, concentrated the organic phase, and purified by flash to give compound 8c (400 mg). LCMS (ESI-MS) m / z: 401.15 [M+H] + .

[0158] Step 3: Preparation of Compound 8

[0159] Compound 8c (200 mg, 0.497 mmol), compound 1e (91.67 mg, 0.497 mmol), cesium carbonate (324.14 mg, 0.995 mmol), and DMF (3 mL) were added. After microwave reaction at 130 °C for 2 hours, LCMS showed the presence of products, and TLC (MeOH:DCM = 10%) showed new spots. The mixture was quenched in water, extracted with EA, washed three times with sodium chloride aqueous solution, concentrated, flash purified, and purified by Prep HPLC (formic acid system) to obtain compound 8 (7 mg). LCMS (ESI-MS) m / z: 505.42 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.77(d,J=8.7Hz,1H),7.58(s,1H),7.46(d,J=8.5Hz,1H),7.10( s,1H),5.82(q,J=7.1Hz,1H),3.87(t,J=7.6Hz,2H),3.58(d,J=7.1Hz,2H),3.47(d,J=7. 0Hz,3H),3.25(s,3H),2.76(s,2H),2.37(t,J=6.4Hz,3H),1.95(d,J=11.2Hz,1H),1.81( d,J=7.2Hz,3H),1.61(d,J=30.6Hz,4H),1.42(d,J=12.8Hz,1H),0.81(d,J=12.0Hz,1H).

[0160] Example 9: (R)-4-(2,4-dichlorobenzyl)-2-(3-(1-(2-hydroxyethyl)piperidin-3-yl)azacyclobutane-1-yl)pyrrolo[2,3,4-de]quinazolin-5(4H)-one (9)

[0161] Step 1: Preparation of compound 9c

[0162] Compound 9a (872 mg, 3.39 mmol), acetonitrile (50 mL), and DIEA (657.6 mg, 5.088 mmol) were reacted with compound 9b (656.85 mg, 3.7 mmol) under an ice-water bath for 2 h to give compound 9c (1.1 g, 2.87 mmol). LCMS (ESI-MS) m / z = 396.13 [M+H] + .

[0163] Step 2: Preparation of compound 9d

[0164] Compound 9c (396 mg, 0.998 mmol) was dissolved in THF (10 mL) and methanol (2 mL), and lithium hydroxide (71.73 mg, 2.995 mmol) was added at room temperature. The reaction was allowed to proceed overnight. The pH was adjusted to 5 with 2N hydrochloric acid aqueous solution. The mixture was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 9d (380 mg, 0.99 mmol), which was used directly in the next step. LCMS (ESI-MS) m / z = 382.10 [M+H] + .

[0165] Step 3: Preparation of compound 9e

[0166] Compound 9d (380 mg, 0.993 mmol) was added to a suspension in DCM (30 mL) with DMAP (182 mg, 1.49 mmol), and the solution became clear. Triethylamine (1.38 mL, 9.9 mmol) was added, followed by dropwise addition of phosphorus oxychloride (761 mg, 4.966 mmol) at room temperature. The reaction was carried out for 1 h. Saturated ammonium chloride was added, and the mixture was extracted with DCM, dried over anhydrous sodium sulfate, and distilled under reduced pressure to give compound 9e (93 mg, 0.255 mmol). LCMS (ESI-MS) m / z = 364.15 [M+H] +

[0167] Step 4: Preparation of Compound 9

[0168] Compound 9e (360 mg, 0.987 mmol) and compound 1e (236.54 mg, 1.284 mmol) were dissolved in DMF (5 mL) and DIEA (255 mg, 1.975 mmol) were added. The reaction was carried out at 110 °C for 3 h. Saturated sodium chloride aqueous solution was added, and the mixture was extracted with EA. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Preparative HPLC purification (ammonia system) yielded compound 9 (24 mg, 0.047 mmol). LCMS (ESI-MS) m / z = 512.40 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ7.80(t,J=7.7Hz,1H),7.70–7.54(m,3H),7.50-7.30(m 2H),4.96(s,2H),4.34(s,1H),4.15-4.04(m,2H),3.79(s,2H),3.52-3.42(m,2H),2.85-2.70(m,2 H),2.46-2.40(m,1H),2.39-2.30(m,2H),2.05–1.85(m,2H),1.75-1.50(m,3H),1.20-1.35(m,2H).

[0169] Example 10: (R)-2-(3-(1-(4-(2,4-dichlorobenzyl)-4,5-dihydropyrrolo[2,3,4-de]quinazolin-2-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (10)

[0170] Step 1: Preparation of compound 10a

[0171] Compound 9c (500 mg, 1.26 mmol), THF (5 mL). Nitrogen was purged three times at room temperature, and lithium aluminum hydride (1.0 mL, 2.5 mmol) was added dropwise under dry ice and ethyl acetate bath. The reaction was allowed to proceed for 1 h, then allowed to proceed for another 1 h at room temperature. Saturated ammonium chloride aqueous solution was added, and the mixture was extracted with EA, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (EA:PE = 30%) to give compound 10a (360 mg). LCMS (ESI-MS) m / z = 368.16 [M+H] + .

[0172] Step 2: Preparation of compound 10b

[0173] Compound 10a (100 mg, 0.27 mmol) was dissolved in DCM (15 mL). The solution was purged with nitrogen at room temperature, and triphenylphosphine (142.3 mg, 0.543 mmol) and DIAD (115.19 mg, 0.57 mmol) were added under ice-water bath conditions. The reaction was allowed to proceed for 3 h at room temperature. Methanol was added, and the solution was concentrated under reduced pressure. The solution was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 20%) to give compound 10b (95 mg). LCMS (ESI-MS) m / z = 350.09 [M+H] +

[0174] Step 3: Preparation of Compound 10

[0175] Compound 1e (210 mg, 1.14 mmol) was dissolved in ethanol (3 mL) by sonication. Compound 10b (200 mg, 0.57 mmol) was dissolved in DIEA (221.17 mg, 1.711 mmol). The reaction was carried out at 60 °C for 3 h, followed by at 80 °C for 5 h. The mixture was concentrated under reduced pressure. Column purification yielded compound 10 (33 mg). LCMS (ESI-MS) m / z = 249.68 [M / 2+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.67(d,J=2.1Hz,1H),7.47(t,J=7.7Hz,1H),7.42(dd,J=8.3,2.1Hz,1 H),7.36(d,J=8.4Hz,1H),7.01(d,J=8.2Hz,1H),6.97(d,J=7.2Hz,1H),4.83(s,2H),4.70(s,2 H),4.32(s,1H),4.07–3.98(m,2H),3.76-3.68(m,2H),3.50-3.42(m,2H),2.80-2.70(m,2H),2 .37–2.31(m,2H),2.04–1.85(m,2H),1.70-1.54(m,4H),1.50-1.35(m,1H),0.85–0.77(m,1H).

[0176] Example 11: 2-[(3S)-3-[1-(7-{[(1R)-1-(2,4-methyl)ethyl]amino}[1,3]thiazo[5,4-d]pyrimidin-5-yl)azacyclobutane-3-yl]hexahydropyridin-1-yl]ethanol (11)

[0177] Step 1: Preparation of compound 11b

[0178] Compound 3b (369 mg, 1.9 mmol) and compound 11a (400 mg, 1.9 mmol) were dissolved in acetonitrile (10 mL), and DIEA (501.84 mg, 3.88 mmol) was added. The mixture was reacted at room temperature for 3 h. The mixture was extracted with ethyl acetate after adding saturated sodium chloride aqueous solution. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 11b (690 mg, 1.9 mmol) was given and used directly in the next step. LCMS (ESI-MS) m / z = 359.15 [M+H] +

[0179] Step 2: Preparation of Compound 11

[0180] Compound 11b (150 mg, 0.417 mmol) and compound 1e (76.86 mg, 0.417 mmol) were dissolved in DMF (2 mL) and DIEA (107.8 mg, 0.83 mmol) were added. The reaction was carried out at 110 °C for 3 h. A saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under reduced pressure. Compound 11 (10 mg) was prepared using a preparative HPLC (ammonium bicarbonate) system. LCMS (ESI-MS) m / z = 507.30 [M+H] +1 H NMR (400MHz, DMSO-d6) δ8.75(s,1H),8.56(d,J=7.1Hz,1H),7.56–7.50(m,2H),7.32-7.37(m,1H),5.62-5.48(m,1H),4.33(s,1H),3.96–3. 76(m,2H),3.62(s,1H),3.50-3.40(m,2H),2.80-2.70(m,2H),2.40-2 .20(m,3H),1.94-1.82(m,1H),1.63–1.34(m,8H),0.80-0.68(s,1H).

[0181] Example 12: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-2-(trifluoromethyl)-1H-imidazo[4,5-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (12)

[0182] Step 1: Preparation of compound 12b

[0183] Compound 12a (1 g, 3.95 mmol), compound 3b (0.90 g, 4.745 mmol), and DIEA (0.72 g, 5.5 mmol) were dissolved in NMP (5 mL), microwaved at 180 °C for 2 hours, extracted with ethyl acetate (50 mL), washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Flash purification (0–30% EA / PE) yielded compound 12b (1.3 g, 3.59 mmol). LCMS (ESI-MS) m / z: 361.09 [M+H] +

[0184] Step 2: Preparation of compound 12c

[0185] Compound 12b (450 mg, 1.2 mmol) was dissolved in dioxane (4 mL), and trifluoroacetic acid (1 mL, 13 mmol) was added. The mixture was reacted in a microwave at 120 °C for 8 hours. After the reaction was complete, saturated ammonium bicarbonate was added to adjust the pH to 6-7. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0–30% EA / PE) to give compound 12c (230 mg, 0.52 mmol). LCMS (ESI-MS) m / z: 439.12 [M+H] +

[0186] Step 3: Preparation of Compound 12

[0187] Compound 12c (140 mg, 0.318 mmol) was dissolved in CH3CN (7 mL), and compound 1e (87.94 mg, 0.477 mmol) and K2CO3 (131.90 mg, 0.954 mmol) were added. The mixture was stirred in a microwave oven at 75 °C for 6 hours. After the reaction was complete, the reaction solution was filtered, concentrated under reduced pressure, and purified by prep-HPLC to give compound 12 (80 mg, 0.147 mmol). LCMS (ESI-MS) m / z: 543.43 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.86(s,1H),7.72(d,J=8.6Hz,1H),7.61(q,J=1.6Hz,1H),7.49(dd,J=8.6,2.2Hz ,1H),6.04(q,J=7.0Hz,1H),4.32(t,J=5.5Hz,1H),4.10–3.97(m,2H),3.79(t,J=7.4Hz,1H),3.64(s,1H) ,3.47(q,J=6.0Hz,2H),2.72(d,J=10.0Hz,2H),2.53(s,1H),2.38–2.31(m,2H),1.99(d,J=7.0Hz,3H),1. 91(t,J=11.1Hz,1H),1.63(dq,J=21.8,11.9,11.5Hz,4H),1.41(d,J=12.7Hz,1H),0.81(d,J=12.6Hz,1H).

[0188] Example 13: 2-((R)-3-(1-(1-(R)-1-(2,4-dichlorophenyl)ethyl)-1H-imidazo[4,5-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethane-1-ol (13)

[0189] The synthesis route is shown in Example 12. LCMS (ESI-MS) m / z: 238.1 [M / 2+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.44(s,1H),7.68–7.62(m,2H),7.51–7.40(m,2H),6.02 (q,J=6.8Hz,1H),4.33(t,J=5.2Hz,1H),4.02(q,J=8.2Hz,2H),3.70(q,J=7.6Hz, 2H),3.47(q,J=6.0Hz,2H),2.74(d,J=9.6Hz,2H),2.34(t,J=6.2Hz,2H),1.90(d, J=7.0Hz,4H),1.73–1.53(m,5H),1.42(t,J=12.0Hz,1H),0.81(d,J=12.2Hz,1H).

[0190] Example 14: 2-((R)-3-(1-(6-((R)-1-(2,4-dichlorophenyl)ethyl)-8-(trifluoromethyl)imidazo[1,5-a]pyrimidin-3-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (14)

[0191] Step 1: Preparation of compound 14b

[0192] Compound 14a (2.3 g, 9.996 mmol) was dissolved in THF (20 mL), and the mixture was purged with nitrogen at room temperature. Sodium hydride (0.52 g, 12.995 mmol) was added in an ice-water bath, and the reaction was continued for 30 min. Iodomethane (0.97 mL, 11.995 mmol) was added dropwise, and the reaction was continued at room temperature for 2 h. A saturated ammonium chloride aqueous solution was added, and the pH was adjusted to 6 with 2N hydrochloric acid aqueous solution. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Crude compound 14b (2.5 g) was obtained and used directly in the next step without further purification. LCMS (ESI-MS) m / z = 247.13 [M+H] + .

[0193] Step 2: Preparation of compound 14c

[0194] Compound 14b (2.4 g, 9.7 mmol) was added sequentially, dissolved in THF (20 mL), water (5 mL), and lithium hydroxide (0.47 g, 19 mmol). The reaction was carried out at room temperature for 20 h. The solvent was removed by vacuum distillation, the pH was adjusted to 5 with 2N hydrochloric acid aqueous solution, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 14c (2.1 g), which was used directly in the next step. LCMS (ESI-MS) m / z = 219.060 [M+H] + .

[0195] Step 3: Preparation of compound 14e

[0196] Compound 14c (1179 mg, 5.38 mmol) was dissolved in DMF (5 mL), and HATU (2232.5 mg, 5.87 mmol), DIEA (1581 mg, 12 mmol), and (5-bromopyrimidin-2-yl)methylamine 14d (920 mg, 4.89 mmol) were added. The mixture was reacted at room temperature for 2 h. Saturated sodium chloride aqueous solution was added, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 14e (1.6 g, 4.1 mmol). LCMS (ESI-MS) m / z = 388.17 [M+H] + .

[0197] Step 4: Preparation of compound 14f

[0198] Compound 14e (1.5 g, 3.855 mmol) was dissolved in phosphorus oxychloride (15 mL) and reacted at 90 °C for 2 h. The solution was concentrated under reduced pressure, water was added, and the solution was adjusted to neutral with saturated sodium bicarbonate. Extraction was performed using DCM. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was then performed by silica gel column chromatography (EA:PE = 30% and MeOH:DCM = 5%), followed by slurry preparation at EA:PE = 10%. The solid was collected to give compound 14f (1.32 g, 3.557 mmol). LCMS (ESI-MS) m / z = 370.10 [M+H] + .

[0199] Step 5: Preparation of 14g of compound

[0200] Compound 14f (1.3 g, 3.5 mmol) was suspended in acetonitrile (3 mL) and purged with nitrogen at room temperature. NIS (0.95 g, 4.2 mmol) and TFA (0.054 mL, 0.70 mmol) were added under ice-water bath conditions. The reaction was allowed to proceed for 15 min. A saturated aqueous sodium sulfite solution was added, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 14f (1.3 g). LCMS (ESI-MS) m / z = 496.10 [M+H] + .

[0201] Step 6: Preparation of compound 14h

[0202] 14 g (496 mg, 0.998 mmol) of compound was dissolved in DMF (3 mL), and cuprous iodide (380 mg, 1.198 mmol), hexamethylphosphonic triamine (214.6 mg, 1.198 mmol), and methyl fluorosulfonyl difluoroacetate (766.96 mg, 3.99 mmol) were added. The mixture was purged with nitrogen at room temperature and reacted at 100 °C for 2 h. After cooling to room temperature, saturated sodium chloride aqueous solution was added, and the mixture was extracted three times with EA. The organic phase was dried, and the solvent was removed by vacuum distillation. The solution was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 20%) to give compound 14 h (420 mg, 0.957 mmol). LCMS (ESI-MS) m / z = 438.17 [M+H] + .

[0203] Step 7: Preparation of Compound 14

[0204] Compound 14h (50 mg, 0.114 mmol) and compound 1e (31.48 mg, 0.171 mmol) were dissolved in dioxane (3 mL), and RuPhos (7.97 mg, 0.017 mmol), RuPhosPdG3 (14 mg, 0.017 mmol), and cesium carbonate (55.66 mg, 0.17 mmol) were added. The mixture was purged with nitrogen at room temperature and reacted at 105 °C for 5 h. The mixture was then concentrated under reduced pressure. Compound 14 (6 mg) was purified by silica gel column chromatography. LCMS (ESI-MS) m / z = 542.46 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.20-8.15(m,1H),7.66-7.62(m,1H),7.42-7.36(m,1H),7. 32-7.27(m,1H),7.24-7.17(m,1H),5.01-4.92(m,1H),4.37(s,1H),4.04–3.80(m,2 H),3.62(t,J=6.9Hz,1H),3.57–3.51(m,1H),3.52-3.43(m,2H),2.76(s,2H),2.38( s,2H),2.02–1.90(m,2H),1.75-1.55(m,6H),1.50-1.35(m,2H),0.87-0.80(m,1H).

[0205] Example 15: (4-(((R)-1-(2,4-dichlorophenyl)ethyl)amino)-2-(3-((((R)-1-(2-hydroxyethyl)piperidin-3-yl)azacyclobutane-1-yl)pyrimidin-5-yl)dimethylphosphine oxide (15)

[0206] Step 1: Preparation of compound 15b

[0207] Compound 15a (1 g, 3.6 mmol), compound 3b (0.76 g, 4.0 mmol), DIEA (0.94 g, 7.27 mmol), and isopropanol (10 mL) were reacted at 80 °C for 5 hours. LC-MS showed the reaction was complete. The reaction was quenched with water, extracted with EA, washed three times with sodium chloride aqueous solution, and the organic phase was concentrated. The crude product was flash purified to give compound 15b (1.4 g). LC-MS (ESI-MS) m / z: 380.16 [M+H] + .

[0208] Step 2: Preparation of compound 15c

[0209] Compound 15b (1 g, 2.3 mmol), dimethylphosphine oxide (0.36 g, 4.668 mmol), Pd2(dba)3 (0.21 g, 0.23 mmol), 4,5-bisdiphenylphosphine-9,9-dimethyloxanthracene (0.27 g, 0.467 mmol), and potassium phosphate (1.07 g, 4.668 mmol) were added to a single-necked flask. Nitrogen gas was replaced with dioxane (10 mL), and the reaction was carried out at 100 °C for 2 hours. The reaction was then quenched with water, extracted with EA, washed three times with sodium chloride aqueous solution, and the organic phase was concentrated. The crude product was Flash purified to give compound 15c (200 mg). LCMS (ESI-MS) m / z: 378.15 [M+H] + .

[0210] Step 3: Preparation of Compound 15

[0211] Compound 15c (200 mg, 0.528 mmol), compound 1e (146 mg, 0.792 mmol), potassium carbonate (146.0 mg, 1.056 mmol), and DMF (5 mL) were stirred at 70 °C for 2 hours. The reaction proceeded for 2 hours. After cooling, the mixture was filtered, washed once with methanol, and the filtrates were combined to obtain the crude product. Prep HPLC (formic acid system) purification yielded compound 15 (110 mg). LCMS (ESI-MS) m / z: 526.18 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=6.2Hz,1H),7.94(d,J=8.2Hz,1H),7.55(d,J=2.1Hz,1H ),7.37(dd,J=8.4,2.1Hz,1H),7.32(d,J=8.4Hz,1H),5.30(s,1H),4.31(s,1H),3.88(d,J =8.7Hz,1H),3.60(s,2H),3.44(s,2H),2.74–2.64(m,2H),2.31(s,3H),1.88(d,J=11.2Hz ,1H),1.65(d,J=13.5Hz,7H),1.56(d,J=12.9Hz,3H),1.35(d,J=7.0Hz,5H),0.72(s,1H).

[0212] Example 16: 2-[(3R)-3-[1-(5-{[(1R)-1-(2,4-dichlorophenyl)ethyl](methyl)amino}imidazo[3,2-a]pyrimidin-7-yl)azacyclobutane-3-yl]hexahydropyridin-1-yl]ethanol-1-ol (16)

[0213] Step 1: Preparation of compound 16b

[0214] Compound 16a (2 g, 11 mmol) was dissolved in DCM (30 mL), and Boc₂O (3.1 mL, 14 mmol) and DIEA (2.7 g, 21 mmol) were added. After stirring at room temperature for 2 hours, compound 16b (3.6 g) was purified by Flash column chromatography. LCMS (ESI-MS) m / z: 234.08 [M-55] + .

[0215] Step 2: Preparation of compound 16c

[0216] Take a 100 mL flask, add compound 16b (3.6 g, 12 mmol), DMF (25 mL), and under nitrogen protection, add NaH (0.99 g, 25 mmol) with stirring in an ice bath. Continue stirring for 20 min, then add iodomethane (2.5 mL, 31 mmol), and allow the mixture to return to room temperature for 1 hour. LCMS monitoring showed the reaction was complete. Quench the reaction mixture with water (40 mL), extract three times with EA (20 mL * 3), wash with saturated sodium chloride aqueous solution (40 mL), dry with anhydrous sodium sulfate, and purify by Flash column chromatography to obtain compound 16c (3.1 g). LCMS (ESI-MS) m / z: 248.12 [M-55] + .

[0217] Step 3: Preparation of compound 16d

[0218] Compound 16c (3.1 g, 10 mmol) was dissolved in DCM (40 mL), and TFA (20 mL) was added. The mixture was reacted under nitrogen protection at room temperature for 3 hours until complete. The solution was evaporated to dryness, and then DCM (20 mL) was added. The mixture was washed twice with saturated K₂CO₃ aqueous solution (20 mL x 2), then washed with saturated sodium chloride aqueous solution (20 mL). The solution was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The solution was then used directly in the next reaction. LCMS (ESI-MS) m / z: 204.10 [M+H] + .

[0219] Step 4: Preparation of compound 16e

[0220] Take 15 mL of the sealed tube, add 16d (100 mg, 0.49 mmol), DMF (2 mL), 5,7-dichloroimidazolo[3,2-a]pyrimidine (92 mg, 0.49 mmol), and NaHCO3 (82 mg, 0.98 mmol), and heat at 70 °C for 5 hours under nitrogen protection. Monitor the reaction by LC-MS. Once the starting material has reacted completely, pour the reaction solution into water (20 mL), extract three times with EA (10 mL * 3), wash with saturated sodium chloride aqueous solution (20 mL), dry with anhydrous sodium sulfate, filter, evaporate the solvent, and purify by Flash column chromatography to obtain compound 16e (120 mg). LC-MS (ESI-MS) m / z: 355.25 [M + H] + .

[0221] Step 5: Preparation of Compound 16

[0222] Compound 16e (140 mg, 0.39 mmol), DMF (3 mL), 2-[(3R)-3-(azacyclobutan-3-yl)hexahydropyridin-1-yl]ethanol-1-ol (110 mg, 0.59 mmol), and NaHCO3 (99 mg, 1.2 mmol) were reacted at 120 °C for 4 hours under nitrogen protection. After the reaction was complete, the mixture was poured into water (30 mL), extracted three times with EA (10 mL * 3), washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The mixture was then prepared by HPLC (ammonium bicarbonate system) to give compound 16 (44 mg). LCMS (ESI-MS) m / z: 252.28 [1 / 2 M + H] + . 1 H NMR (400MHz, DMSO-d6) δ7.63(d,J=8.5Hz,1H),7.56(d,J=2.2Hz,1H),7.44(dd,J=8.5,2.2Hz,1H),7.32(d,J=1. 7Hz,1H),7.23(d,J=1.7Hz,1H),5.38(s,1H),5.23(q,J=6.8Hz,1H),4.36(t,J=5.3Hz,1H),4.05–3.91(m,2H),3. 66(ddd,J=23.6,8.4,5.9Hz,2H),3.47(d,J=5.1Hz,2H),2.82–2.71(m,2H),2.69(s,3H),2.43(d,J=7.3Hz,1H),2 .34(t,J=6.4Hz,2H),1.95–1.84(m,1H),1.62(t,J=8.8Hz,6H),1.50–1.36(m,1H),0.79(q,J=12.7,10.4Hz,1H).

[0223] Example 17: (R)-2-(4-(1-(5-((1-(2,4-dichlorophenyl)ethyl)amino)-2-methylimidazo[1,2-a]pyrimidin-7-yl)azacyclobutane-3-yl)piperidin-1-yl)ethane-1-ol (17)

[0224] Step 1: Preparation of compound 17b

[0225] Compound 17a (2.5 g, 16 mmol) and bromoacetone (3.3 g, 0.080 mmol) were dissolved in acetone (15 mL). The mixture was microwaved at 100 °C for 3 hours. The reaction was quenched with sodium bicarbonate solution, extracted with dichloromethane / methanol (10:1), dried, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to give compound 17b (0.80 g). LCMS (ESI-MS) m / z: 180.13 [M+H] + .

[0226] Step 2: Preparation of compound 17c

[0227] Compound 17b (0.50 g, 2.8 mmol) and sodium iodide (3.3 g, 8.4 mmol) were dissolved in acetonitrile (10 mL), and trimethylchlorosilane (0.91 g, 8.4 mmol) was added at room temperature. After reacting at 90 °C for 6 hours, the solution was concentrated and purified by silica gel column chromatography to give compound 17c (0.40 g). LCMS (ESI-MS) m / z: 166.14 [M+H] + .

[0228] Step 3: Preparation of compound 17d

[0229] Compound 17c (0.40 g, 2.4 mmol) was dissolved in phosphorus oxychloride (5.0 mL) and purged with nitrogen. The reaction was carried out at 100 °C for 4 hours. The reaction was quenched with sodium bicarbonate aqueous solution, extracted with DCM, dried and filtered, the filtrate was concentrated, and purified by silica gel column chromatography to give compound 17d (0.20 g). LCMS (ESI-MS) m / z: 202.06 [M+H] + .

[0230] Step 4: Preparation of compound 17e

[0231] Compound 17d (0.18 g, 0.89 mmol), (R)-1-(2,4-dichlorophenyl)ethylamine (0.17 g, 0.89 mmol) was reacted in acetonitrile (5.0 mL), followed by the addition of N,N-diisopropylethylamine (0.35 g, 2.7 mmol). The mixture was reacted at 100 °C for 6 hours, concentrated, and purified by flash to give compound 17e (0.20 g). LCMS (ESI-MS) m / z: 355.27 [M+H]+ .

[0232] Step 5: Preparation of Compound 17

[0233] Compound 17e (0.15 g, 0.34 mmol) and 2-[(3R)-3-(azacyclobutan-3-yl)hexahydropyridin-1-yl]ethane-1-ol (0.16 g, 0.84 mmol) were dissolved in acetonitrile (4.0 mL), and potassium carbonate (0.17 g, 1.3 mmol, 3.0 mL) was added at room temperature. The reaction was carried out in a microwave oven at 120 °C for 1.5 h. The reaction solution was concentrated, and the crude product was purified by preparative liquid chromatography (ammonium bicarbonate) to give compound 17 (55 mg). LCMS (ESI-MS) m / z: 503.40 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.66(s,1H),7.58(d,J=6.8Hz,1H),7.49(d,J=5.8Hz,2H),7.43(d,J=8.8 Hz,1H),5.01–4.93(m,1H),4.64(s,1H),4.34(s,1H),3.88(t,J=8.2Hz,1H),3.83–3.76(m,1H),3 .61–3.56(m,1H),3.47(s,3H),2.74(d,J=13.0Hz,2H),2.35(q,J=9.2,7.8Hz,3H),2.17(s,3H),1 .92–1.84(m,1H),1.62(d,J=20.2Hz,4H),1.54(d,J=6.8Hz,3H),1.43(s,1H),0.81–0.72(m,1H).

[0234] Example 18: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-3-(1-methyl-1H-1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (18)

[0235] Step 1: Preparation of compound 18b

[0236] Compound 18a (200 mg, 1.2 mmol) was dissolved in THF (16 mL) under nitrogen atmosphere in an ice bath. Isopropylmagnesium chloride-lithium chloride (2.8 mL, 3.7 mmol) was added, and the mixture was stirred in an ice bath for 1 hour. Tri-n-butyltin chloride (0.67 mL, 2.5 mmol) was then added, and the mixture was stirred for another 1 hour. After the reaction was complete, a saturated potassium fluoride solution (30 mL) was added, and the mixture was stirred for half an hour. EA (50 mL) was added, and the mixture was stirred. The solid was filtered off, and the organic phase was washed three times with saturated sodium chloride (50 mL). The mixture was dried, filtered, and concentrated under reduced pressure to give compound 18b (960 mg). LCMS (ESI-MS) m / z: 374.38 [M+H] + .

[0237] Step 2: Preparation of Compound 18

[0238] Compound 18c (200 mg, 0.33 mmol), compound 18b (300 mg, 0.40 mmol), tetrakis(triphenylphosphine)palladium (58 mg, 0.050 mmol), and CuI (9.5 mg, 0.050 mmol) were dissolved in DMF (4.0 mL) and reacted under nitrogen atmosphere at 100 °C for 1 hour. The mixture was cooled to room temperature and quenched by stirring with saturated potassium fluoride for 1 hour. The mixture was extracted with DCM, washed twice with saturated potassium fluoride solution, washed twice with saturated brine, dried, and concentrated under reduced pressure. The solution was purified by Flash column chromatography, followed by prep-HPLC purification, and lyophilized to give compound 18 (56 mg). LCMS (ESI-MS) m / z: 278.82 [M / 2+1] + . 1 HNMR(400MHz,DMSO-d6)δ8.56(s,1H),7.91(s,1H),7.62(d,J=2.0Hz,1H),7.47–7.38(m,2H),6.30(q ,J=7.0Hz,1H),4.31(t,J=5.4Hz,1H),4.18(dd,J=15.9,8.1Hz,2H),3.95(s,3H),3.89(d,J=12.4Hz, 2H),3.47(q,J=6.0Hz,2H),2.75(t,J=11.0Hz,2H),2.55(d,J=7.8Hz,1H),2.35(t,J=6.3Hz,2H),2.0 3–1.80(m,4H),1.66(ddd,J=34.8,23.0,12.1Hz,4H),1.42(d,J=12.1Hz,1H),0.83(d,J=10.4Hz,1H).

[0239] Example 19: 2-[(3R)-3-(1-{1-[(1R)-1-(2,4-dichlorophenyl)ethyl]-3-(trifluoromethyl)pyrazolo[4,3-c][1,2]diazin-6-yl}azacyclobutane-3-yl)hexahydropyridin-1-yl]ethanol-1-ol (19)

[0240] Step 1: Preparation of compound 19b

[0241] Compound 19a (1.0 g, 3.6 mmol), (1S)-1-(2,4-dichlorophenyl)ethanol-1-ol (0.68 g, 3.6 mmol), and triphenylphosphine (1.1 g, 4.3 mmol) were dissolved in THF (60 mL), and DIAD (0.87 g, 4.3 mmol) was added. The mixture was stirred at room temperature for 2 hours. After 2 hours, the reaction was monitored by LCMS to indicate completion. The mixture was quenched with water, extracted twice with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by Flash column chromatography to give compound 19b (770 mg). LCMS (ESI-MS) m / z: 453.21 [M+H] +

[0242] Step 2: Preparation of compound 19c

[0243] Compound 19b (500 mg, 1.1 mmol), methyl 2,2-difluoro-2-(fluorodioxy-λ6-thioalkyl)acetate (420 mg, 2.2 mmol), HMPA (0.77 mL, 4.4 mmol), and CuI (210 mg, 1.1 mmol) were dissolved in DMF (5.0 mL). The mixture was stirred at 110 °C for 4 hours under nitrogen protection, and the product was monitored by LCMS. The mixture was filtered, and the filtrate was dispersed in water, extracted twice with ethyl acetate, and washed twice with water with the organic phase. The solution was dried, concentrated, and purified by Flash column chromatography to give compound 19c (350 mg). LCMS (ESI-MS) m / z: 395.23 [M+H] + .

[0244] Step 3: Preparation of Compound 19

[0245] Compound 19c (150 mg, 0.38 mmol), 2-[(3R)-3-(azacyclobutan-3-yl)hexahydropyridin-1-yl]ethanol-1-ol (100 mg, 0.57 mmol), and DIEA (150 mg, 1.1 mmol) were dissolved in DMF (6.0 mL). After stirring at 145 °C for 4 hours, the product was monitored by LCMS. The mixture was concentrated and purified by preparative liquid chromatography to give compound 19 (47 mg). LCMS (ESI-MS) m / z: 543.5 [M+H] + .1 H NMR (400MHz, DMSO-d6) δ7.66(t,J=1.2Hz,1H),7.48(d,J=1.5Hz,2H),6.74(s,1H),6.24(q,J=6.8Hz,1H ),4.33(t,J=5.4Hz,1H),4.15(q,J=7.9Hz,2H),3.83(t,J=7.2Hz,2H),3.47(q,J=6.0Hz,2H),2.76(t,J= 11.9Hz,2H),2.60(d,J=7.9Hz,1H),2.35(t,J=6.3Hz,2H),1.92(t,J=11.3Hz,1H),1.83(d,J=6.9Hz,3H ),1.70(q,J=11.5,10.4Hz,3H),1.60(d,J=15.5Hz,1H),1.43(d,J=12.2Hz,1H),0.85(d,J=12.1Hz,1H).

[0246] Example 20: 2-((R)-3-(1-(3-cyclopropyl-1-(R)-1-(2,4-dichlorophenyl)ethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol-1-ol (20)

[0247] Step 1: Preparation of Compound 20

[0248] Compound 20a (0.30 g, 0.50 mmol), cyclopropylboronic acid (0.060 g, 0.75 mmol), and sodium carbonate (0.16 g, 1.5 mmol) were dissolved in dioxane (6.0 mL) and purged with nitrogen. [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (0.040 g, 0.05 mmol) was added at room temperature, purged with nitrogen, and reacted at 100 °C for 12 hours. The reaction was quenched with water, extracted with EA, dried and filtered, and the filtrate was concentrated and purified by liquid chromatography (ammonium bicarbonate) to give compound 20 (29 mg). LCMS (ESI-MS) m / z: 515.40 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.72(s,1H),7.63–7.58(m,1H),7.41(s,2H),6.15(q,J=7.2Hz,1H),4.32(t,J=5.4 Hz,1H),4.12(dt,J=13.8,8.4Hz,2H),3.83(t,J=8.4Hz,2H),3.48(q,J=6.0Hz,2H),2.79–2.71(m,2H),2.5 8–2.52(m,1H),2.35(t,J=6.4Hz,2H),2.22–2.15(m,1H),1.91(t,J=11.0Hz,1H),1.77(d,J=7.0Hz,3H),1. 72–1.57(m,4H),1.46–1.38(m,1H),1.19–1.11(m,2H),0.99(dd,J=8.6,3.2Hz,2H),0.82(d,J=12.4Hz,1H).

[0249] Example 21: 5-((R)-1-(2,4-dichlorophenethyl)amino)-7-(3-((R)-1-(2-hydroxyethyl)piperidin-3-yl)azacyclobut-1-yl)imidazo[1,2-a]pyrimidine-2-carboxynitrile (21)

[0250] Step 1: Preparation of compound 21c

[0251] Compound 21a (1.0 g, 6.1 mmol) was dissolved in acetone (6.0 mL), and compound 21b (1.6 g, 9.1 mmol) was added. The mixture was microwaved at 100 °C for 2 hours. The reaction was quenched with 20 mL of saturated sodium bicarbonate aqueous solution, concentrated under vacuum at 45 °C using a diaphragm pump, and extracted with EA (20 mL * 3). The organic phase was washed once with saturated brine (20 mL), dried over anhydrous Na₂SO₄ powder, filtered, and the filtrate was evaporated to dryness to obtain the crude product (1.6 g). Purification by silica gel column chromatography yielded compound 21c (330 mg). LCMS (ESI-MS) m / z: 224.1 [M + H] + .

[0252] Step 2: Preparation of compound 21d

[0253] Compound 21c (330 mg, 1.5 mmol) was dissolved in ACN (4.0 mL), and sodium iodide (660 mg, 4.4 mmol) and TMSCl (480 mg, 4.4 mmol) were added. The mixture was reacted at 90 °C for 2 hours under nitrogen protection. The reaction solution was cooled to room temperature and concentrated under vacuum at 45 °C using a diaphragm pump to obtain compound 21d (360 mg). The crude product was directly used in the next step. LCMS (ESI-MS) m / z: 210.1 [M+H] + .

[0254] Step 2: Preparation of compound 21e

[0255] Compound 21d (360 mg, 1.5 mmol) was reacted with phosphorus oxychloride (4.0 mL) at 100 °C for 2 h. TLC (PE:EA = 3:1) monitored the formation of a new spot. The reaction was cooled to room temperature, concentrated under vacuum at 45 °C using a diaphragm pump, and quenched with 50 mL of saturated sodium bicarbonate in an ice bath. Extraction was performed with EA (50 mL * 3). The organic phase was washed once with saturated brine (20 mL), dried over anhydrous Na₂SO₄ powder, filtered, and the filtrate was evaporated to dryness to give 600 mg of crude product. Silica gel column chromatography yielded compound 21e (210 mg). LCMS (ESI-MS) m / z: 246.0 [M + H] + .

[0256] Step 3: Preparation of 21g of compound

[0257] Compound 21e (210 mg, 0.81 mmol) was dissolved in 3.0 mL of a container. Compound 21f (160 mg, 0.81 mmol) and DIEA (210 mg, 1.60 mmol) were added. After heating at 90 °C for 2 hours, the mixture was dissolved and diluted with methanol. TLC (PE:EA = 1:1) showed the disappearance of the starting material and the formation of a new spot. LCMS detected the target product. The reaction solution was brought to room temperature and quenched with 20 mL of saturated ammonium chloride solution. After vacuum concentration at 45 °C, the mixture was extracted with EA (20 mL * 3). The organic phase was washed once with 20 mL of saturated brine, dried with anhydrous Na₂SO₄ powder, filtered, and the filtrate was evaporated to dryness to obtain the crude product (600 mg). Silica gel column chromatography was used to purify compound 21 g (190 mg). LCMS (ESI-MS) m / z: 399.2 [M + H] + .

[0258] Step 4: Preparation of compound 21h

[0259] Compound 21 g (190 mg, 0.81 mmol) was dissolved in methanol (2.0 mL), and ammonia water (2.0 mL) was added. The mixture was heated at 80 °C for 4 hours. After the reaction solution was cooled to room temperature, it was concentrated under vacuum at 45 °C using a diaphragm pump to give compound 21 h (200 mg). LCMS (ESI-MS) m / z: 384.2 [M+H] + .

[0260] Step 5: Preparation of compound 21j

[0261] Compound 21h (100 mg, 0.26 mmol) was dissolved in acetonitrile (2.0 mL), and compound 21i (48 mg, 0.26 mmol) and potassium carbonate (110 mg, 0.78 mmol) were added. The mixture was reacted in a microwave oven at 150 °C for 18 h. The reaction solution was directly mixed and purified by silica gel column chromatography to obtain compound 21j (60 mg). LCMS (ESI-MS) m / z: 267.0 [M / 2+H] + .

[0262] Step 6: Preparation of Compound 21

[0263] Compound 21j (60 mg, 0.11 mmol) was dissolved in DCM (1.0 mL), and TEA (0.078 mL, 0.56 mmol) was added. TFAA (47 mg, 0.23 mmol) was added under ice bath conditions. After stirring at room temperature for 2 hours, the target product was detected by LCMS. 50 mL of water was added to the reaction solution, and the mixture was extracted with a 1:9 MeOH:DCM mixture (50 mL x 3). The organic phase was washed once with saturated brine (20 mL), dried over anhydrous Na₂SO₄ powder, filtered, and the filtrate was evaporated to dryness to obtain the crude product. The crude product was dissolved in methanol (2.0 mL) and directly sent to the preparation to obtain compound (4.3 mg). LCMS (ESI-MS) m / z: 257.7 [M / 2+H] + . 1H NMR (400MHz, Methanol-d4) δ8.35(s,1H),7.54(d,J=2.0Hz,1H),7.45(d,J=8.5Hz,1H),7.3 4(dd,J=8.5,1.9Hz,2H),5.10(q,J=6.6Hz,2H),4.79(s,1H),4.55(s,1H),4.08(t,J=8.4Hz ,2H),3.77(dd,J=8.7,5.4Hz,2H),3.72(t,J=6.0Hz,2H),3.49–3.46(m,1H),3.14–3.12(m, 1H), 2.66 (s, 2H), 2.48 (s, 2H), 1.77 (d, J = 11.0Hz, 4H), 1.64 (d, J = 6.8Hz, 3H), 1.29 (s, 1H).

[0264] Example 22: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-3-(oxecyclobutane-3-ylethynyl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (22)

[0265] Step 1: Preparation of compound 22c

[0266] Compound 22a (1.0 g, 3.6 mmol), (1S)-1-(2,4-dichlorophenyl)ethanol-1-ol (1.0 g, 5.3 mmol), and triphenylphosphine (1.9 g, 7.1 mmol) were dissolved in anhydrous THF (15 mL). DIAD (1.4 g, 7.1 mmol) was slowly added under an inert atmosphere in an ice-water bath, and the reaction was maintained at this temperature for 1.5 h. After the reaction was complete, water was added and the mixture was extracted with ethyl acetate (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification by Flash column chromatography yielded compound 22c (690 mg). LCMS (ESI-MS) m / z: 453.20 [M+H] + .

[0267] Step 2: Preparation of compound 22e

[0268] Compound 22c (1.2 g, 2.6 mmol), 2-[(3R)-3-(azacyclobutan-3-yl)hexahydropyridin-1-yl]ethanol-1-ol 22d (0.73 g, 4.0 mmol), and DIEA (0.68 g, 5.3 mmol) were dissolved in anhydrous DMA (5.0 mL) and reacted in an oil bath at 150 °C for 2 h. After the reaction was complete, water was added and the mixture was extracted with ethyl acetate (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash column chromatography to give compound 22e (580 mg). LCMS (ESI-MS) m / z: 601.53 [M+H] + .

[0269] Step 3: Preparation of Compound 22

[0270] Compound 22e (300 mg, 0.50 mmol), bis(triphenylphosphine)palladium chloride (70 mg, 0.10 mmol), cuprous iodide (48 mg, 0.25 mmol), and triethylamine (150 mg, 1.5 mmol) were dissolved in anhydrous THF (5.0 mL). The mixture was purged with nitrogen three times at room temperature. 3-ethyloxetane 22f (57 mg, 0.70 mmol) was added under ice-water bath conditions, and the mixture was reacted in an oil bath at 40 °C for 1 h. After complete reaction, water was added and the mixture was extracted with dichloromethane (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by flash column chromatography, and prepared in liquid chromatography (ammonium bicarbonate) to give compound 22 (78 mg). LCMS (ESI-MS) m / z: 556.49 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.63(t,J=1.2Hz,1H),7.46(d,J=1.3Hz,2H),6.65(s,1H),6. 13(q,J=6.8Hz,1H),4.83(dd,J=8.5,5.5Hz,2H),4.65(dd,J=7.0,5.5Hz,2H),4.37–4. 20(m,2H),4.12(q,J=8.3Hz,2H),3.80(dd,J=8.2,5.9Hz,2H),3.47(q,J=6.0Hz,2H), 2.75(t,J=11.3Hz,2H),2.62–2.51(m,1H),2.35(t,J=6.3Hz,2H),2.01–1.29(m,10H).

[0271] Example 23: 2-((R)-3-(1-(R)-1-(2,4-dichlorophenyl)ethyl)-3-methoxy-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol-1-ol (23)

[0272] Step 1: Preparation of compound 23b

[0273] (R)-6-chloro-1-(1-(2,4-dichlorophenyl)ethyl)-3-iodo-1H-pyrazolo[3,4-b]pyrazine 23a (1.0 g, 2.2 mmol) and (R)-2-(3-(azacyclobutan-3-yl)piperidin-1-yl)ethanol-1-ol (2.0 g, 11 mmol) were dissolved in acetonitrile (30 mL), and cesium carbonate (3.6 g, 11 mmol) was added under ice bath conditions. The mixture was stirred overnight at room temperature. The solution was quenched with water, extracted with ethyl acetate, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to give compound 23b (660 mg). LCMS (ESI-MS) m / z: 601.44 [M+H] +

[0274] Step 2: Preparation of compound 23c

[0275] Compound 23b (0.66 g, 1.1 mmol) was dissolved in DMF (8.0 mL), and imidazole (0.30 g, 4.4 mmol) and tert-butyldimethylchlorosilane (0.33 g, 2.2 mmol) were added in an ice bath. The mixture was stirred overnight at room temperature. The solution was quenched in water, extracted with methyl tert-butyl ether, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to give compound 23c (500 mg). LCMS (ESI-MS) m / z: 715.62 [M+H] +

[0276] Step 3: Preparation of compound 23d

[0277] Compound 23c (0.12 g, 0.17 mmol), sodium tert-butoxide (0.030 g, 0.25 mmol), and methanol (0.07 mL, 1.7 mmol) were dissolved in dioxane (4.0 mL) and purged with nitrogen. 2-Di-tert-butylphosphine-2',4',6'-triisopropyl-3,6-dimethoxy-1,1'-biphenyl (0.010 g, 0.030 mmol) and 2-(di-tert-butylphosphine)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl(2-amino-1,1'-biphenyl-2-yl)palladium (0.020 g, 0.030 mmol) were added at room temperature and purged with nitrogen. The reaction was carried out at 100 °C for 8 hours. The solution was quenched in water, extracted with ethyl acetate, dried over the organic phase, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 23d (66 mg). LCMS (ESI-MS) m / z: 619.71 [M+H] +

[0278] Step 4: Preparation of Compound 23

[0279] Compound 23d (0.070 g, 0.11 mmol) was dissolved in dichloromethane (1.2 mL), and trifluoroacetic acid (0.60 mL) was added under ice bath conditions. The mixture was stirred at room temperature for 16 hours. The reaction was quenched with ice-cold aqueous sodium bicarbonate solution, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by preparative liquid chromatography (ammonium bicarbonate) to give compound 23 (19 mg). LCMS (ESI-MS) m / z: 505.54 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.70(s,1H),7.60(d,J=2.2Hz,1H),7.48(d,J=8.6Hz,1H),7.39(dd,J=8.6,2.2Hz ,1H),6.11(d,J=7.0Hz,1H),4.32(t,J=5.8Hz,1H),4.12(dd,J=13.8,8.6Hz,2H),4.01(s,3H),3.86–3.80( m,2H),3.49(t,J=5.8Hz,2H),2.77–2.71(m,2H),2.60–2.53(m,1H),2.35(t,J=6.4Hz,2H),1.95–1.90(m, 1H), 1.76 (d, J = 7.0Hz, 3H), 1.63 (dt, J = 22.8, 11.4Hz, 4H), 1.42 (d, J = 12.2Hz, 1H), 0.82 (d, J = 11.8Hz, 1H).

[0280] Example 24: 2-[(3R)-3-(1-{1-[(1R)-1-(2,4-dichlorophenyl)ethyl]-3-(1,3-oxazol-5-yl)pyrazolo[4,3-b]pyrazin-6-yl}azacyclobutane-3-yl)hexahydropyridin-1-yl]ethanol-1-ol (24)

[0281] Step 1: Preparation of compound 24c

[0282] 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1,3-oxazole 24b (82 mg, 0.42 mmol), 4-(3-((R)-1-(6-(3-((R)-1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-3-yl)azacyclobutane-1-yl)-3-iodo-1H-pyrazolo[3,4-b]pyrazin-1-yl)ethyl)-2-chlorophenyl)oxazole 24a (200 mg, 0.25 mmol), Pd(dppf)Cl2 (41 mg, 0.056 mmol), and potassium carbonate (77 mg, 0.56 mmol) were dissolved in dioxane (10 mL) and water (0.50 mL). The mixture was stirred at 100 °C for 2 hours under nitrogen protection, and the reaction was monitored by LCMS until complete. The mixture was concentrated and purified by Flash column chromatography to give compound 24c (170 mg). LCMS (ESI-MS) m / z: 656.71 [M+H] +

[0283] Step 2: Preparation of Compound 24

[0284] Compound 24c (170 mg, 0.26 mmol) was dissolved in DCM (2.0 mL), and TFA (6.0 mL, 78 mmol) was added. The mixture was reacted at room temperature for 4 hours, then concentrated. The crude product was purified by preparative HPLC to give compound 24 (65 mg). LCMS (ESI-MS) m / z: 542.52 [M+H] + . 1 HNMR(400MHz,DMSO-d6)δ8.51(s,1H),7.92(s,1H),7.71(s,1H),7.61(d,J=2.1Hz,1H),7.48– 7.38(m,2H),6.27(q,J=6.9Hz,1H),4.31(s,1H),4.17(d,J=9.5Hz,2H),3.88(s,2H),3.46(q,J =6.0Hz,2H),2.74(s,2H),2.54(s,1H),2.34(t,J=6.2Hz,2H),1.91(t,J=11.2Hz,1H),1.84(d ,J=7.1Hz,3H),1.66(dt,J=22.0,12.4Hz,4H),1.41(d,J=12.4Hz,1H),0.83(t,J=11.2Hz,1H).

[0285] Example 25: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-3-(trifluoromethoxy)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azatidine-3-yl)piperidin-1-yl)ethane-1-ol (25)

[0286] Step 1: Preparation of compound 25c

[0287] Compound 25a (4.0 g, 8.8 mmol), compound 25b (2.4 g, 13 mmol), and cesium carbonate (17 g, 53 mmol) were dissolved in acetonitrile and stirred at room temperature for 8 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by Flash column chromatography to give compound 25c (4.7 g). LCMS (ESI-MS) m / z: 601.45 [M+H] + .

[0288] Step 2: Preparation of compound 25d

[0289] Compound 25c (1.0 g, 1.7 mmol) was dissolved in DMF (20 mL), and imidazole (0.57 g, 8.3 mmol) and TBSCI (0.50 g, 3.3 mmol) were added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with methyl tert-butyl ether, washed five times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Compound 25d (1.0 g) was purified by Flash column chromatography. LCMS (ESI-MS) m / z: 715.63 [M+H] + .

[0290] Step 3: Preparation of compound 25e

[0291] Compound 25d (600 mg, 0.84 mmol), potassium hydroxide (120 mg, 2.2 mmol), t-BuBrettPhos Pd G3 (130 mg, 0.15 mmol), and t-BuBrettPhos (97 mg, 0.20 mmol) were dissolved in dioxane (30 mL) and water (6.0 mL). The mixture was stirred at 80 °C for 2 hours under argon atmosphere. After the reaction was complete, the crude product was concentrated under reduced pressure and purified by Flash column chromatography to give compound 25e (450 mg). LCMS (ESI-MS) m / z: 605.69 [M+H] + .

[0292] Step 4: Preparation of 25g of compound

[0293] Compound 25e (450 mg, 0.74 mmol) was dissolved in DMF (22 mL), and 1-(trifluoromethyl)-1,2-benzyl-3(1H)-one 25f (540 mg, 1.7 mmol) and cesium carbonate (970 mg, 3.0 mmol) were added. The mixture was stirred overnight at room temperature, extracted with methyl tert-butyl ether, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by Flash column chromatography to give compound 25 g (100 mg). LCMS (ESI-MS) m / z: 673.75 [M+H] + .

[0294] Step 5: Preparation of Compound 25

[0295] Compound 25 g (150 mg, 0.22 mmol), triethylamine trifluoride (140 mg, 0.89 mmol), was dissolved in tetrahydrofuran (7.5 mL), stirred overnight at room temperature, concentrated under reduced pressure, and the crude product was purified by prep-HPLC and lyophilized to give compound 25 (17 mg). LCMS (ESI-MS) m / z: 559.61 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ7.91(s,1H),7.63–7.59(m,1H),7.41(d,J=1.3Hz,2H),6.20(q,J =7.0Hz,1H),4.29(s,1H),4.18(s,2H),3.88(s,2H),3.46(d,J=5.1Hz,2H),2.72(s,2H), 2.54(d,J=8.1Hz,1H),2.33(t,J=6.3Hz,2H),1.90(t,J=11.0Hz,1H),1.76(d,J=7.0Hz,3 H), 1.63 (td, J = 24.6, 22.7, 13.1Hz, 4H), 1.40 (d, J = 12.7Hz, 1H), 0.81 (d, J = 10.3Hz, 1H).

[0296] Example 26: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-3-((1-(trifluoromethyl)-1H-1,2,4-triazol-3-yl)ethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (26)

[0297] Step 1: Preparation of compound 26b

[0298] 3-Iodo-1H-1,2,4-thiazole 26a (1000 mg, 5.1 mmol), tetrabutylammonium bromide (9.9 mg, 0.031 mmol), purged three times with nitrogen at room temperature, cooled to 0 °C, and sodium hydride (310 mg, 7.7 mmol) was added. The reaction was allowed to proceed for 10 min, then dibromodifluoroane (1600 mg, 7.7 mmol) was added dropwise, and the mixture was slowly heated to room temperature and reacted for 16 h. A saturated aqueous sodium chloride solution was added to the system, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 26b (400 mg). LCMS (ESI-MS) m / z = 324.030 [M+H] + .

[0299] Step 2: Preparation of compound 26d

[0300] Compound 26b (650 mg, 1.3 mmol), compound 26c (350 mg, 1.1 mmol), cuprous iodide (170 mg, 0.54 mmol), triethylamine (0.15 mL, 1.1 mmol), palladium dichloride (150 mg, 0.22 mmol), and tetrahydrofuran (20 mL) were reacted at room temperature with nitrogen purging three times. The reaction was allowed to proceed for 10 h at room temperature. Water was added, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 26d (240 mg). LCMS (ESI-MS) m / z = 696.63 [M+H] + .

[0301] Step 3: Preparation of Compound 26

[0302] Compound 26d (130 mg, 0.19 mmol), cesium fluoride (140 mg, 0.94 mmol), sulfolane (5 mL), and dibenzo-18-crown ether-6 (6.7 mg, 0.019 mmol) were reacted in a microwave oven at 100 °C for 2 h. After cooling to room temperature, a small amount of water was added. Compound 26 (3.0 mg) was prepared by HPLC (acetonitrile / water, ammonium bicarbonate). LCMS (ESI-MS) m / z = 634.66 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ9.46 (s, 1H), 7.97 (s, 1H), 7.62 (d, J = 2.0Hz, 1H), 7.48–7. 39(m,2H),6.35-6.25(m,1H),4.35-4.26(m,1H),4.24-4.09(m,2H),3.95-3.79(m ,2H),3.52-3.40(m,1H),2.80-2.68(m,2H),2.38-2.31(m,2H),2.01-1.87(m,3H) ,1.83(d,J=7.0Hz,3H),1.73–1.56(m,4H),1.45-1.35(m,1H),0.86-0.75(m,1H).

[0303] Example 27: 2-((R)-3-(1-(1-((R)-1-(2,4-dichlorophenyl)ethyl)-3-((1-(difluoromethyl)-1H-1,2,4-triazol-3-yl)ethynyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethanol (27)

[0304] Step 1: Preparation of compound 27b

[0305] 3-Iodo-1H-1,2,4-thiazole 27a (1000 mg, 5.1 mmol), tetrabutylammonium bromide (9.9 mg, 0.031 mmol), purged three times with nitrogen at room temperature, cooled to 0 °C, and sodium hydride (310 mg, 7.7 mmol) was added. The reaction was allowed to proceed for 10 min, then dibromodifluoroane (1600 mg, 7.7 mmol) was added dropwise, and the mixture was slowly heated to room temperature and reacted for 16 h. The system was then extracted with saturated sodium chloride aqueous solution, combined with anhydrous sodium sulfate, dried over filtration, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 27b (400 mg). LCMS (ESI-MS) m / z = 324.030 [M+H] + .

[0306] Step 2: Preparation of compound 27d

[0307] Compound 27b (650 mg, 1.3 mmol), compound 27c (350 mg, 1.1 mmol), cuprous iodide (170 mg, 0.54 mmol), triethylamine (0.15 mL, 1.1 mmol), palladium dichloride (150 mg, 0.22 mmol), and tetrahydrofuran (20 mL) were reacted under nitrogen purging three times at room temperature for 10 h. Water was added to the system, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 27d (240 mg) was purified by silica gel column chromatography. LCMS (ESI-MS) m / z = 696.63 [M+H] + .

[0308] Step 3: Preparation of Compound 27

[0309] Compound 27d (130 mg, 0.19 mmol), cesium fluoride (140 mg, 0.94 mmol), sulfolane (5.0 mL), and dibenzo-18-crown ether-6 (6.7 mg, 0.019 mmol) were reacted in a microwave at 100 °C for 2 h. After cooling to room temperature, a small amount of water was added. Compound 27 (5.0 mg) was prepared directly by HPLC (acetonitrile / water, ammonium bicarbonate). LCMS (ESI-MS) m / z = 616.64 [M+H] +1 H NMR (400MHz, DMSO-d6) δ9.18 (s, 1H), 8.20-7.10 (m, 2H), 7.62 (d, J = 2.1Hz, 1H), 7. 49–7.41(m,2H),6.34-6.25(m,1H),4.35-4.25(m,1H),4.24-4.10(m,2H),3.95-3. 80(s,2H),3.51-3.40(m,2H),2.76-2.66(m,2H),2.36-2.26(m,2H),2.00–1.91(m ,2H),1.90-1.81(m,3H),1.75-1.54(m,4H),1.45-1.36(m,1H),0.86-0.75(m,1H).

[0310] Bioactivity test

[0311] Experiment 1: In vitro activity determination of the small molecule CCR4 inhibitor compound of this invention

[0312] 1. Experimental Objective

[0313] This experiment tested the inhibitory effect of the compound of this invention on the CCR4 receptor using high-throughput real-time fluorescence spectrometry (FLIPR). The inhibitory effect was determined based on the half-maximal inhibitory concentration (IC50). 50 To evaluate the in vitro activity of the compound.

[0314] 2. Experimental Methods

[0315] 2.1 Experimental Materials

[0316] 2.2 Experimental Procedure

[0317] (1) Using an Echo pipetting workstation, serially dilute the test compound 3-fold to 10 fractions in 100% DMSO. Transfer 900 nL of the compound to the target plate. Add 30 μL of assay buffer to the compound plate; (2) Remove the cell culture plate from the incubator, discard the culture medium, and add 20 μL of assay buffer. Dispense 20 μL of 2X Fluo-4 Direct™ rinse-free loading buffer into a 384-well cell culture plate; (3) Transfer 10 μL of the compound from the compound plate to the cell culture plate, bringing the final volume to 50 μL; (4) Start the FLIPRTETRA system; (5) Add 10 μL of 6×EC 80 (6) Transfer the concentration of the agonist reference compound to the cell plate; (7) Read the fluorescence signal; (8) For the antagonist test, calculate the “maximum-minimum” from reading 1 to the maximum allowed value, and calculate the “maximum” from reading 1 to the maximum allowed value of 130; (9) Analyze the data.

[0318] 2.3 Experimental Results

[0319] Table 1 Results of in vitro activity experiments of the compounds of the present invention

[0320] Conclusion: The compounds of this invention have significant inhibitory activity against the CCR4 receptor.

[0321] Experiment 2: In vivo pharmacodynamic experiment of the compound of this invention in mice.

[0322] I. Liver Cancer Model

[0323] 1. Modeling and Drug Administration: Eight-week-old female immunodeficient mice (CB17-SCID), with an average weight of 20g, were purchased. After a 3-day acclimatization period, the experiment began. The mice were divided into three groups: a model group, an FLX475 group, and a compound group (examples), with eight mice in each group. Humanized Hepg2 liver cancer cells were cultured before the experiment, and each mouse was subcutaneously injected with 1*10... 7 Once the tumors reached a certain size, each group was administered the drug via gavage and injection at a dose of 10 mL / kg, once daily; the model group was administered the same volume of solvent via gavage / injection for 28 consecutive days. Specific design details are shown in Table 2.

[0324] Table 2: In vivo efficacy test protocols for compounds

[0325] 2. Experimental drug: Compound 2 from Example 2.

[0326] 3. Drug preparation: Dissolve an appropriate amount of the compound in 30% polyethylene glycol (PEG400) using vortex sonication, add 70% physiological saline, and prepare a clear and transparent solution of 3 mg / mL.

[0327] 4. Detection indicators and methods: The size of the mouse tumor and its weight were observed daily. The tumor size was measured three times a week. At the endpoint, the mouse was dissected and the tumor was removed. The endpoint tumor size was recorded.

[0328] 5. Experimental results and conclusions: After transplantation of humanized Hepg2 tumor cells, the tumor size of mice in the model control group continued to increase with the number of days, while the tumor volume of compound 2 in Example 2 began to decrease significantly after day 14.

[0329] Experiment 3: Effect of the compound of this invention on hERG channel current

[0330] 1. Experimental Objective

[0331] The cardiac safety of the compounds of this invention was evaluated by detecting the effect of the compounds on hERG channel current using manual patch-clamp technique.

[0332] 2. Experimental Methods

[0333] 2.1 Experimental Materials

[0334] Table 3

[0335] 2.2 Experimental Procedure

[0336] 1) HEK-293 cell line stably expressing hERG potassium channels was cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg / mL G418 at 37°C and 5% carbon dioxide.

[0337] 2) Cell passage: Remove the old culture medium and wash once with PBS, then add 1 mL TrypLE TM Incubate with Express solution at 37°C for approximately 0.5 min. Once cells detach from the bottom of the dish, add approximately 5 mL of preheated (37°C) complete culture medium. Gently pipette the cell suspension to separate any aggregated cells. Transfer the cell suspension to sterile centrifuge tubes and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, seed cells into 6 cm cell culture dishes at a density of 2.5 × 10⁶ cells per dish. 5 Cells (final volume: 5 mL).

[0338] 3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0339] 4) Patch-clamp assay: Cells were subjected to TrypLE assay before the experiment. TMExpress separation, 4×10 3 Cells were seeded onto coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, experiments were performed. The voltage stimulation protocol for whole-cell patch-clamp recording of hERG potassium currents was as follows: After whole-cell sealing, the cell membrane voltage was clamped at -80 mV. The clamp voltage was depolarized from -80 mV to -50 mV and maintained for 0.5 s (as a leakage current detection), then stepped to 30 mV and maintained for 2.5 s, and then rapidly restored to -50 mV and maintained for 4 s to elicit the tail current of the hERG channel. Data were collected every 10 s to observe the effect of the drug on the hERG tail current. A 0.5 s -50 mV stimulation was used as the leakage current detection. Experimental data were acquired using an IPA amplifier (Sutter Instrument) and stored in Sutter Patch software.

[0340] Once the hERG current recorded in whole cells stabilized, drug administration began. Each drug concentration was administered for 5 minutes (or until the current stabilized) before moving to the next concentration. One or more concentrations were measured for each test compound. A coverslip containing cells was placed in the recording bath of an inverted microscope. The working solution of the test compound and the external solution without the compound were administered sequentially from low to high concentration through the recording bath using gravity perfusion, while a peristaltic pump was used for fluid replacement during recording. The current detected in the external solution without the compound for each cell served as its control group. Each concentration was measured independently in duplicate using at least two cells. All electrophysiological experiments were performed at room temperature.

[0341] 2.3 Data Analysis

[0342] First, the peak tail current compound and the peak tail current control of the blank solvent treatment group were normalized (Peak tail current compound / Peak tail current control). Then, the inhibition rate (1-(Peak tail current compound / Peak tail current control) corresponding to each drug concentration was calculated. The mean, standard deviation (SD), and standard error (SE) of the inhibition rate at each concentration were calculated. The data are expressed as Mean ± SE and saved in Excel. Y = 1 / (1+10) ^ ((LogIC 50 -X)*HillSlope), use the above equation to calculate the IC for each compound. 50The values ​​were calculated, and a nonlinear fit was performed on the concentration-effect curve, where IC50 was used. 50 This is the half-inhibitory concentration (IC50). 50 The calculations and curve fitting were performed using GraphPad Prism software.

[0343] 2.4 Experimental Results

[0344] Table 4

Claims

1. The compound represented by formula III or its pharmaceutically acceptable salt, in: Indicates a single or double bond; ring A is a 6-membered heteroaromatic ring, where Z 1 For C, Z 3 Z 4 Each can be independently represented as C or N, Z 2 Z 6 Each can be independently represented as N or CH, Z 5 For N or CR z Z 2 ~Z 6 There are 1, 2, or 3 N; R z For H or C, R connected to it 1 and Z 4 Forming ring C; R 3 -H or -CH3; R 6 Or R 7 Each can be independently -H, halogen, -CN, hydroxyl, or -C. 1-3 Alkyl or -C 1-3 Halogenated alkyl groups; X is NR 2 or CR 2a R 2b ; R 2 -H, -C 1-3 Alkyl, or R 2 and the N and R connected to it 1 Z 3 Z 4 Form ring B; R 2a R 2b Each is independently -H, -C 1-3 Alkyl groups or C, R groups connected to them as bonds 1 Z 3 Z 4 Form ring B; R 1 -H, -OC 1-3 Alkyl, -C 1-3 Alkyl, -P(O)(C 1-3 alkyl)2, or R 1 and Z 4 Z 5 Forming rings C and / or R 1 and X, Z 3 Z 4 Form ring B; Rings B and C are each independently 5- or 6-membered rings, and each ring B and C are independently and optionally bounded by one or more -R groups within the range allowed by the valence. 8 replace, R 8 Selected from =O, halogen, -C 1-3 Alkyl, -C 1-3 Halogenated alkyl groups, -OC 1-3 Alkyl, -OC 1-3 Halogenated alkyl groups, -CN, -C 2-4 Alkyne group, 5-8 membered heteroaryl group, or 3-8 membered cycloalkyl group; The 5-8 nucleotide heteroaryl group is optionally surrounded by one or more -C groups within the range allowed by the valence. 1-3 Alkyl substitution; The -C 2-4 The alkynyl group is optionally replaced by a 4-8-membered heterocyclic alkyl group or a 5-8-membered heteroaryl group, wherein the 5-8-membered heteroaryl group is optionally replaced by one or more -C groups within the range permitted by the valence. 1-3 Alkyl or -C 1-3 Halogenated alkyl substitution.

2. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, Z 2 ~Z 6 There is one N and Z 5 N; preferably, Z 1 Z 3 Z 4 For C, Z 2 Z 6 For CH, Z 5 For N; or Z 2 ~Z 6 There are 2 N's; preferably, Z 4 Z 6 For N, or Z 2 Z 5 For N, or Z 3 Z 5 For N, or Z 2 Z 6 For N, or Z 5 Z 6 For N; more preferably, for Z 1 Z 3 For C, Z 5 For CR z Z 2 For CH, Z 4 Z 6 For N; or Z 1 Z 3 Z 4 For C, Z 5 For CR z Z 2 Z 6 For N; or Z 1 Z 3 For C, Z 4 Z 6 For CH, Z 2 Z 5 For N; or Z 1 Z 3 Z 4 For C, Z 6 For CH, Z 2 Z 5 For N; or Z 1 Z 4 For C, Z 2 Z 6 For CH, Z 3 Z 5 For N; or Z 1 Z 3 Z 4 For C, Z 5 For CR z Z 2 Z 6 For N; or Z 1 Z 3 Z 4 For C, Z 2 For CH, Z 5 Z 6 For N; or Z 2 ~Z 6 There are 3 N's; preferably, Z 1 Z 3 For C, Z 5 For CR z Z 2 Z 4 Z 6 Let N be the number of elements in the array.

3. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, X is NR 2 R 2 -H or -CH3 or R 2 and the N and R connected to it 1 Z 3 Z 4 Form ring B; or X is CR 2a R 2b R 2a R 2b The key is C and R connected to it. 1 Z 3 Z 4 Ring B is formed.

4. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, R 1 -H, -OMe, -Me, -Et, -CH2OH or -P(O)Me2; and / or Ring A is selected from the following structures: Preferably, ring A has the following connection method:

5. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, R 1 and X, Z 3 Z 4 Ring B is formed, wherein ring B is a 5-membered heterocycle containing 1 or 2 N atoms; preferably, ring B is a 5-membered heterocycle containing 2 N atoms; more preferably, ring B is selected from the following structures: More preferably, ring A and ring B are selected from the following structures: or R 1 and Z 4 Z 5 A ring C is formed, wherein ring C is a 5- or 6-membered ring containing 0, 1, or 2 atoms optionally selected from N, O, or S atoms; preferably, ring C is selected from the following structures: More preferably, the ring A and C are selected from the following structures: or R 1 and Z 4 Z 5 Forming a ring C and connecting with X and Z 3 Z 4 Ring B is formed, wherein ring B is a 5-membered heterocyclic alkane containing one nitrogen atom, and ring C is a 6-membered cyclic alkane; preferably, ring B and ring C are selected from the following structures: More preferably, the fused ring formed by rings A, B, and C has the following structure:

6. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, R 6 Or R 7 Each is independently -Cl or -F; preferably R 6 and R 7 -Cl; and / or R 8 Selected from =O, -F, -Cl, -Me, -Et, -halomethyl, -OMe, -OEt, -O-halomethyl, -CN, -C≡CH, 5-membered heteroaryl, or 3-4-membered cycloalkyl; wherein the 5-membered heteroaryl is optionally substituted with one or more methyl groups within the range allowed by the valence; wherein the -C≡CH is optionally substituted with one or more 4-5-membered heterocycloalkyl or 5-membered heteroaryl, wherein the 5-membered heteroaryl is optionally substituted with one or more -C groups within the range allowed by the valence. 1-3 Halogenated alkyl substitution; preferably, R 8 Selected from =O, -F, -Cl, -CF3, -OMe, -Me, cyclopropyl, -CN, -OCF3, 7. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, Having structures as shown in equations Ⅲ-a, Ⅲ-b, Ⅲ-c, Ⅲ-d, Ⅲ-e, Ⅲ-f, Ⅲ-g, Ⅲ-h, Ⅲ-i, Ⅲ-j, Ⅲ-k, and Ⅲ-l: or It has the structure shown in formulas IV-a, IV-b, IV-c, IV-d, IV-e, IV-f, IV-g, IV-h, and IV-i: Among them, R 1 R 2 R 6 R 7 R 8 Each is independent of the claims 1.

8. The compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The compound is selected from the following compounds or their pharmaceutically acceptable salts:

9. A pharmaceutical composition, characterized in that, It includes a therapeutically effective amount of the compound of any one of claims 1-8 or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers and / or excipients.

10. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1-9, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for the prevention and / or treatment of CCR4-mediated diseases; Preferably, the CCR4-mediated diseases include inflammatory diseases, autoimmune diseases, or cancer; More preferably, 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 stomach cancer.

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