Isatoic anhydride compounds and preparation method and use thereof

Abstract

The present invention discloses isatoic anhydride compounds as selective inhibitors of histone deacetylase 6 (HDAC6), and uses thereof in the preparation of drugs for treating HDAC6-related diseases; specifically discloses compounds represented by formula (I) and pharmaceutically acceptable salts thereof. The compounds of the present invention have high inhibitory activity and high HDAC enzyme subtype selectivity against HDAC6, and also 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 specifically relates to a class of isatoic anhydride compounds as histone deacetylase 6 (HDAC6) selective inhibitors, and use thereof in preparing drugs for treating HDAC6-related diseases. Background Art

[0002] Histone deacetylases (HDACs) can catalyze the deacetylation of histones and non-histone proteins, and together with histone acetyltransferases (HATs), regulate the intracellular acetylation level, thereby regulating gene expression. At present, there are 18 subtypes of mammalian HDACs, 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] There are five histone deacetylase inhibitors (HDACi) currently 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 side effects, such as nausea, vomiting, bone marrow suppression, etc.; (2) genotoxicity; (3) poor pharmacokinetic properties, low bioavailability, short half-life, etc. The above 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, HDACs subtype selective inhibitors have become a research hotspot in this field, among which HDAC6 subtype inhibitors have attracted much attention. HDAC6 involves a wide range of diseases, including tumors, neurodegenerative diseases, neuropsychiatric diseases, inflammation, autoimmune response, tumors and bacterial infections.

[0006] Pharmacological studies have shown that HDAC6 selective inhibitors can effectively inhibit the proliferation of glioblastoma cells and induce their apoptosis, and inhibition of HDAC6 can enhance the sensitivity of glioblastoma cells to temozolomide, suggesting that the two have a synergistic effect. 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 ubiquitinated misfolded proteins with high affinity and promote their degradation. Therefore, the combined treatment 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 pro-apoptotic signals of the unfolded protein response and endoplasmic reticulum stress, and finally overcame the endoplasmic reticulum stress tolerance, reduced the viability of TMZ-resistant glioma cells and induced apoptosis.

[0007] Similar to brain glioma, Alzheimer's disease (AD) is also in urgent need of effective therapeutic drugs. Studies have found that the expression level of HDAC6 in the hippocampus (increased by 91%) and cortex (increased by 52%) of AD brains is significantly increased, 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 tau T231 phosphorylation, which is a key regulatory site of tau function. HDAC6 inhibition improves cognition in AD mice. The above studies show that centrally targeted HDAC6 inhibitors are expected to be developed into new, highly effective and low-toxic AD therapeutics.

[0008] Existing drugs for schizophrenia, depression, and bipolar disorder have disadvantages such as large side effects (such as extrapyramidal side effects and weight gain), slow onset, and poor patient compliance, which are mainly related to the drugs acting on dopamine and serotonin receptors. At present, there is an urgent need for innovative drugs for schizophrenia, depression, and bipolar disorder that act on new targets and new mechanisms to circumvent the shortcomings of existing drugs. Studies have found that a large number of histone modifications (acetylation) in the promoter region of schizophrenia susceptibility genes are significantly abnormal. The latest positron emission tomography (PET) results show that histone deacetylase expression 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, have lower anxiety levels 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 the side effects of existing drugs such as extrapyramidal and weight gain.

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

[0010] In one aspect, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0011]

[0012] in,

[0013] X is selected from NH or a single bond;

[0014] T1 is selected from CH or N;

[0015] T2 is selected from CH or N;

[0016] R1 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 alkyl;

[0017] R2 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 alkyl;

[0018] m is selected from 1 or 2;

[0019] n is selected from 0, 1 or 2.

[0020] In some embodiments of the present invention, the above structural unit Selected from: Best

[0021] In some embodiments of the present invention, when R1 or R2 is halogen, the halogen is selected from fluorine, chlorine or bromine, preferably fluorine or chlorine.

[0022] In some embodiments of the present invention, when R1 or R2 is C 1-6 When the C 1-6 The alkyl group is selected from methyl, ethyl, n-propyl or isopropyl, preferably methyl.

[0023] In some embodiments of the present invention, when R1 or R2 is C 1-6 When the C 1-6 The alkoxy group is selected from methoxy, ethoxy, n-propoxy or isopropoxy, preferably methoxy.

[0024] In some embodiments of the present invention, when R1 or R2 is a halogen-substituted C 1-6 When it is alkyl, the halogen is selected from fluorine, chlorine or bromine, preferably fluorine.

[0025] In some embodiments of the present invention, when R1 or R2 is a halogen-substituted C 1-6 When the C 1-6 The alkyl group is selected from methyl, ethyl, n-propyl or isopropyl, preferably methyl.

[0026] In some embodiments of the present invention, when R1 or R2 is a halogen-substituted C 1-6 When alkyl, the halogen-substituted C 1-6 The alkyl group is selected from -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2 or -CF2CF3, preferably -CF3.

[0027] In some embodiments of the present invention, R1 is selected from hydrogen, fluorine, chlorine, nitro, methyl, methoxy or -CF3.

[0028] In some embodiments of the present invention, R2 is selected from hydrogen, fluorine, chlorine, nitro, methyl, methoxy or -CF3.

[0029] In some embodiments of the present invention, the above structural unit Selected from:

[0030]

[0031] In some embodiments of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from:

[0032] wherein R1, R2, m and n are as defined in any of the previous schemes.

[0033] In another aspect, the present invention provides the following compounds and pharmaceutically acceptable salts thereof, selected from:

[0034]

[0035]

[0036] In another aspect, the present invention provides a compound represented by formula (II),

[0037]

[0038] in,

[0039] X is selected from NH or a single bond;

[0040] T1 is selected from CH or N;

[0041] T2 is selected from CH or N;

[0042] R1 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 Alkyl; R2 is selected from hydrogen, halogen, hydroxyl, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 Alkyl; R3 is C 1-6 alkyl;

[0043] m is selected from 1 or 2;

[0044] n is selected from 0, 1 or 2.

[0045] In some embodiments of the present invention, the compound of formula (II) is selected from:

[0046]

[0047] On the other hand, the present invention provides a method for preparing the compound of formula (I), including scheme 1 and scheme 2.

[0048] Solution 1

[0049]

[0050] Wherein, R1, R2, m and n are defined as in any of the previous schemes, and X is a single bond.

[0051] Compound (III) and compound (IV) are subjected to substitution reaction to obtain compound (II), and then compound (II) is subjected to hydroxylation reaction to obtain compound (I).

[0052] Solution 2

[0053]

[0054] wherein R1, R2, m and n are as defined in any of the previous schemes, and X is NH.

[0055] Compound (III) is subjected to a substitution reaction to obtain compound (V), compound (V) is further subjected to a substitution reaction with compound (VI) to obtain compound (II), and compound (II) is subjected to a hydroxylation reaction to obtain compound (I).

[0056] In another aspect, 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.

[0057] The pharmaceutically acceptable carrier (pharmaceutical excipient) can 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 to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can 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 can include one or more of the following excipients: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0058] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0059] 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, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft 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, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0060] In another aspect, the present invention provides use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating HDAC6-related diseases.

[0061] In another aspect, the present invention provides use of the above-mentioned pharmaceutical composition in preparing a drug for treating HDAC6-related diseases.

[0062] In some embodiments of the present invention, the HDAC6-related disease is cancer, a neurological disease or an autoimmune disease.

[0063] In some embodiments of the invention, the cancer comprises 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 tumors, gestational trophoblastic tumors, glioblastoma, myeloma, neuroblastic CNS tumors, monocytic leukemia, B cell leukemia, T cell leukemia, B cell lymphoma, T cell lymphoma and mast cell tumors, and combinations thereof.

[0064] In some embodiments of the present invention, the neurological disease comprises a neurodegenerative disease, a neuropsychiatric disease, a peripheral neuropathy, and combinations thereof.

[0065] In some embodiments of the invention, the autoimmune disease comprises psoriasis, inflammatory diseases, conditions treatable by immunomodulation.

[0066] In some embodiments of the invention, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Rett's syndrome, pain.

[0067] In some embodiments of the present invention, the neuropsychiatric disease is schizophrenia, depression, bipolar disorder, anxiety disorder.

[0068] In some embodiments of the present invention, the peripheral neuropathy includes Charcot-Marie-Tooth disease and giant axial neuropathy.

[0069] In some embodiments of the invention, the conditions treated by immunomodulation include multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergies, asthma, allergic rhinitis, inflammatory bowel disease.

[0070] The beneficial effects of the present invention are as follows: as a novel histone deacetylase 6 (HDAC6) selective inhibitor, the compound of the present invention has significant in vitro activity (high inhibitory activity on HDAC6 enzyme) and high enzyme subtype selectivity (weak inhibition on HDAC1); in addition, the compound of the present invention shows ideal pharmacokinetic characteristics, higher blood-brain barrier permeability and good safety.

[0071] Definition and Description

[0072] 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 to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0073] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0074] The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, 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 invention contain relatively basic functional groups, 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, such as 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, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and 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 basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

[0075] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0076] In addition to the form of salts, compounds provided by the present invention also exist in prodrug forms. Prodrugs of compounds described herein easily undergo chemical changes under physiological conditions to be converted into compounds of the present invention. In addition, prodrugs can be converted to compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0077] Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0078] 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, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0079] The term "substituted" or "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted 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 aromatic groups. Unless otherwise specified, the type and number of substituents can be any on the basis of chemically achievable.

[0080] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0081] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When the listed substituent does not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0082] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0083] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0084] The term "C 1-6 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. 1-6 Alkyl groups include C 1-5 , C 1-4 , C 1-3 , C1-2 , C 2-6 , C 2-4 , C6 and C5 alkyl, etc. 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, and the like.

[0085] The term "C 1-6 "Alkoxy" refers to those alkyl groups containing 1 to 6 carbon atoms which are attached to the rest of the molecule via an oxygen atom. 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, etc. 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, and the like.

[0086] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0087] The term "halogen-substituted C 1-6 "Alkyl" refers to the above-mentioned "C 1-6 "Alkyl" is substituted by one or more halogen groups. Exemplary halogen-substituted alkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. DETAILED DESCRIPTION

[0088] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. All technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0089] The starting materials in the examples of the present invention are known and can be purchased on the market, or can be synthesized by or according to methods known in the art.

[0090] The abbreviations used in this application are as follows:

[0091] CDCl3 denotes deuterated chloroform; DMSO denotes dimethyl sulfoxide; DIEA denotes N,N-diisopropylethylamine; DMSO-d6 denotes dimethyl sulfoxide-d6; TLC denotes preparative silica thin layer chromatography; eq denotes equivalent; PE denotes petroleum ether; EA denotes ethyl acetate; DCM denotes dichloromethane; MeOH denotes methanol; THF denotes tetrahydrofuran; DMF denotes N,N-dimethylformamide.

[0092] Example 1: Synthesis of Compound ANDD-1

[0093]

[0094] Step 1: Add compound 1-2 (0.42 g, 1.84 mmol, 1.5 eq) to a solution of compound 1-1 (0.20 g, 1.23 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL), add DIEA (0.48 g, 3.69 mmol, 3.0 eq), stir, and heat to 90 ° C for 3 hours. Cool to room temperature, pour the reactant 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 is purified by column chromatography (PE / EA = 5:1) to obtain intermediate 1-3 (0.28 g, off-white solid, yield 73%).

[0095] MS ESI calcd for C17H13NO5 [M+H] + 312, measured value 312.

[0096] Step 2: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 1-3 (0.28 g, 0.90 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-1 (0.15 g, white solid, yield 54%).

[0097] 1 H NMR (400MHz, DMSO-d6) δ5.45(s,2H),7.32-7.39(m,3H),7.90-8.00(m,4H),8.05-8.10(m,1H),9.00(s,1H),11.18(s,1H).

[0098] MS ESI calcd for C16H12N2O5 [M+H] +313, measured value 313.

[0099] Example 2: Synthesis of Compound ANDD-2

[0100]

[0101] Step 1: Compound 2-2 (0.34 g, 1.35 mmol, 1.5 eq) and DIEA (0.35 g, 2.70 mmol, 3.0 eq) were added to a solution of compound 2-1 (0.20 g, 0.90 mmol, 1.0 eq) in N, N-dimethylacetamide (2 mL) in sequence, stirred, heated to 120 ° C for 12 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 2-3 (100 mg, white solid, yield 28%).

[0102] MS ESI calcd for C21H21NO7 [M+H] + 400, measured value 400.

[0103] Step 2: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 2-3 (100 mg, 0.25 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-2 (55 mg, white solid, yield 55%).

[0104] 1 H NMR (400MHz, DMSO-d6) δ2.17-2.27(m,2H),2.65(t,J=7.2Hz,2H),3.75(s,3H),3.86(s,2H),4.34(t,J=7.2 Hz,2H),7.05(d,J=8.0Hz,2H),7.45(s,1H)7.71(d,J=8.0Hz,2H),7.81(s,1H),9.02(s,1H),11.18(s,1H).

[0105] MS ESI calcd for C20H20N2O7 [M+H] + 401, measured value 401.

[0106] Example 3: Synthesis of Compound ANDD-3

[0107]

[0108] Step 1: At 0-5°C, under nitrogen protection, compound 3-2 (0.36 g, 1.92 mmol, 2.0 eq) and sodium hydride (25.00 mg, 1.05 mmol, 1.1 eq) were added to a DMSO (2 mL) solution of compound 3-1 (0.20 g, 0.96 mmol, 1.0 eq) in sequence, and stirred at room temperature for 2 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (10 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 = 5:1) to obtain intermediate 3-3 (0.11 g, yellow solid, yield 36%).

[0109] 1 H NMR (400MHz, CDCl3) δ3.95 (t, J = 7.2 Hz, 2H), 4.15 (t, J = 7.2 Hz, 2H), 8.11 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.94 (s, 1H).

[0110] Step 2: Methyl p-aminobenzoate (0.08 g, 0.52 mmol, 1.5 eq) and DIEA (0.14 g, 2.88 mmol, 3.0 eq) were added to a solution of intermediate 3-3 (0.11 g, 0.21 mmol, 1.0 eq) in N, N-dimethylacetamide (2 mL) in sequence, stirred, heated to 120 ° C for 12 hours. After cooling to room temperature, the reactant 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 3-4 (0.08 g, yellow solid, yield 59%).

[0111] MS ESI calcd for C17H14N4O7 [M+H] + 386, measured value 386.

[0112] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 3-4 (80 mg, 0.25 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 16 hours. The reactant was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-3 (40 mg, yellow solid, yield 50%).

[0113] 1 H NMR (400MHz, DMSO-d6) δ3.68(t,J=7.2Hz,2H),3.92(t,J=7.2Hz,2H),6.88(d,J=8.0Hz,2H),7.61(d, J=8.0Hz,2H),8.11(d,J=8.0Hz,1H),8.51(d,J=8.0Hz,1H),8.94(s,1H),9.03(s,1H),11.16(s,1H).

[0114] MS ESI calcd for C17H14N4O7 [M+H] + 387, measured value 387.

[0115] Example 4: Synthesis of Compound ANDD-4

[0116]

[0117] Step 1: At 0-5°C, under nitrogen protection, compound 3-2 (0.41 g, 2.20 mmol, 2.0 eq) and sodium hydride (29.00 mg, 1.21 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 4-1 (0.20 g, 1.10 mmol, 1.0 eq) in sequence, stirred, and reacted at room temperature for 2 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL) and extracted with EA (10 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 = 5:1) to obtain intermediate 4-2 (100.00 mg, off-white solid, yield 31%).

[0118] 1 H NMR (400MHz, CDCl3) δ3.95 (t, J = 6.8 Hz, 2H), 4.15 (t, J = 6.8 Hz, 2H), 7.21-7.25 (m, 1H), 7.65-7.81 (m, 2H).

[0119] Step 2: Compound 4-3 (0.08 g, 0.52 mmol, 1.5 eq) and DIEA (134.00 mg, 1.04 mmol, 3.0 eq) were added to a solution of intermediate 4-2 (100.00 mg, 0.35 mmol, 1.0 eq) in N, N-dimethylacetamide (2 mL) in sequence, stirred, heated to 120 ° C for 17 hours, cooled to room temperature, poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), combined organic layers, 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 4-4 (80.00 mg, white solid, yield 61%).

[0120] MS ESI calcd for C19H17FN2O5 [M+H] + 373, measured value 373.

[0121] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 4-4 (80.00 mg, 0.22 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 18 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-4 (35.00 mg, off-white solid, yield 43%).

[0122] 1 H NMR(400MHz, DMSO-d6)δ2.85(t,J=6.8Hz,2H),3.78(t,J=6.8Hz,2H),7.20-7.28(m,1H),7 .35(d,J=8.0Hz,2H),7,65-7.84(m,2H),7.91(d,J=8.0Hz,2H),8.94(s,1H),11.17(s,1H).

[0123] MS ESI calcd for C18H16FN3O5 [M+H] + 374, measured value 374.

[0124] Example 5: Synthesis of Compound ANDD-5

[0125]

[0126] Step 1: At 0-5°C, under nitrogen protection, compound 3-2 (0.13 g, 1.34 mmol, 1.5 eq) and sodium hydride (24.00 mg, 0.99 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 5-1 (200.00 mg, 0.90 mmol, 1.0 eq) in sequence, stirred, and reacted at room temperature for 5 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (10 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 = 5:1) to obtain intermediate 5-2 (115 mg, off-white solid, yield 35%).

[0127] 1 H NMR (400MHz, CDCl3) 3.74 (s, 3H), 3.88 (s, 3H), 3.93 (t, J = 6.8 Hz, 2H), 4.15 (t, J = 6.8 Hz, 2H), 7.55 (s, 1H), 7.78 (s, 1H).

[0128] Step 2: Compound 5-3 (86.77 mg, 0.52 mmol, 1.5 eq) and DIEA (135.00 mg, 1.05 mmol, 3.0 eq) were added to a solution of intermediate 5-2 (115.00 mg, 0.35 mmol, 1.0 eq) in N, N-dimethylacetamide (2 mL) in sequence, stirred, heated to 90 ° C for 14 hours, cooled to room temperature, poured into water (20 mL), extracted with ethyl acetate (20 mL × 3), combined organic layers, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (DCM / MeOH = 20: 1) to obtain intermediate 5-4 (90.00 mg, off-white solid, yield 62%).

[0129] MS ESI calcd for C20H21N3O7 [M+H] + 416, measured value 416.

[0130] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 5-4 (90.00 mg, 0.22 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 10 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-5 (47.00 mg, light yellow solid, yield 52%).

[0131] 1 H NMR(400MHz,DMSO-d6)δ2.87(t,J=6.8Hz,2H),3.72-3.79(m,5H),3.84(s,3H),4.13-4.17(m,3H) ,7.50(s,1H),7,69-7.78(m,2H),8.16(d,J=8.0Hz,1H),8.86(s,1H),9.06(s,1H),11.21(s,1H).

[0132] MS ESI calcd for C19H20N4O7 [M+H] + 417, measured value 417.

[0133] Example 6: Synthesis of Compound ANDD-6

[0134]

[0135] Step 1: Compound 6-2 (380.00 mg, 1.65 mmol, 1.5 eq) and DIEA (426.00 mg, 3.30 mmol, 3.0 eq) were added to a DMSO (2 mL) solution of compound 6-1 (200.00 mg, 1.10 mmol, 1.0 eq) in sequence, stirred, heated to 80 ° C for 13 hours. Cooled to room temperature, the reactant 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 = 5:1) to obtain intermediate 6-3 (212.00 mg, white solid, yield 58%).

[0136] MS ESI calcd for C16H11FN2O5 [M+H] + 331, measured value 331.

[0137] Step 2: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 6-3 (212.00 mg, 0.64 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 18 hours. The reactant was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-6 (165.00 mg, white solid, yield 78%).

[0138] 1H NMR (400MHz, DMSO-d6) δ5.25(s,2H),7.50-7.61(m,2H),7.72(d,J=8.0Hz,1H),7.83-7.94(m,2H),8.19(d,J=8.0Hz,1H),8.92(s,1H),11.21(s,1H).

[0139] MS ESI calcd for C15H10FN3O5 [M+H] + 332, measured value 332.

[0140] Example 7: Synthesis of Compound ANDD-7

[0141]

[0142] Step 1: Compound 6-2 (380.00 mg, 1.65 mmol, 1.5 eq) and DIEA (426.00 mg, 3.30 mmol, 3.0 eq) were added to a DMSO (2 mL) solution of compound 7-1 (180.00 mg, 1.10 mmol, 1.0 eq) in sequence, stirred, heated to 80 ° C for 12 hours. After cooling to room temperature, the reactant 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 = 5:1) to obtain intermediate 7-2 (205.00 mg, white solid, yield 66%).

[0143] MS ESI calcd for C16H12N2O5 [M+H] + 313, measured value 313.

[0144] Step 2: At 0-5°C, MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 7-2 (205.00 mg, 0.66 mmol, 1.0 eq) in THF (2 ml) in sequence and stirred at room temperature for 17 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-7 (144.00 mg, off-white solid, yield 70%).

[0145] 1H NMR (400MHz, DMSO-d6) δ5.25 (s, 2H), 7.30-7.41 (m, 1H), 7.72 (d, J = 8.0Hz, 1H), 7.85-7.93 (m, 2H), 8.05-8.18 (m, 2H), 8.89 (s, 1H), 11.20 (s, 1H).

[0146] MS ESI calcd for C15H11N3O5 [M+H] + 314, measured value 314.

[0147] Example 8: Synthesis of Compound ANDD-8

[0148]

[0149] Step 1: Compound 6-2 (380.00 mg, 1.65 mmol, 1.5 eq) and DIEA (426.00 mg, 3.30 mmol, 3.0 eq) were added to a DMSO (2 mL) solution of compound 8-1 (212.00 mg, 1.10 mmol, 1.0 eq) in sequence, stirred, heated to 90 ° C for 15 hours. Cooled 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 = 5: 1) to obtain intermediate 8-2 (195.00 mg, off-white solid, yield 52%).

[0150] MS ESI calcd for C17H14N2O6 [M+H] + 343, measured value 343.

[0151] Step 2: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 8-2 (195.00 mg, 0.57 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 13 hours. The reactant was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-8 (124.00 mg, off-white solid, yield 64%).

[0152] 1H NMR (400MHz, DMSO-d6) δ3.79 (s, 3H), 5.23 (s, 2H), 7.72 (d, J = 8.0 Hz, 1H), 7.78-7.83 (m, 2H), 8.59 (d, J = 8.0 Hz 1H), 8.90 (s, 1H), 11.21 (s, 1H).

[0153] MS ESI calcd for C16H13N3O6 [M+H] + 344, measured value 344.

[0154] Example 9: Synthesis of Compound ANDD-9

[0155]

[0156] Step 1: At 0°C, under nitrogen protection, compound 3-2 (0.63 g, 3.39 mmol, 2.0 eq) and sodium hydride (45.00 mg, 1.86 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 9-1 (300.00 mg, 1.69 mmol, 1.0 eq) in sequence, stirred, and reacted at room temperature for 3 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (10 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 = 5:1) to obtain intermediate 9-2 (153.58 mg, white solid, yield 32%).

[0157] 1 H NMR (400MHz, CDCl3) δ2.43 (s, 3H), 3.94 (t, J = 7.2Hz, 2H), 4.15 (t, J = 7.2Hz, 2H), 7.65 (d, J = 8.0Hz, 1H), 7.83 (d, J = 8.0Hz, 1H), 8.11 (s, 1H).

[0158] Step 2: Compound 9-3 (123.00 mg, 0.81 mmol, 1.5 eq) and DIEA (209.00 mg, 1.62 mmol, 3.0 eq) were added to a solution of intermediate 9-2 (153.58 mg, 0.54 mmol, 1.0 eq) in N, N-dimethylacetamide (2 mL) in sequence, stirred, heated to 110 ° C for 12 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 9-4 (112.00 mg, white solid, yield 58%).

[0159] MS ESI calcd for C18H17N3O5 [M+H] + 356, measured value 356.

[0160] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 9-4 (112.00 mg, 0.32 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 20 hours. The reactant was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-9 (78.00 mg, gray solid, yield 70%).

[0161] 1 H NMR (400MHz, DMSO-d6) δ2.42(s,3H),3.69(t,J=7.2Hz,2H),3.91(t,J=7.2Hz,2H),6.91(d,J=8.0Hz, 1H),7.66(d,J=8.0Hz,1H),7.78-7.89(m,3H),8.10(s,1H),8.33(s,1H),9.03(s,1H),11.19(s,1H).

[0162] MS ESI calcd for C17H16N4O5 [M+H] + 357, measured value 357.

[0163] Example 10: Synthesis of Compound ANDD-10

[0164]

[0165] Step 1: At 0°C, under nitrogen protection, compound 3-2 (0.53 g, 2.84 mmol, 1.5 eq) and sodium hydride (50.00 mg, 0.99 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 10-1 (400.00 mg, 1.90 mmol, 1.0 eq) in sequence, and reacted at room temperature for 5 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (10 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 = 5:1) to obtain intermediate 10-2 (168.00 mg, off-white solid, yield 28%).

[0166] 1H NMR (400MHz, CDCl3) 2.14 (s, 3H), 3.94 (t, J = 7.2Hz, 2H), 4.15 (t, J = 7.2Hz, 2H), 7.80 (s, 1H), 7.87 (s, 1H).

[0167] Step 2: Add compound 5-3 (131.50 mg, 0.79 mmol, 1.5 eq) and DIEA (201.00 mg, 1.59 mmol, 3.0 eq) to a solution of intermediate 10-2 (168.00 mg, 0.53 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL), stir, and heat to 110 ° C for 12 hours. Cool to room temperature, pour the reactant 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 is purified by column chromatography (DCM / MeOH = 20: 1) to obtain intermediate 10-3 (103.00 mg, white solid, yield 48%).

[0168] MS ESI calcd for C19H18ClN3O5 [M+H] + 404, measured value 404.

[0169] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 10-3 (103.00 mg, 0.25 mmol, 1.0 eq) in THF (2 ml) at 0-5 ° C, and stirred at room temperature for 17 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10: 1) to obtain compound ANDD-10 (68.00 mg, light yellow solid, yield 66%).

[0170] 1 H NMR (400MHz, DMSO-d6) δ2.14(s,3H),2.86(t,J=7.2Hz,2H),3.79(t,J=7.2Hz,2H),4.16( s,2H),7.71-7.89(m,3H),8.21(d,J=8.0Hz,1H),8.88(s,1H),9.04(s,1H),11.20(s,1H).

[0171] MS ESI calcd for C18H17ClN4O5 [M+H] + 405, measured value 405.

[0172] Example 11: Synthesis of Compound ANDD-11

[0173]

[0174] Step 1: Same as step 1 in Example 9.

[0175] Step 2: Add compound 11-1 (121.20 mg, 0.80 mmol, 1.5 eq) and DIEA (209.00 mg, 1.62 mmol, 3.0 eq) to a solution of intermediate 9-2 (155.00 mg, 0.54 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL), stir, heat to 100 ° C and react for 15 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 is purified by column chromatography (PE / EA = 1: 1) to obtain intermediate 11-2 (86.50 mg, white solid, yield 45%).

[0176] MS ESI calcd for C17H16N4O5 [M+H] + 357, measured value 357.

[0177] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 11-2 (86.50 mg, 0.24 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-11 (58.00 mg, white solid, yield 67%).

[0178] 1 H NMR (400MHz, DMSO-d6) δ2.42(s,3H),3.69(t,J=7.2Hz,2H),3.90(t,J=7.2Hz,2H),7.01(s,1H), 7.65(d,J=8.0Hz,1H),7.76-7.88(m,2H),8.09(s,1H),8.64(s,2H),9.06(s,1H),11.23(s,1H).

[0179] MS ESI calcd for C16H15N5O5 [M+H] + 358, measured value 358.

[0180] Example 12: Synthesis of Compound ANDD-12

[0181]

[0182] Step 1: At 0°C, under nitrogen protection, compound 3-2 (560.00 mg, 3.00 mmol, 1.5 eq) and sodium hydride (53.00 mg, 2.20 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 12-1 (400.00 mg, 2.00 mmol, 1.0 eq) in sequence, stirred, and slowly warmed to room temperature for 2 hours. The reactant was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (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 = 5:1) to obtain intermediate 12-2 (189.00 mg, off-white solid, yield 31%).

[0183] 1 H NMR (400MHz, CDCl3) 2.14 (s, 3H), 3.94 (t, J = 7.2Hz, 2H), 4.15 (t, J = 7.2Hz, 2H), 7.80 (s, 1H), 7.87 (s, 1H).

[0184] Step 2: Compound 11-1 (142.30 mg, 0.93 mmol, 1.5 eq) and DIEA (240.00 mg, 1.86 mmol, 3.0 eq) were added to a solution of intermediate 12-2 (189.00 mg, 0.62 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL) in sequence, stirred, and heated to 120 ° C for 12 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 (DCM / MeOH = 20: 1) to obtain intermediate 12-3 (92.00 mg, white solid, yield 39%).

[0185] MS ESI calcd for C16H12F2N4O5 [M+H] + 379, measured value 379.

[0186] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 12-3 (92.00 mg, 0.24 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 16 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-12 (48.00 mg, light yellow solid, yield 52%).

[0187] 1 H NMR (400MHz, DMSO-d6) δ3.70(t,J=7.2Hz,2H),3.91(t,J=7.2Hz,2H),7.02(s,1H),7.91(s,1H),7.97(s,1H),8.65(s,2H),9.07(s,1H),11.23(s,1H).

[0188] MS ESI calcd for C15H11F2N5O5 [M+H] + 380, actual measured value 380.

[0189] Example 13: Synthesis of Compound ANDD-13

[0190]

[0191] Step 1: Same as step 1 in Example 10.

[0192] Step 2: Compound 11-1 (72.20 mg, 0.47 mmol, 1.5 eq) and DIEA (120.00 mg, 0.93 mmol, 3.0 eq) were added to a solution of intermediate 10-2 (100.00 mg, 0.31 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL) in sequence, stirred, heated to 100 ° C and reacted for 18 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 (DCM / MeOH = 20: 1) to obtain intermediate 13-1 (82.00 mg, white solid, yield 67%).

[0193] MS ESI calcd for C17H15ClN4O5 [M+H] + 391, measured value 391.

[0194] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 13-1 (82.00 mg, 0.21 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 20 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-13 (36.00 mg, light yellow solid, yield 44%).

[0195] 1 H NMR (400MHz, DMSO-d6) δ2.18(s,3H),3.71(t,J=7.2Hz,2H),3.92(t,J=7.2Hz,2H) ,7.03(s,1H),7.81(s,1H),7.88(s,1H),8.67(s,2H),9.05(s,1H),11.22(s,1H).

[0196] MS ESI calcd for C16H14ClN5O5 [M+H] + 392, measured value 392.

[0197] Example 14: Synthesis of Compound ANDD-14

[0198]

[0199] Step 1: At 0°C, under nitrogen protection, compound 3-2 (560.00 mg, 3.00 mmol, 1.5 eq) and sodium hydride (53.00 mg, 2.20 mmol, 1.1 eq) were added to a DMF (2 mL) solution of compound 14-1 (462.00 mg, 2.00 mmol, 1.0 eq) in sequence, stirred, and slowly warmed to room temperature for 3 hours. The reaction solution was poured into a saturated ammonium chloride solution (10 mL), extracted with EA (20 mL × 2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 5:1) to obtain intermediate 14-2 (219.00 mg, off-white solid, yield 32%).

[0200] 1 H NMR (400MHz, CDCl3) 2.14 (s, 3H), 3.94 (t, J = 7.2Hz, 2H), 4.15 (t, J = 7.2Hz, 2H), 7.80 (s, 1H), 7.87 (s, 1H).

[0201] Step 2: Compound 11-1 (150.00 mg, 0.97 mmol, 1.5 eq) and DIEA (252.00 mg, 1.95 mmol, 3.0 eq) were added to a solution of intermediate 14-2 (219.00 mg, 0.65 mmol, 1.0 eq) in N,N-dimethylacetamide (2 mL) in sequence, stirred, and heated to 120 ° C 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 (DCM / MeOH = 20: 1) to obtain intermediate 14-3 (122.00 mg, white solid, yield 46%).

[0202] MS ESI calcd for C17H13F3N4O5 [M+H] + 411, measured value 411.

[0203] Step 3: MeOH (2 ml), sodium hydroxide solid (3.0 eq) and hydroxylamine aqueous solution (50% aqueous solution, 10 eq) were added to a solution of intermediate 14-3 (122.00 mg, 0.30 mmol, 1.0 eq) in THF (2 ml) at 0-5 °C and stirred at room temperature for 10 hours. The reaction solution was acidified to pH = 4 with acetic acid (10% aqueous solution) and concentrated under reduced pressure to remove most of the solvent. The residue was purified by column chromatography (DCM / MeOH = 10:1) to obtain compound ANDD-14 (85.00 mg, light yellow solid, yield 70%).

[0204] 1 H NMR (400MHz, DMSO-d6) δ3.72(t,J=7.2Hz,2H),3.93(t,J=7.2Hz,2H),7.04(s,1H),7.80( d,J=8Hz,1H),7.95(d,J=8Hz,1H),8.44(s,1H),8.65(s,2H),9.05(s,1H),11.24(s,1H).

[0205] MS ESI calcd for C16H12F3N5O5 [M+H] + 412, measured value 412.

[0206] Example 15: Test of the inhibitory activity of compounds on HDAC

[0207] Material:

[0208] HDAC Assay Buffer (BPS Cat. No. 50031)

[0209] HDAC Assay Developer (BPS Cat. No. 50030)

[0210] HDAC substrate 3 (BPS No. 50037)

[0211] The test compound is the compound of the present invention, with a storage concentration of 10 mM, the dissolving reagent is DMSO, the test range is 0.001-30 μM and 0.0001-3 μM, and the intermediate diluent is HDAC assay buffer containing 10% DMSO.

[0212] Enzymes and substrates are listed in Table 1.

[0213] Table 1

[0214] Determination Table of contents# 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

[0215] Test method:

[0216] All test compounds were dissolved in DMSO, and the test compounds were further prepared into diluents, which were HDAC assay buffer containing 10% DMSO; 5 μl of the diluent was added to 50 μl of the reactants so that the final concentration of DMSO in all reactants was 1%. The compounds were pre-incubated at room temperature for 30 minutes in a mixture containing HDAC assay buffer, 5 μg BSA, HDAC enzyme (see Table 1) and test compounds, in duplicate. After 30 minutes, the enzymatic reaction was initiated by adding a final concentration of 10 μM HDAC substrate (see Table 1). The enzymatic reaction was carried out at 37°C for 30 minutes.

[0217] After the enzymatic reaction, 50 μl of 2x HDAC developer was added to each well containing HDAC enzyme, and the plate was incubated for another 15 min at room temperature.

[0218] Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at excitation at 360 nm and emission at 460 nm.

[0219] Data Analysis:

[0220] HDAC activity was measured in duplicate at each concentration. Fluorescence intensity data were analyzed using computer software GraphpadPrism. In the absence of compound, the fluorescence intensity (F t ) is defined as 100% activity. In the absence of HDAC, the fluorescence intensity (F b ) was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (FF b ) / (F t -F b ), where F = fluorescence intensity in the presence of the compound.

[0221] Then use the formula Y = B + (TB) / 1 + 10 ((LogEC50-X)×Hill斜率) Nonlinear regression analysis of the generated Sigmoidal dose response curves plots the % activity values ​​against a range of compound concentrations, where Y = percent activity, B = minimum percent activity, T = maximum percent activity, X = logarithm of compound, Hill slope = slope factor or Hill coefficient. IC 50 Values ​​were determined from the concentrations that produced half-maximal percent activity.

[0222] The test results are shown in Table 2.

[0223] Table 2 Inhibitory activity and enzyme subtype selectivity of the compounds of the present invention against HDAC6

[0224]

[0225] As shown in Table 2, the compounds of the present invention have significant inhibitory activity against HDAC6 (IC 50 :0.9~26nM), and weak inhibitory activity on HDAC1 (IC 50 :380~2560nM); The compounds of the present invention show good selectivity for the inhibitory activity of HDAC6 / HDAC1, with a selectivity between 53 and 577 times.

[0226] Example 16: Pharmacokinetic properties of compounds

[0227] The pharmacokinetic properties of some compounds of the present invention, ANDD-1, ANDD-6, ANDD-7 and ANDD-14, were tested by single IV and PO administration to male SD rats. The drug concentrations of the compounds in rat plasma and brain samples were determined by LC-MS / MS, and the pharmacokinetic parameters, oral bioavailability and blood-brain barrier permeability (Brain / Plasma) of the test compounds were calculated. The test results are shown in Table 3.

[0228] Table 3 Pharmacokinetic results of some compounds of the present invention in rats

[0229]

[0230]

[0231] Compounds ANDD-1, ANDD-6, ANDD-7 and ANDD-14 have high bioavailability (31% to 76%), high brain permeability (B / P: 2.8-4.5) and good pharmacokinetic properties.

[0232] Example 17: Compound bacterial reverse mutation experiment

[0233] The histidine auxotrophic Salmonella typhimurium reverse mutation test was used to study whether some of the compounds ANDD-1, ANDD-6, ANDD-7 and ANDD-14 of the present invention cause gene mutations, so as to evaluate their potential mutagenicity.

[0234] (1) Preparation method

[0235] Before use, accurately weigh 0.0303 g of the test sample and completely dissolve it in a certain volume of solvent DMSO under sterile and ultrasonic conditions to prepare a solution with the highest concentration of 100000.0 μg / mL, and then dilute it at a ratio of 1:2 (v / v) into 9 solutions with concentrations of 33333.0, 11111.0, 3704.0, 1235.0, 412.0, 137.0, 46.0 and 15.0 μg / mL.

[0236] (2) Negative control DMSO

[0237] (3) Positive control

[0238]

[0239] (4) Test strains

[0240] The histidine auxotrophic mutant strains TA98 and TA100 of Salmonella typhimurium were purchased from MolTox.

[0241] (5) Metabolic Activation System

[0242] The metabolic activation system (S9) was purchased from MolTox, with a specification of 2 mL / bottle, batch number 2548, and a protein content of 38.5 mg / mL. It was liver homogenate of SD male rats induced with polychlorinated biphenyls (Aroclor 1254) at 500 mg / kg.

[0243] Before use, S9, coenzyme II, glucose-6-phosphate, etc. form the liver microsomal enzyme system (S9 mixed solution).

[0244] (6) Formal test

[0245] The formal experiment consisted of two parallel experiments with or without the metabolic activation system. Using the standard plate incorporation method, 500 μL of molten top culture medium containing 0.6% agar, 0.5% NaCl, 0.5 mM biotin and 0.5 mM histidine was mixed with the following substances:

[0246] 20 μL test solution (or negative / positive control);

[0247] 25 μL overnight culture solution;

[0248] 100 μL S9 mixed solution or 0.2 M sodium phosphate buffer (pH = 7.4).

[0249] After the mixture is shaken, it is spread on the pre-prepared VB bottom culture medium, solidified at room temperature, and incubated upside down in a 37°C incubator for 72 hours to observe the results. In the formal test, each strain has a negative and positive control group, and each group is cultured in parallel with 2 wells / group.

[0250] (7) Experimental results

[0251] Compounds ANDD-1, ANDD-6, ANDD-7 and ANDD-14 did not cause any significant increase in colony reversion at all tested doses, whether in the experimental system without S9 or with S9, and all tested compounds were negative in the Ames test (contaminant mutagenicity test), indicating that the compounds of the present invention are non-mutagenic and have good safety.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, X is selected from NH or a single bond; T1 is selected from CH or N; T2 is selected from CH or N; R1 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 alkyl; R2 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 alkyl; m is selected from 1 or 2; n is selected from 0, 1 or 2.

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

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein: Structural unit Selected from: Any one of .

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

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, characterized in that: When R1 or R2 is halogen, the halogen is selected from any one of fluorine, chlorine or bromine; Or, when R1 or R2 is C 1-6 When the C 1-6 The alkyl group is selected from any one of methyl, ethyl, n-propyl or isopropyl; Or, when R1 or R2 is C 1-6 When the C 1-6 The alkoxy group is selected from any one of methoxy, ethoxy, n-propoxy or isopropoxy; Or, when R1 or R2 is a halogen-substituted C 1-6 When it is alkyl, the halogen is selected from any one of fluorine, chlorine or bromine; or, when R1 or R2 is a halogen-substituted C 1-6 When the C 1-6 The alkyl group is selected from any one of methyl, ethyl, n-propyl or isopropyl.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, characterized in that: When R1 or R2 is halogen substituted C 1-6 When alkyl, the halogen-substituted C 1-6 The alkyl group is selected from any one of -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2 or -CF2CF3.

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, characterized in that: When R1 or R2 is halogen substituted C 1-6 When alkyl, the halogen-substituted C 1-6 The alkyl group is selected from -CF3.

8. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from hydrogen, fluorine, chlorine, nitro, methyl, methoxy or -CF3; Or, R2 is selected from hydrogen, fluorine, chlorine, nitro, methyl, methoxy or -CF3.

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

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

11. A compound represented by formula (II), in, X is selected from NH or a single bond; T1 is selected from CH or N; T2 is selected from CH or N; R1 is selected from hydrogen, halogen, hydroxy, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 Alkyl; R2 is selected from hydrogen, halogen, hydroxyl, nitro, C 1-6 Alkyl, C 1-6 Alkoxy or halogen substituted C 1-6 Alkyl; R3 is C 1-6 alkyl; m is selected from 1 or 2; n is selected from 0, 1 or 2; And the compound represented by formula (II) is not:

12. The compound according to claim 11, which is selected from: And the compound represented by formula (II-1) is not:

13. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

14. Use of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof or the composition according to claim 13 in the preparation of a medicament for treating HDAC6-related diseases.

15. The use according to claim 14, characterized in that The HDAC6-related disease is cancer, neuropsychiatric disease or autoimmune disease; the neuropsychiatric disease is neurodegenerative disease, mental disease, peripheral neuropathy, and Its combination.

16. The use according to claim 15, characterized in that The cancer is 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, gestational trophoblastic tumor, glioblastoma, myeloma, neuroblastic CNS tumor, monocytic leukemia, B cell leukemia, T cell leukemia, B cell lymphoma, T cell lymphoma and mast cell tumor, and combinations thereof; Or, the autoimmune disease is an inflammatory disease, a condition treated by immunomodulation; the condition treated by immunomodulation is multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergy, asthma, inflammatory bowel disease.

17. The use according to claim 15, characterized in that The neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, and Rett's syndrome; Or, the mental illness is schizophrenia, depression, bipolar disorder, anxiety disorder; Or, the peripheral neuropathy is Charcot-Marie-Tooth disease or giant axial neuropathy.

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

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