Aryl pyrimidine-based HDAC6 selective inhibitor, its preparation method and uses

Abstract

The present invention discloses arylpyrimidine compounds which are selective inhibitors of histone deacetylase 6 (HDAC6), and their use in the preparation of drugs for treating HDAC6-related diseases; specifically, compounds of formula (I) and their pharmaceutically acceptable salts are disclosed. The compounds of the present invention have high inhibitory activity against HDAC6 and high selectivity for HDAC enzyme subtypes, and at the same time have ideal pharmacokinetic characteristics, high blood-brain barrier permeability and good safety.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a class of arylpyrimidine compounds as selective inhibitors of histone deacetylase 6 (HDAC6), and their use in the preparation of drugs for treating HDAC6-related diseases. Background Art

[0002] Histone deacetylases (HDACs) can catalyze the deacetylation process of histones and non-histones, and jointly regulate the intracellular acetylation level with histone acetyltransferases (HATs), thereby regulating gene expression. Currently, 18 subtypes of mammalian HDACs are known, which are divided into four categories: Class I (HDAC1, HDAC2, HDAC3, HDAC8); Class II is further divided into two subfamilies, IIa (HDAC4, HDAC5, HDAC7, HDAC9) and IIb (HDAC6, HDAC10); Class III (Sirt1-Sirt7); Class IV (HDAC11).

[0003] Currently, there are 5 histone deacetylase inhibitors (HDACi) on the market, namely vorinostat, belinostat, panobinostat, romidepsin and chidamide. The first three are broad-spectrum inhibitors, and the latter two selectively act on Class I subtypes. Vorinostat and romidepsin are used to treat cutaneous T-cell lymphoma (CTCL), belinostat and chidamide are used to treat relapsed and refractory peripheral T-cell lymphoma (PTCL), and panobinostat is used in combination with bortezomib and dexamethasone to treat multiple myeloma (MM).

[0004] Although the above HDAC inhibitors have achieved good clinical efficacy, broad-spectrum HDAC inhibitors generally have the following disadvantages: (1) strong toxic and side effects, such as nausea, vomiting, bone marrow suppression, etc.; (2) genotoxicity; (3) poor pharmacokinetic properties, low bioavailability, short half-life, etc. These disadvantages not only cause inconvenience to cancer patients, but also hinder the application of broad-spectrum HDAC inhibitors in fields other than cancer treatment.

[0005] Currently, subtype-selective inhibitors of HDACs have become a research hotspot in this field. Among them, HDAC6 subtype inhibitors have attracted much attention. The disease fields involved in HDAC6 are very extensive, including tumors, neurodegenerative diseases, neuropsychiatric diseases, inflammation, autoimmune responses, tumors and bacterial infections, etc.

[0006] Pharmacological studies have shown that HDAC6 selective inhibitors can effectively inhibit the proliferation of glioblastoma cells and induce their apoptosis, and inhibiting HDAC6 can enhance the sensitivity of glioblastoma cells to temozolomide, suggesting a synergistic effect between the two. Specifically, HDAC6 can regulate the activity of its acetylated substrates such as heat shock proteins, which, as molecular chaperones, can participate in the repair of misfolded and unfolded proteins; at the same time, the ZnF-UBP domain of the HDAC6 protein can bind to ubiquitinated misfolded proteins with high affinity and promote their degradation. Therefore, the combination therapy of the HDAC6 inhibitor Tubastatin A and temozolomide (TMZ) reversed the ratio between HDAC6 and p97 / VCP, weakened the activation of heat shock proteins, triggered the apoptotic signals of the unfolded protein response and endoplasmic reticulum stress, and ultimately overcome endoplasmic reticulum stress tolerance, reduced the viability of TMZ-resistant glioma cells and induced apoptosis.

[0007] Similar to glioblastoma, there is also an urgent clinical need for effective therapeutic drugs for Alzheimer's disease (AD). Studies have found that the expression level of HDAC6 is significantly increased in the hippocampus (91% increase) and cortex (52% increase) of the AD brain, and tubulin acetylation is reduced in neurons containing NFTs. HDAC6 is involved in the process of tau hyperphosphorylation. HDAC6 interacts with tau in human brain tissue, and this interaction is mediated by the microtubule-binding domain on tau and the SE14 domain on HDAC6. Inhibition of HDAC6 does not affect its interaction with tau, but weakens the phosphorylation of T231 on tau, which is a key regulatory site for tau function. Inhibition of HDAC6 improves the cognition of AD mice. The above studies suggest that centrally targeted HDAC6 inhibitors are promising to be developed into new, highly effective and low-toxic AD therapeutic drugs.

[0008] Existing antipsychotic, antidepressant, and bipolar disorder drugs have drawbacks such as severe side effects (e.g., extrapyramidal side effects, weight gain), slow onset, and poor patient compliance, which are mainly related to the drugs' action on dopamine and serotonin receptors. Currently, there is an urgent need for innovative drugs for schizophrenia, depression, and bipolar disorder that act on new targets and mechanisms to avoid the drawbacks of existing drugs. Studies have found that histone modification (acetylation) in the promoter regions of a large number of schizophrenia susceptibility genes is significantly abnormal. The latest positron emission tomography (PET) results show that the expression of histone deacetylase is abnormal in multiple brain regions of schizophrenia patients, suggesting that abnormal histone modification in the brain is closely related to the occurrence of schizophrenia. Mice with HDAC6 gene knockout are more active in the open field test, less anxious in the elevated plus maze test, and show stronger antidepressant effects in the tail suspension test and forced swimming test. Therefore, highly selective HDAC6 inhibitors can be used to treat schizophrenia, depression, bipolar disorder, etc., and can avoid side effects such as extrapyramidal system and weight gain of existing drugs.

[0009] Therefore, it is necessary to further develop HDAC6 inhibitors with novel structures, high activity, and high selectivity to overcome the drawbacks of existing or traditional HDAC inhibitors such as nausea, vomiting, bone marrow suppression, mutagenicity, and QT interval prolongation. Summary of the Invention

[0010] On the one hand, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0011]

[0012] Wherein,

[0013] X is selected from CH or N;

[0014] Y is selected from CH or N;

[0015] R is selected from halogen, cyano, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, C 3-8 cycloalkyl, 3-8 membered heterocycloalkyl, phenyl, 5-12 membered heteroaryl;

[0016] The 3-8 membered heterocycloalkyl and 5-12 membered heteroaryl each contain 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from NH, O, S, and N;

[0017] The C 3-8 cycloalkyl, 3-8 membered heterocycloalkyl, phenyl, and 5-12 membered heteroaryl are optionally substituted by 1, 2, or 3 R1;

[0018] Each of said R1s is independently selected from hydrogen, halogen, hydroxy, cyano, C 1-6 alkyl or C 1-6 alkoxy;

[0019] n is selected from 1 or 2.

[0020] In some embodiments of the present invention, when R is halogen, said halogen is selected from fluorine, chlorine or bromine, preferably fluorine or chlorine.

[0021] In some embodiments of the present invention, when R is C 1-6 alkyl, said C 1-6 alkyl is selected from methyl, ethyl, n-propyl or isopropyl, preferably methyl.

[0022] In some embodiments of the present invention, when R is C 1-6 alkoxy, said C 1-6 alkoxy is selected from methoxy, ethoxy, n-propoxy or isopropoxy, preferably methoxy.

[0023] In some embodiments of the present invention, when R is halogen-substituted C 1-6 alkyl, said halogen is selected from fluorine, chlorine or bromine, preferably fluorine.

[0024] In some embodiments of the present invention, when R is halogen-substituted C 1-6 alkyl, said C 1-6 alkyl is selected from methyl, ethyl, n-propyl or isopropyl, preferably methyl.

[0025] In some embodiments of the present invention, when R is halogen-substituted C 1-6 alkyl, said halogen-substituted C 1-6 alkyl is selected from -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2 or -CF2CF3, preferably -CF3.

[0026] In some embodiments of the present invention, when R is C 1-6 alkoxy, said C 1-6 alkoxy is selected from methoxy, ethoxy, n-propoxy or isopropoxy, preferably methoxy.

[0027] In some embodiments of the present invention, when R is C 3-8 cycloalkyl optionally substituted by 1, 2 or 3 R1s, said C 3-8 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclopropyl or cyclohexyl.

[0028] In some embodiments of the present invention, when R is a 3-8 membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3-8 membered heterocycloalkyl contains 1 or 2 heteroatoms or heteroatom groups selected from NH, O and N.

[0029] In some embodiments of the present invention, when R is a 3-8 membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3-8 membered heterocycloalkyl is a 5-6 membered heterocycloalkyl.

[0030] In some embodiments of the present invention, when R is a 3-8 membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3-8 membered heterocycloalkyl is selected from tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, piperazinyl or morpholinyl, preferably piperazinyl.

[0031] In some embodiments of the present invention, when R is a 5-12 membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5-12 membered heteroaryl contains 1 or 2 heteroatoms or heteroatom groups selected from NH, O, S and N.

[0032] In some embodiments of the present invention, when R is a 5-12 membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5-12 membered heteroaryl is a 5-6 membered heteroaryl.

[0033] In some embodiments of the present invention, when R is a 5-12 membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5-12 membered heteroaryl is selected from pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl or pyrazinyl, preferably pyridyl.

[0034] In some embodiments of the present invention, R is selected from fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, phenyl, pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl or pyrazinyl, preferably fluorine, chlorine, methyl, methoxy, -CF3, piperazinyl, phenyl, furyl, thienyl or pyridyl.

[0035] In some embodiments of the present invention, each R1 is independently selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy or isopropoxy, preferably hydrogen, fluorine, chlorine, methyl or methoxy.

[0036] In some embodiments of the present invention, the above structural unit is selected from:

[0037]

[0038] In some embodiments of the present invention, the compounds of formula (I) are selected from:

[0039]

[0040] wherein R is as defined in any of the previous embodiments.

[0041] On the other hand, the present invention provides the following compounds and their pharmaceutically acceptable salts, selected from:

[0042]

[0043]

[0044] On the other hand, the present invention provides the compounds of formula (II),

[0045]

[0046] wherein,

[0047] X is selected from CH or N;

[0048] Y is selected from CH or N;

[0049] R is selected from halogen, cyano, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, C 3-8 cycloalkyl, 3-8 membered heterocycloalkyl, phenyl, 5-12 membered heteroaryl;

[0050] The 3-8 membered heterocycloalkyl and 5-12 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms or heteroatom groups independently selected from NH, O, S and N;

[0051] The C 3-8 cycloalkyl, 3-8 membered heterocycloalkyl, phenyl and 5-12 membered heteroaryl are optionally substituted by 1, 2 or 3 R1;

[0052] Each R1 is independently selected from hydrogen, halogen, hydroxyl, cyano, C 1-6 alkyl or C 1-6 alkoxy;

[0053] R2 is C 1-6 alkyl;

[0054] n is selected from 1 or 2.

[0055] In some embodiments of the present invention, the compounds of formula (II) are selected from:

[0056]

[0057] On the other hand, the present invention provides a method for preparing a compound of formula (I), comprising: preparing compound (II) by a substitution reaction of compound (III) and compound (IV), and preparing compound (I) by an oximation reaction of compound (II).

[0058]

[0059] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

[0060] The pharmaceutically acceptable carrier (pharmaceutical excipient) may be those excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method for the active ingredient to dissolve at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the administration of the composition. The pharmaceutical excipient may be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient may include one or more of the following excipients: binder, suspending agent, emulsifier, diluent, filler, granulating agent, adhesive, disintegrant, lubricant, anti-adhesive agent, glidant, wetting agent, gelling agent, absorption retardant, dissolution inhibitor, enhancer, adsorbent, buffer, chelating agent, preservative, coloring agent, flavoring agent and sweetening agent.

[0061] The pharmaceutical composition of the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilization processes.

[0062] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra - arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled - release or delayed - release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft gelatin capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, injectable solutions, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injectable suspensions and injectable emulsions. Examples of other suitable preparations of the said pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols: such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0063] On the other hand, the present invention provides the use of the above - mentioned compound or its pharmaceutically acceptable salt in the preparation of a medicament for treating HDAC6 - related diseases.

[0064] On the other hand, the present invention provides the use of the above - mentioned pharmaceutical composition in the preparation of a medicament for treating HDAC6 - related diseases.

[0065] In some embodiments of the present invention, the HDAC6 - related diseases are cancers, neurological diseases or autoimmune diseases.

[0066] In some embodiments of the present invention, the cancers include ovarian cancer, colon cancer, breast cancer, liver cancer, pancreatic cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, cervical cancer, prostate cancer, lung cancer, melanoma, germ cell tumor, gestational trophoblastic tumor, glioblastoma, myeloma, neuroblastoma - derived CNS tumor, monocytic leukemia, B - cell - derived leukemia, T - cell - derived leukemia, B - cell - derived lymphoma, T - cell - derived lymphoma and mast cell - derived tumors, and combinations thereof.

[0067] In some embodiments of the present invention, the neurological diseases include neurodegenerative diseases, neuropsychiatric diseases, peripheral neuropathies, and combinations thereof.

[0068] In some embodiments of the present invention, the autoimmune diseases include psoriasis, inflammatory diseases, conditions treated by immunomodulation.

[0069] In some embodiments of the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Rett syndrome, pain.

[0070] In some embodiments of the present invention, the neuropsychiatric diseases are schizophrenia, depression, bipolar disorder, anxiety disorder.

[0071] In some embodiments of the present invention, the peripheral neuropathies include Charcot-Marie-Tooth disease, giant axonal neuropathy.

[0072] In some embodiments of the present invention, the conditions treated by immunomodulation include multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergy, asthma, allergic rhinitis, inflammatory bowel disease.

[0073] The beneficial effects of the present invention are as follows: As a novel selective inhibitor of histone deacetylase 6 (HDAC6), the compounds of the present invention have significant in vitro activity (high inhibitory activity against HDAC6 enzyme) and high enzyme subtype selectivity (weak inhibition against HDAC1); in addition, the compounds of the present invention show ideal pharmacokinetic characteristics, high blood-brain barrier permeability and good safety.

[0074] Definitions and Explanations

[0075] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0076] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0077] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared from the compounds with specific substituents found in the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, the base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either base or acid addition salts.

[0078] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of appropriate bases or acids in water or an organic solvent or a mixture of both.

[0079] In addition to the salt form, the compounds provided by the present invention also exist in prodrug forms. The prodrugs of the compounds described herein are readily chemically changed under physiological conditions to convert into the compounds of the present invention. In addition, the prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in the in vivo environment.

[0080] Certain compounds of the present invention can exist in non-solvated form or solvated form, including hydrate form. Generally, the solvated form and the non-solvated form are equivalent and both are included within the scope of the present invention.

[0081] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereoisomer-enriched mixtures, all of which mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of the present invention.

[0082] "Optional" or "optionally" means that the subsequent described event or circumstance may but does not necessarily occur, and this description includes both the case where the described event or circumstance occurs and the case where the described event or circumstance does not occur.

[0083] The term "substituted" or "substitution" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the resulting compound is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are replaced. Keto substitution does not occur on an aromatic group. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of being chemically achievable.

[0084] When any variable (e.g., R1) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 - 2 R1s, the group may optionally be substituted with up to two R1s, and R1 in each case has independent options. In addition, combinations of substituents and / or their variants are only permitted if such combinations result in a stable compound.

[0085] When a substituent is absent, it means that the substituent is not present. For example, when Z is absent in A-Z, it means that the structure is actually A. When it is not specified which atom of a listed substituent is bonded to the group being substituted, such a substituent can be bonded through any of its atoms. For example, a pyridyl group as a substituent can be bonded to the group being substituted through any carbon atom on the pyridine ring.

[0086] The term "C 1-6 alkyl" is used to denote a straight-chain or branched-chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C 1-6 alkyl includes C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4, C6 and C5 alkyl groups, etc. C 1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.

[0087] The term "C 1-6 alkoxy" refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through an oxygen atom. The C 1-6 alkoxy includes C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C6, C5, C4 and C3 alkoxy groups, etc. C 1-6 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentoxy (including n-pentoxy, isopentoxy and neopentoxy), hexyloxy, etc.

[0088] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0089] The term "halogen-substituted C 1-6 alkyl" means that the above-mentioned "C 1-6 alkyl" is substituted by one or more halogen groups. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethylethyl, etc.

[0090] The term "C 3-8 cycloalkyl" refers to a saturated cyclic hydrocarbon group composed of 3 to 8 carbon atoms, which is a monocyclic and bicyclic system, where the bicyclic system includes spiro, fused and bridged rings. The C 3-8 cycloalkyl includes C 4-5 and C 5-6 cycloalkyl groups, etc. C 3-8 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0091] The term "3-8 membered heterocycloalkyl", either alone or in combination with other terms, respectively represents a saturated cyclic group composed of 3 to 8 ring atoms, where 1, 2, 3 or 4 of the ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, where the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O)p , where p is 1 or 2). It includes monocyclic, bicyclic and tricyclic systems, and the bicyclic and tricyclic systems include spirocycles, fused rings and bridged rings. In addition, for the "3- to 8-membered heterocycloalkyl", the heteroatom can occupy the connection position between the heterocycloalkyl and the rest of the molecule. The 3- to 8-membered heterocycloalkyl includes 5- to 8-membered, 5- to 6-membered, 5- to 7-membered, 3- to 6-membered, 3-membered, 4-membered, 5-membered and 6-membered heterocycloalkyls, etc. Examples of the 3- to 8-membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuryl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxolanyl, dithiolanyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxepanyl, etc.

[0092] The term "5- to 12-membered heteroaryl" refers to a cyclic group composed of 5 to 12 ring atoms with a conjugated π-electron system, in which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. It can be a monocyclic, fused bicyclic or fused tricyclic system, and each ring is aromatic. Among them, the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O) p, where p is 1 or 2). The 5- to 12-membered heteroaryl can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5- to 12-membered heteroaryl includes 5- to 8-membered, 5- to 7-membered, 5- to 6-membered, 5-membered, and 6-membered heteroaryls, etc. Examples of the 5- to 12-membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, etc.), furyl (including 2-furyl, 3-furyl, etc.), thienyl (including 2-thienyl, 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, etc.), benzothiazolyl (including 5-benzothiazolyl, etc.), purinyl, benzimidazolyl (including 2-benzimidazolyl, etc.), benzoxazolyl, indolyl (including 5-indolyl, etc.), isoquinolinyl (including 1-isoquinolinyl, 5-isoquinolinyl, etc.), quinoxalinyl (including 2-quinoxalinyl, 5-quinoxalinyl, etc.), or quinolinyl (including 3-quinolinyl, 6-quinolinyl, etc.). Detailed Description of the Invention

[0093] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples described herein. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0094] The starting materials in the examples of the present invention are known and can be purchased on the market, or can be synthesized by methods known in the art or according to such methods.

[0095] The abbreviations used in this application are specifically represented as follows:

[0096] CDCl3 represents deuterated chloroform; DMSO represents dimethyl sulfoxide; DIEA represents N,N-diisopropylethylamine; DMSO-d6 represents dimethyl sulfoxide-d6; TLC represents preparative silica gel thin layer chromatography; eq represents equivalent; PE represents petroleum ether; EA represents ethyl acetate; DCM represents dichloromethane; MeOH represents methanol; THF represents tetrahydrofuran.

[0097] Example 1: Synthesis of Compound AND-1

[0098]

[0099] Step 1: Compound 1-2 (369 mg, 1.61 mmol, 1.5 eq) and DIEA (418 mg, 3.24 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of Compound 1-1 (200 mg, 1.08 mmol, 1.0 eq). The mixture was stirred and heated to 60 °C for reaction for 6 hours. After cooling to room temperature, the reaction mixture was poured into water (20 mL), and extracted with ethyl acetate (20 mL × 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain Intermediate 1-3 (180 mg, white solid, yield 49.7%).

[0100] 1 H NMR (400 MHz, DMSO-d6) 2.43 (s, 3H), 3.85 (s, 3H), 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.59 - 7.69 (m, 1H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.04 (brs, 1H), 9.21 (s, 1H), 11.18 (s, 1H).

[0101] MS ESI calculated value for C19H18N4O2 [M + H] + 335, found 335.

[0102] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of Intermediate 1-3 (180 mg, 0.54 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain Compound AND-1 (100.4 mg, white solid, yield 55%).

[0103] 1 H NMR (400 MHz, CDCl3) 2.43 (s, 3H), 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.59 - 7.69 (m, 1H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.21 (s, 1H).

[0104] MS ESI calculated value: C18H17N5O2 [M+H] + 336, measured value 336.

[0105] Example 2: Synthesis of Compound AND-2

[0106]

[0107] Step 1: Compound 2-2 (0.30 g, 1.26 mmol, 1.5 eq) and DIEA (0.33 g, 2.52 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of Compound 2-1 (0.20 g, 0.84 mmol, 1.0 eq). The mixture was stirred and heated to 120 °C for reaction for 12 hours. After cooling to room temperature, the reaction solution was poured into water (20 mL), and extracted with ethyl acetate (20 mL × 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain intermediate 2-3 (100 mg, white solid, yield 29.7%).

[0108] 1 1H NMR (400 MHz, CDCl3) δ 3.85 (s, 3H), 4.46 (s, 2H), 6.90 - 6.95 (m, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.57 - 7.65 (m, 2H), 7.98 (d, J = 8.0 Hz, 2H), 8.02 - 8.12 (m, 1H), 8.49 - 8.64 (m, 2H), 9.21 (s, 1H).

[0109] MS ESI calculated value: C23H20N4O3 [M+H] + 401, measured value 401.

[0110] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 2-3 (100 mg, 0.25 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain Compound AND-2 (65 mg, white solid, yield 65%).

[0111] 11H NMR (400 MHz, DMSO-d6) δ 4.46 (s, 2H), 6.90 - 6.95 (m, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.57 - 7.65 (m, 2H), 7.98 (d, J = 8.0 Hz, 2H), 8.02 - 8.12 (m, 1H), 8.49 - 8.64 (m, 2H), 9.04 (brs, 1H), 9.21 (s, 1H), 11.18 (s, 1H).

[0112] MS ESI calcd for C22H19N5O3 [M+H] + + 402, found 402.

[0113] Example 3: Synthesis of Compound AND-3

[0114]

[0115] Step 1: Compound 1-2 (0.28 g, 1.21 mmol, 1.5 eq) and DIEA (0.31 g, 2.43 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of Compound 3-1 (0.20 g, 0.81 mmol, 1.0 eq, prepared according to the method of Eur J Med Chem, 2021, 209:112871). The mixture was stirred and heated to 100 °C for 17 h. The reaction mixture was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to give Intermediate 3-3 (100 mg, off-white solid, yield 30.8%).

[0116] 1 1H NMR (400 MHz, CDCl3) δ 3.85 (s, 3H), 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.49 - 7.54 (m, 3H), 7.55 (s, 1H), 7.57 - 7.94 (m, 3H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.21 (s, 1H).

[0117] MS ESI calcd for C24H20N4O2 [M+H] + + 397, found 397.

[0118] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 3 - 3 (100 mg, 0.25 mmol, 1.0 eq), and stirred at room temperature for 19 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND - 3 (55 mg, off - white solid, yield 55.4%).

[0119] 1 H NMR (400 MHz, DMSO - d6) 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.49 - 7.54 (m, 3H), 7.55 (s, 1H), 7.57 - 7.94 (m, 3H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.04 (brs, 1H), 9.21 (s, 1H), 11.18 (s, 1H).

[0120] MS ESI calculated value for C23H19N5O3 [M + H] + 398, found 398.

[0121] Example 4: Synthesis of compound AND - 4

[0122]

[0123] Step 1: Compound 1 - 2 (0.26 g, 1.13 mmol, 1.5 eq) and DIEA (0.29 g, 2.25 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 4 - 1 (0.20 g, 0.75 mmol, 1.0 eq, prepared according to the method of Lett Org Chem, 2021, 18(8): 617 - 624), stirred, and heated to 100 °C for reaction for 14 hours. After cooling to room temperature, the reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain intermediate 4 - 3 (110 mg, white solid, yield 35.4%).

[0124] MS ESI calculated value for C24H19FN4O2 [M + H] + 415, found 415.

[0125] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 4-3 (110 mg, 0.27 mmol, 1.0 eq), and the mixture was stirred at room temperature for 13 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-4 (65 mg, white solid, yield 58.0%).

[0126] 1 H NMR (400 MHz, DMSO-d6) 4.45 (s, 2H), 7.28 - 7.35 (m, 4H), 7.55 (s, 1H), 7.57 - 7.86 (m, 3H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.03 (brs, 1H), 9.20 (s, 1H), 11.16 (s, 1H).

[0127] MS ESI calculated value C23H18FN5O2 [M + H] + 416, found 416.

[0128] Example 5: Synthesis of compound AND-5

[0129]

[0130] Step 1: Compound 1-2 (0.13 g, 0.56 mmol, 1.5 eq) and DIEA (0.14 g, 1.11 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 5-1 (0.10 g, 0.37 mmol, 1.0 eq), and the mixture was stirred and heated to 100 °C for reaction for 15 hours. After cooling to room temperature, the reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 1:1) to obtain intermediate 5-3 (45 mg, white solid, yield 29.2%).

[0131] 11H NMR (400 MHz, CDCl3): δ 2.25 (s, 3H), 2.40 (t, J =?, 4H), 3.65 (t, J =?, 4H), 3.85 (s, 3H), 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.57 (m, 1H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 (m, 1H), 8.64 (m, 1H), 9.21 (s, 1H).

[0132] MS ESI calculated for C24H27N5O2 [M+H]+ + 418, found 418.

[0133] Step 2: At 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a solution of intermediate 5-3 (45 mg, 0.11 mmol, 1.0 eq) in THF (2 ml). The mixture was stirred at room temperature for 17 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-5 (27 mg, white solid, yield 58.7%).

[0134] 1 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3H), 2.40 (t, J =?, 4H), 3.65 (t, J =?, 4H), 4.45 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.55 (s, 1H), 7.57 - 7.67 (m, 1H), 7.98 (d, J = 8.0 Hz, 2H), 8.49 - 8.64 (m, 2H), 9.04 (brs, 1H), 9.22 (s, 1H), 11.17 (s, 1H).

[0135] MS ESI calculated for C23H26N6O2 [M+H]+ + 419, found 419.

[0136] Example 6: Synthesis of compound AND-6

[0137]

[0138] Step 1: Compound 6-2 (0.17 g, 0.73 mmol, 1.5 eq) and DIEA (0.19 g, 1.47 mmol, 3.0 eq) were successively added to a solution of compound 6-1 (0.10 g, 0.49 mmol, 1.0 eq) in DMSO (2 mL). The mixture was stirred and heated to 80 °C for 13 h. After cooling to room temperature, the reaction mixture was poured into water (20 mL), and extracted with ethyl acetate (20 mL×3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to give intermediate 6-3 (42 mg, white solid, yield 24.1%).

[0139] MS ESI calculated value for C17H14ClN5O2 [M+H] + 356, found 356.

[0140] Step 2: Under the condition of 0-5 °C, MeOH (2 mL), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a solution of intermediate 6-3 (42 mg, 0.12 mmol, 1.0 eq) in THF (2 mL). The mixture was stirred at room temperature for 16 h. The reaction mixture was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to give compound AND-6 (25 mg, off-white solid, yield 58.4%).

[0141] 1 1H NMR (400 MHz, DMSO-d6) δ 4.74 (s, 2H), 6.87 (s, 1H), 7.55 - 7.69 (m, 2H), 8.31 - 8.45 (m, 2H), 8.62 - 8.70 (m, 1H), 8.87 (s, 1H), 9.04 (brs, 1H), 9.23 (s, 1H), 11.15 (s, 1H).

[0142] MS ESI calculated value for C16H13ClN6O2 [M+H] + 357, found 357.

[0143] Example 7: Synthesis of Compound AND-7

[0144]

[0145] Step 1: Compound 6-2 (0.37 g, 1.61 mmol, 1.5 eq) and DIEA (0.42 g, 3.24 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 7-1 (0.20 g, 1.08 mmol, 1.0 eq). The mixture was stirred and heated to 80 °C for reaction for 19 h. After cooling to room temperature, the reaction solution was poured into water (20 mL), and extracted with ethyl acetate (20 mL × 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain intermediate 7-3 (0.22 g, pale yellow oil, yield 60.8%).

[0146] MS ESI calculated value for C18H17N5O2 [M+H] + 336, found 336.

[0147] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 7-3 (0.22 g, 0.65 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 h. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-7 (160 mg, off-white solid, yield 73.2%).

[0148] 1 H NMR (400 MHz, DMSO-d6) 2.46 (s, 3H), 4.74 (s, 2H), 6.87 (s, 1H), 7.45 - 7.69 (m, 2H), 8.18 - 8.25 (m, 1H), 8.45 - 8.70 (m, 2H), 8.87 (s, 1H), 9.01 (brs, 1H), 9.22 (s, 1H), 11.15 (s, 1H).

[0149] MS ESI calculated value for C17H16N6O2 [M+H] + 337, found 337.

[0150] Example 8: Synthesis of Compound AND-8

[0151]

[0152] Step 1: Compound 6-2 (0.28 g, 1.22 mmol, 1.5 eq) and DIEA (0.31 g, 2.43 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 8-1 (0.20 g, 0.81 mmol, 1.0 eq). The mixture was stirred and heated to 90 °C for 14 h. After cooling to room temperature, the reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain intermediate 8-3 (0.25 g, white solid, yield 77.9%).

[0153] MS ESI calculated value for C24H20N4O2 [M+H] + 397, found 397.

[0154] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 8-3 (0.25 g, 0.63 mmol, 1.0 eq). The mixture was stirred at room temperature for 18 h. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-8 (150 mg, white solid, yield 60%).

[0155] 1 H NMR (400 MHz, DMSO-d6) 4.73 (s, 2H), 7.48 - 7.54 (m, 6H), 7.65 - 7.71 (m, 1H), 7.85 (s, 1H), 7.91 - 7.96 (m, 4H), 8.16 (m, 1H), 8.85 (s, 1H), 9.02 (brs, 1H), 11.17 (s, 1H).

[0156] MS ESI calculated value for C23H19N5O2 [M+H] + 398, found 398.

[0157] Example 9: Synthesis of Compound AND-9

[0158]

[0159] Step 1: Compound 6-2 (0.25 g, 1.08 mmol, 1.5 eq), DIEA (0.28 g, 2.16 mmol, 3.0 eq) were successively added to DMSO (2 mL) of compound 9-1 (0.20 g, 0.72 mmol, 1.0 eq, prepared according to the method of Eur J Med Chem, 2021, 209:112871). Stir the mixture and heat it to 70 °C for reaction for 17 hours. Cool to room temperature, pour the reaction solution into water (20 mL), extract with ethyl acetate (20 mL × 3), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate. The residue was purified by column chromatography (PE / EA = 1:1) to obtain intermediate 9-3 (0.23 g, off-white solid, yield 75%).

[0160] MS ESI calculated value for C24H21N5O3 [M+H] + 428, found 428.

[0161] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were successively added to the THF (2 ml) solution of intermediate 9-3 (0.23 g, 0.54 mmol, 1.0 eq). Stir at room temperature for 16 hours. Acidify the reaction solution with acetic acid (10% aqueous solution) to pH = 4, and concentrate under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 20:1) to obtain compound AND-9 (175 mg, white solid, yield 75.7%).

[0162] 1 1H NMR (400 MHz, DMSO-d6) δ 3.80 (s, 3H), 4.74 (s, 2H), 6.87 (s, 1H), 7.04 (m, 1H), 7.25 (s, 1H), 7.46 - 7.55 (m, 3H), 7.62 - 7.69 (m, 1H), 8.16 - 8.18 (m, 1H), 8.42 - 8.45 (m, 1H), 8.70 - 8.75 (m, 1H), 8.87 (s, 1H), 9.03 (brs, 1H), 9.25 (s, 1H), 11.18 (s, 1H).

[0163] MS ESI calculated value for C23H20N6O3 [M+H] + 429, found 429.

[0164] Example 10: Synthesis of Compound AND-10

[0165]

[0166] Step 1: Compound 6-2 (0.26 g, 1.15 mmol, 1.5 eq) and DIEA (0.29 g, 2.16 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 10-1 (0.20 g, 0.76 mmol, 1.0 eq, prepared according to the method of Bioorg Chem, 2020, 99, 103805). The mixture was stirred and heated to 100 °C for 8 hours. After cooling to room temperature, the reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 2:1) to obtain intermediate 10-3 (0.26 g, white solid, yield 82%).

[0167] MS ESI calculated value for C24H21N5O2 [M+H] + 412, found 412.

[0168] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 10-3 (0.26 g, 0.63 mmol, 1.0 eq). The mixture was stirred at room temperature for 11 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 20:1) to obtain compound AND-10 (155 mg, off-white solid, yield 59.7%).

[0169] 1 1H NMR (400 MHz, DMSO-d6) δ 2.31 (s, 3H), 4.74 (s, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.55 - 7.59 (m, 3H), 7.69 (d, J = 8.0 Hz, 1H), 8.03 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.42 - 8.45 (m, 1H), 8.65 - 8.70 (m, 1H), 8.87 (s, 1H), 9.04 (brs, 1H), 9.25 (s, 1H), 11.18 (s, 1H).

[0170] MS ESI calculated value for C23H20N6O2 [M+H] + 413, found 413.

[0171] Example 11: Synthesis of Compound AND-11

[0172]

[0173] Step 1: Compound 1-2 (0.28 g, 1.20 mmol, 1.5 eq), DIEA (0.31 g, 2.40 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 11-1 (0.20 g, 0.80 mmol, 1.0 eq, prepared according to the method of Eur J Med Chem, 2021, 209: 112871). Stir and heat to 90 °C for reaction for 13 hours. Cool to room temperature, pour the reaction solution into water (20 mL), extract with ethyl acetate (20 mL × 3), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate. The residue was purified by column chromatography (PE / EA = 1:1) to obtain intermediate 11-3 (0.24 g, light yellow solid, yield 75%).

[0174] MS ESI calculated value for C23H19N5O2 [M+H] + 398, found 398.

[0175] Step 2: Under the condition of 0-5 °C, MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 11-3 (0.24 g, 0.60 mmol, 1.0 eq). Stir at room temperature for 13 hours. Acidify the reaction solution with acetic acid (10% aqueous solution) to pH = 4, and concentrate under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-11 (150 mg, off-white solid, yield 62.8%).

[0176] 1 H NMR (400 MHz, DMSO-d6)) δ 4.38 (s, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.55 - 7.59 (m, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.92 - 8.03 (m, 1H), 8.42 - 8.45 (m, 2H), 8.70 - 8.74 (m, 2H), 8.99 (s, 1H), 9.03 (brs, 1H), 9.24 (s, 1H), 11.19 (s, 1H).

[0177] MS ESI calculated value for C22H18N6O2 [M+H] + 399, found 399.

[0178] Example 12: Synthesis of Compound AND-12

[0179]

[0180] Step 1: Compound 6-2 (0.29 g, 1.25 mmol, 1.5 eq) and DIEA (0.33 g, 2.40 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 12-1 (0.20 g, 0.84 mmol, 1.0 eq). The mixture was stirred and heated to 70 °C for 18 h. After cooling to room temperature, the reaction mixture was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), and the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 1:1) to give intermediate 12-3 (0.24 g, white solid, yield 72%).

[0181] MS ESI calculated value for C19H15F3N4O2 [M+H] + 389, found 389.

[0182] Step 2: Under the condition of 0-5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 12-3 (0.24 g, 0.62 mmol, 1.0 eq). The mixture was stirred at room temperature for 17 h. The reaction mixture was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to give compound AND-12 (170 mg, white solid, yield 70.3%).

[0183] 1 H NMR (400 MHz, DMSO-d6) δ 4.72 (s, 2H), 7.40 (s, 1H), 7.45 - 7.55 (m, 3H), 7.69 (d, J = 8.0 Hz, 1H), 8.14 - 8.24 (m, 3H), 8.86 (s, 1H), 9.05 (brs, 1H), 11.16 (s, 1H).

[0184] MS ESI calculated value for C18H14F3N5O2 [M+H] + 390, found 390.

[0185] Example 13: Synthesis of Compound AND-13

[0186]

[0187] Step 1: Compound 1-2 (0.27 g, 1.18 mmol, 1.5 eq), DIEA (0.31 g, 2.37 mmol, 3.0 eq) were successively added to a DMSO (2 mL) solution of compound 13-1 (0.20 g, 0.79 mmol, 1.0 eq), stirred, and heated to 70 °C for reaction for 19 h. After cooling to room temperature, the reaction solution was poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE / EA = 1:1) to obtain intermediate 13-3 (0.22 g, white solid, yield 69.4%).

[0188] MS ESI calculated value for C23H19N3O2S [M+H] + 402, found 402.

[0189] Step 2: Under the condition of 0 - 5 °C, MeOH (2 ml), solid sodium hydroxide (3.0 eq), and aqueous hydroxylamine solution (50% aqueous solution, 10 eq) were successively added to a THF (2 ml) solution of intermediate 13-3 (0.22 g, 0.55 mmol, 1.0 eq), and stirred at room temperature for 15 h. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution), and most of the solvent was removed by concentration under reduced pressure. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound AND-13 (152 mg, white solid, yield 68.7%).

[0190] 1 H NMR (400 MHz, DMSO-d6) 4.33 (s, 2H), 7.19 - 7.28 (m, 3H), 7.54 (s, 1H), 7.56 - 7.65 (m, 2H), 7.84 - 7.97 (m, 3H), 8.48 - 8.63 (m, 2H), 9.02 (brs, 1H), 11.17 (s, 1H).

[0191] MS ESI calculated value for C22H18N4O2S [M+H] + 403, found 403.

[0192] Example 14: Inhibitory Activity Test of the Compound against HDAC

[0193] Materials:

[0194] HDAC assay buffer (BPS catalog number 50031)

[0195] HDAC assay developer (BPS catalog number 50030)

[0196] HDAC Substrate 3 (BPS No. 50037)

[0197] The test compound is the compound of the present invention, with a storage concentration of 10 mM, dissolved in DMSO as the solvent. The test range is 0.001 - 30 μM and 0.0001 - 3 μM, and the intermediate diluent is the HDAC assay buffer containing 10% DMSO.

[0198] The enzyme and substrate are shown in Table 1.

[0199] Table 1

[0200] Determination Catalog# Enzyme batch# Enzyme used (ng) / reaction Substrate HDAC1 50051 200917-2 7.2 10 μM HDAC substrate 3 HDAC6 50006 210721 10 10 μM HDAC substrate 3

[0201] Test method:

[0202] All test compounds were dissolved in DMSO. Further, the test compounds were prepared into diluents, which were the HDAC assay buffer containing 10% DMSO; and 5 μl of the diluent was added to 50 μl of the reactant so that the final concentration of DMSO in all reactants was 1%. The compounds were pre-incubated in a mixture containing HDAC assay buffer, 5 μg BSA, HDAC enzyme (shown in Table 1) and the test compound at room temperature for 30 minutes, in duplicate. After 30 minutes, the enzymatic reaction was initiated by adding the HDAC substrate (shown in Table 1) at a final concentration of 10 μM. The enzymatic reaction was carried out at 37 °C for 30 minutes.

[0203] After the enzymatic reaction, 50 μl of 2x HDAC developer was added to each well containing the HDAC enzyme, and the plate was incubated at room temperature for another 15 minutes.

[0204] The fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at an excitation of 360 nm and an emission of 460 nm.

[0205] Data analysis:

[0206] The HDAC activity assay was performed in duplicate at each concentration. The fluorescence intensity data was analyzed using the computer software GraphPad Prism. In the absence of the compound, the fluorescence intensity (F t ) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescence intensity (F b ) in each data set was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (F - F b ) / (F t - F b ), where F = the fluorescence intensity in the presence of the compound.

[0207] Then use the formula Y = B + (T - B) / 1 + 10((LogEC50-X)×Hill斜率) Nonlinear regression analysis of the resulting sigmoidal dose-response curve plots the values of % activity against a series of compound concentrations, where Y = percentage activity, B = minimum percentage activity, T = maximum percentage activity, X = logarithm of the compound, and Hill slope = slope factor or Hill coefficient. The IC 50 value is determined by the concentration that produces half the maximum percentage activity.

[0208] The test results are shown in Table 2.

[0209] Table 2 Inhibitory activity of the compounds of the present invention against HDAC6 and enzyme subtype selectivity

[0210]

[0211] As can be seen from Table 2, the compounds of the present invention have significant inhibitory activity against HDAC6 (IC 50 : 1.1 - 32 nM), while having weak inhibitory activity against HDAC1 (IC 50 : 278 - 1296 nM); the compounds of the present invention show good selectivity in the inhibitory activity against HDAC6 / HDAC1, with the selectivity ranging from 13 to 665 times.

[0212] Example 15: Pharmacokinetic properties of the compounds

[0213] Evaluate the pharmacokinetic properties of some compounds AND-4, AND-6, AND-11, and AND-12 of the present invention after single IV and PO administration to male SD rats. The drug concentrations of the compounds in rat plasma and brain plasma samples are determined by LC-MS / MS method, and the pharmacokinetic parameters, oral bioavailability, and blood-brain barrier permeability (Brain / Plasma) of the test compounds are calculated. The test results are shown in Table 3.

[0214] Table 3 Pharmacokinetic results of some compounds of the present invention in rats

[0215]

[0216] Compounds AND-4, AND-6, AND-11, and AND-12 have relatively high bioavailability (26% - 58%), high brain permeability (B / P: 2.4 - 4.2), and good pharmacokinetic properties.

[0217] Example 16: Bacterial reverse mutation test of the compounds

[0218] Use the histidine-requiring Salmonella typhimurium reverse mutation test to study whether some compounds AND-4, AND-6, AND-11, and AND-12 of the present invention cause gene mutations to evaluate their potential mutagenicity.

[0219] (1) Preparation method

[0220] Accurately weigh 0.0303 g of the test sample before use, and completely dissolve it in a certain volume of DMSO under sterile and ultrasonic conditions to prepare a solution with a maximum concentration of 100000.0 μg / mL. Then, dilute it to 9 concentrations of 33333.0, 11111.0, 3704.0, 1235.0, 412.0, 137.0, 46.0, and 15.0 μg / mL according to a ratio of 1:2 (v / v).

[0221] (2) Negative control DMSO

[0222] (3) Positive control

[0223]

[0224] (4) Test strains

[0225] The histidine-requiring mutant strains TA98 and TA100 of Salmonella typhimurium were purchased from MolTox.

[0226] (5) Metabolic activation system

[0227] The metabolic activation system (S9) was purchased from MolTox, with a specification of 2 mL / vial, batch number 2548, and a protein content of 38.5 mg / mL. It is a liver homogenate of SD male rats induced by polychlorinated biphenyl (Aroclor 1254) at 500 mg / kg.

[0228] Before use, S9 was combined with coenzyme II, glucose-6-phosphate, etc. to form a liver microsomal enzyme system (S9 mixture).

[0229] (6) Formal test

[0230] The formal test consisted of two parallel experiments with or without a metabolic activation system. Using the standard plate incorporation method, 500 μL of molten top agar medium containing 0.6% agar, 0.5% NaCl, 0.5 mM biotin, and 0.5 mM histidine was mixed with the following substances:

[0231] 20 μL of the test sample solution (or negative / positive control);

[0232] 25 μL of the overnight culture broth;

[0233] 100 μL of the S9 mixture or 0.2 M sodium phosphate buffer (pH = 7.4).

[0234] After shaking the mixture well, spread it evenly on the pre-prepared V-B bottom medium, let it solidify at room temperature, and incubate it upside down in a 37°C incubator for 72 hours, then observe the results. In the formal experiment, each strain is set with negative and positive control groups, and each group is cultured in parallel with 2 wells / group.

[0235] (7) Experimental results

[0236] Compounds AND-4, AND-6, AND-11, and AND-12 did not cause any significant increase in the number of revertant colonies at all tested doses in either the S9-free or S9-supplemented experimental systems. All the tested compounds were negative in the Ames test (detection of pollutant mutagenicity), indicating that the compounds of the present invention have no mutagenicity and exhibit good safety.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein, X is selected from CH or N; Y is selected from CH or N; R is selected from halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, C 3-8 cycloalkyl, 3- to 8-membered heteroalkyl, phenyl, 5- to 12-membered heteroaryl; the 3- to 8-membered heterocycloalkyl and 5- to 12-membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms or heteroatom groups independently selected from NH, O, S and N; The said C 3-8 The cycloalkyl, 3- to 8-membered hetero cycloalkyl, phenyl and 5- to 12-membered heteroaryl are optionally substituted with 1, 2 or 3 R1s; Each of said R1s is independently selected from hydrogen, halogen, hydroxy, C 1-6 alkyl or C 1-6 alkoxy; n is selected from 1 or 2.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from:

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that when R is a halogen, the halogen is selected from fluorine, chlorine or bromine; Or, when R is C 1-6 alkyl, the C 1-6 alkyl is selected from methyl, ethyl, n-propyl or isopropyl; Or, when R is C 1-6 alkoxy, the C 1-6 alkoxy is selected from methoxy, ethoxy, n-propoxy or isopropoxy; Or, when R is a halogen-substituted C 1-6 alkyl group, the halogen is selected from fluorine, chlorine or bromine; Or, when R is a halogen-substituted C 1-6 alkyl, the C 1-6 alkyl is selected from methyl, ethyl, n-propyl or isopropyl; Or, when R is a C 3-8 cycloalkyl optionally substituted by 1, 2 or 3 R1s, the C 3-8 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; 3-8 cycloalkyl, the C 3-8 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; or, when R is a 3- to 8-membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3- to 8-membered heterocycloalkyl contains 1 or 2 heteroatoms or heteroatom groups selected from NH, O and N; or, when R is a 3- to 8-membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3- to 8-membered heterocycloalkyl is a 5- to 6-membered heterocycloalkyl; or, when R is a 5- to 12-membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5- to 12-membered heteroaryl contains 1 or 2 heteroatoms or heteroatom groups selected from NH, O, S and N; or, when R is a 5- to 12-membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5- to 12-membered heteroaryl is a 5- to 6-membered heteroaryl.

4. The compound or its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that, When R is a halogen-substituted C 1-6 alkyl group, the halogen-substituted C 1-6 alkyl group is selected from -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2 or -CF2CF3; or, when R is a 3- to 8-membered heterocycloalkyl optionally substituted by 1, 2 or 3 R1s, the 3- to 8-membered heterocycloalkyl is selected from tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl or morpholinyl; or, when R is a 5- to 12-membered heteroaryl optionally substituted by 1, 2 or 3 R1s, the 5- to 12-membered heteroaryl is selected from pyrrolyl, furanyl, thiophenyl, pyridyl, pyrimidinyl or pyrazinyl.

5. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, R is selected from fluorine, chlorine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, phenyl, pyrrolyl, furanyl, thiophenyl, pyridyl, pyrimidinyl or pyrazinyl.

6. The compound or its pharmaceutically acceptable salt according to claim 1 or 2, wherein, Each R1 is independently selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy or isopropoxy.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, Structural unit Selected from:

8. The following compounds or pharmaceutically acceptable salts thereof, selected from:

9. A compound of formula (II), wherein, X is selected from CH or N; Y is selected from CH or N; R is selected from halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, C 3-8 cycloalkyl, 3-8 membered heteroalkyl, phenyl, 5-12 membered heteroaryl; the 3- to 8-membered heterocycloalkyl and 5- to 12-membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms or heteroatom groups independently selected from NH, O, S and N; The said C 3-8 The cycloalkyl, 3- to 8-membered hetero cycloalkyl, phenyl and 5- to 12-membered heteroaryl are optionally substituted with 1, 2 or 3 R1s; Each of the R1s is independently selected from hydrogen, halogen, hydroxyl group, C 1-6 alkyl or C 1-6 alkoxy group; R2 is C 1-6 alkyl; n is selected from 1 or 2.

10. The compound according to claim 9, which is selected from:

11. A pharmaceutical composition comprising a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 as an active ingredient and a pharmaceutically acceptable carrier.

12. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 or the composition according to claim 11 in the preparation of a medicament for treating HDAC6-related diseases.

13. The use according to claim 12, characterized in that, The HDAC6-related diseases are cancer, neuropsychiatric diseases or autoimmune diseases; the neuropsychiatric diseases are neurodegenerative diseases, psychiatric diseases, peripheral neuropathies, and combinations thereof.

14. The use according to claim 13, wherein, The cancers are ovarian cancer, colon cancer, breast cancer, liver cancer, pancreatic cancer, gallbladder cancer, cervical cancer, prostate cancer, lung cancer, melanoma, gestational trophoblastic tumor, glioblastoma, myeloma, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, and mast cell-derived tumors, and combinations thereof; The autoimmune diseases are inflammatory diseases, conditions treatable by immunomodulation; the conditions treatable by immunomodulation are multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergy, asthma, inflammatory bowel disease.

15. The use according to claim 13, wherein The neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Rett syndrome; The psychiatric diseases are schizophrenia, depression, bipolar disorder, anxiety disorder; The peripheral neuropathies are Charcot-Marie-Tooth disease, giant axonal neuropathy.

16. The use according to claim 14, characterized in that, The inflammatory diseases are osteoarthritis, rheumatoid arthritis, colitis.

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