HDAC Inhibitors, Compositions and Their Applications

Abstract

The present invention discloses a class of HDAC inhibitors and their applications, belonging to the field of medicinal chemistry. The HDAC inhibitor is a compound based on a flavone or flavone mimetic skeleton represented by any one of the general formulas (I)-(III), or a pharmaceutically acceptable salt, deuterated compound or optical isomer thereof. The present invention also discloses a pharmaceutical composition comprising the HDAC inhibitor. The HDAC inhibitor or the HDAC inhibitor composition has HDAC inhibitory activity. Pharmacodynamic experiments show that it can be used to prepare drugs for the treatment and / or prevention of tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease, thereby providing new ideas for the treatment and / or prevention of diseases such as tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a class of HDAC inhibitors, compositions and their applications. Background Art

[0002] Histone deacetylases (HDACs) are important drug targets in epigenetics. The abnormal expression of this enzyme in a variety of malignant tumors can affect the chromatin state by catalyzing the deacetylation of nucleosomal histones, thereby downregulating the expression of tumor suppressor genes and mediating the occurrence and development of tumors; histone deacetylase inhibitors (HDACis) can play an anti-tumor role by intervening in the above epigenetic process. Currently approved HDACis are mainly broad-spectrum inhibitors, including Vorinostat (SAHA), Belinostat, Panobinostat, and Romidespin and Chidamide that inhibit several subtypes simultaneously. Since the above inhibitors act on multiple HDAC subtypes at the same time, they all have side effects such as nausea, vomiting, myelosuppression and QT interval prolongation; at the same time, due to the narrow safety window, it limits their application in the treatment of solid tumors. Therefore, it is urgent to discover HDAC inhibitors with better subtype selectivity to improve the safety of such drugs and obtain the efficacy of solid tumors.

[0003] HDACs mainly include 4 subfamilies, namely classes I, II, III and IV: classes I (HDAC1, 2, 3, 8), II (HDAC4, 5, 6, 7, 9, 10) and class IV HDAC (HDAC11) are all Zn 2+ -dependent metalloenzymes. The current research and development of HDACis mainly targets Zn 2+ -dependent HDAC. Among the many subtypes of HDACs, class I HDACs are closely related to the occurrence and development of tumors and are widely present in various human organs. Studies have confirmed that the HDAC1 subtype in class I HDACs is highly expressed in a variety of malignant tumors, such as endometrial cancer, lung cancer, prostate cancer, breast cancer, colon cancer, pancreatic cancer, blood cancer, etc. Selective inhibition of class I HDACs can not only produce a significant inhibitory effect on tumor cell proliferation, but also obtain a wider safety window than broad-spectrum inhibitors such as SAHA and Panobinostat.

[0004] Among class II HDACs, the HDAC6 subtype has unique structure and substrate specificity, and can mediate the deacetylation of non-histones. Alterations in its expression and function are closely related to various diseases. Research has shown that abnormal expression of HDAC6 can promote cell carcinogenesis and the growth, proliferation, and angiogenesis of tumor cells by regulating signaling pathways such as Ras and EGFR. In addition, its high expression can also enhance the invasion and metastasis of tumor cells, while inhibiting HDAC6 can produce anti-solid tumor efficacy. Meanwhile, HDAC6 is also closely related to the pathological processes of neurodegenerative diseases, inflammation, autoimmune diseases, etc. Gene knockout experiments have confirmed that experimental animals lacking HDAC6 have no obvious phenotypic abnormalities, suggesting that selective inhibition of HDAC6 has excellent safety. In addition to directly intervening in the life activities of tumor cells, selective HDAC6 inhibitors (Histonedeacetylase 6 inhibitors, HDAC6is) can also play an immunotherapeutic role by reducing the phosphorylation level of STAT3 and downregulating the expression of Programmed Death-Ligand 1 (PD-L1), so it can provide more options for tumor immunotherapy. Given the above advantages, subtype-selective HDAC6is are the key direction for the development of new drugs targeting HDACs at present. However, no drugs of this type of inhibitor have been marketed; HDAC6is such as Ricolinostat, Citarinostat, and KA2507 are in clinical research, but their selectivity for HDAC6 is insufficient, and there are still problems with the safety window. Therefore, the discovery of highly selective HDAC6is has important clinical application value.

[0005] Flavonoid natural products and their mimetic skeletons have various pharmacological activities such as antioxidant, antibacterial, and antitumor. So far, there is no research report on the discovery of HDAC inhibitors based on flavonoids or flavonoid mimetic skeletons. The present invention is committed to elucidating a class of HDAC inhibitors based on flavonoids or flavonoid mimetic skeletons and their applications. Summary of the Invention

[0006] Aiming at the problem that "the marketed HDACis have obvious side effects due to the extensive inhibition of multiple HDAC subtypes" at present, and the technical deficiency that the currently clinically studied HDAC6is have poor selectivity for HDAC6, the present invention provides a class of HDAC inhibitors based on flavonoids or flavonoid mimetic skeletons and their uses.

[0007] Multiple activity tests have shown that the compounds based on flavonoids or flavonoid mimetic skeletons provided by the present invention all have HDAC inhibitory activity, and most of these compounds can strongly inhibit HDAC1 and / or HDAC6 and exhibit excellent safety.

[0008] In some specific embodiments of the present invention, the compounds conforming to a specific structural general formula, while strongly inhibiting HDAC6, exhibit significant anti-tumor cell proliferation activity, show excellent selectivity for HDAC6, and have tumor immunotherapy activity; in some other specific embodiments of the present invention, the compounds conforming to a specific structural general formula, while strongly inhibiting HDAC1, exhibit significant anti-tumor cell proliferation activity and have excellent selectivity for HDAC1 / 2 / 3. The compounds with HDAC6 selectivity or HDAC1 / 2 / 3 selectivity also have excellent safety. In addition, pharmacodynamic experiments show that the compounds involved in the present invention can be used to prepare drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention first provides a class of HDAC inhibitors, which are compounds based on flavone or flavone-like skeletons represented by any one of the following general formulas (I)-(III) or pharmaceutically acceptable salts, deuterated compounds or optical isomers thereof:

[0011]

[0012] Wherein, W is a C6-14 aryl, C5-14 heteroaryl, C7-12 aralkyl or C6-12 heteroaralkyl group which is at least substituted by one R1; X is a C6-14 aryl, C5-14 heteroaryl, C7-12 aralkyl or C6-12 heteroaralkyl group which is at least substituted by one R2; it should be noted that when W and X are substituted by two or more R1 and R2 respectively, R1 or R2 at different substitution positions may be the same or different;

[0013] Y is

[0014] Ring A is a benzene ring, pyridine or thiophene which is at least substituted by one R5; it should be noted that when ring A is substituted by two or more R5, R5 at different positions may be the same or different;

[0015] R1 and R2 are each independently selected from hydrogen, halogen, hydroxyl, cyano, carbamoyl, C1-6 alkyl or cycloalkyl, C1-6 alkoxy or C2-6 unsaturated aliphatic hydrocarbon group; R3 is selected from substituted or unsubstituted C6-14 aryl or C5-14 heteroaryl; R4 is selected from hydrogen, C1-6 alkyl or cycloalkyl; R5 is selected from hydrogen, methyl or halogen;

[0016] n1 = 2 - 4; n2 = 0 - 3; n3 = 0 - 3.

[0017] Further embodiment, the HDAC inhibitor is selected from any one of the following compounds or a pharmaceutically acceptable salt or deuterated compound thereof:

[0018] It is understood that the above compounds are only for illustration to make the technical solutions of the present invention clearer. The HDAC inhibitors in the present invention include more than just the above several kinds.

[0019] Further embodiment, when the HDAC inhibitor has a specific structure, it is a selective HDAC6 inhibitor. In some specific embodiments of the present invention, the compound shown in formula (I) based on a flavone or flavone-like skeleton or a pharmaceutically acceptable salt or deuterated compound thereof can be used as a selective HDAC6 inhibitor:

[0020]

[0021] In formula (I), W is a C6-14 aryl, C5-14 heteroaryl, C7-12 aralkyl or C6-12 heteroaralkyl group at least substituted by one R1; ring A is a benzene ring, pyridine or thiophene at least substituted by 1 R5; it is understood that when W is substituted by two or more R1s, the R1s at different substitution positions may be the same or different; when ring A is substituted by two or more R5s, the R5s at different positions may be the same or different from each other.

[0022] R1 is selected from hydrogen, halogen, hydroxyl, cyano, carbamoyl, C1-6 alkyl or cycloalkyl, C1-6 alkoxy or C2-6 unsaturated aliphatic hydrocarbon group; R3 is selected from substituted or unsubstituted C6-14 aryl or C5-14 heteroaryl; R5 is selected from hydrogen, methyl, or halogen; n3 = 0 - 3.

[0023] Further embodiment, when the HDAC inhibitor has some other specific structures, it is selective for HDAC1 and / or HDAC2 and / or HDAC3. In some other specific embodiments of the present invention, the HDAC inhibitor selective for HDAC1 and / or HDAC2 and / or HDAC3 is a compound shown in formula (Ⅲ) based on a flavone or flavone-like skeleton or a pharmaceutically acceptable salt, deuterated compound or optical isomer thereof:

[0024]

[0025] In formula (Ⅲ), Y is

[0026] Ring A is selected from a benzene ring, pyridine or thiophene at least substituted by one R5; it is understood that when ring A is substituted by two or more R5s, the R5s at different positions may be the same or different from each other.

[0027] R3 is selected from substituted or unsubstituted C6-14 aryl or C5-14 heteroaryl; R5 is selected from hydrogen, methyl or halogen; n1 = 2-4; n2 = 0-3.

[0028] The present invention further provides the use of the HDAC inhibitor as described above in the preparation of HDAC inhibitor drugs.

[0029] The present invention further provides the use of the HDAC inhibitor as described above in the preparation of drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

[0030] The present invention further provides an HDAC inhibitor composition, which comprises the HDAC inhibitor as described above and further comprises at least one pharmaceutical carrier or excipient.

[0031] In a further embodiment, the HDAC inhibitor composition further comprises at least one therapeutic agent.

[0032] In a further embodiment, the dosage form of the HDAC inhibitor composition is any clinically or pharmaceutically acceptable dosage form.

[0033] The present invention further provides the use of the HDAC inhibitor composition as described above in the preparation of drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

[0034] In some specific embodiments of the present invention, the dosage of the HDAC inhibitor in the present invention is 1 mg - 1000 mg / day. It can be understood that the specific dosage is not limited to this range and can be adjusted according to the severity of the disease or the type of dosage form.

[0035] In addition, unless otherwise defined, the scientific terms mentioned in the present invention have the same meaning as generally understood by those skilled in the art to which the subject matter claimed in the present invention belongs. The following scientific terms are described.

[0036] Among them, the flavone skeleton refers to a parent nucleus having a 2-phenylchromone structure, and its benzene ring can be substituted by substituents; the flavone mimetic skeleton refers to a parent nucleus obtained by replacing one or two benzene rings in the flavone skeleton with C5-C6 aryl heterocycles, and its benzene ring or aryl heterocycle can be substituted by substituents.

[0037] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0038] "C6-14 aryl" refers to a fully carbon single ring containing 6-14 carbon atoms and having a completely conjugated π electron system. Specific examples that can be mentioned include, but are not limited to: benzene ring, naphthalene ring, anthracene ring, etc.

[0039] "C5-14 heteroaryl" refers to a non-all-carbon monocyclic or fused polycyclic group having 5 to 14 ring atoms and a completely conjugated π-electron system. Specific examples that may be mentioned include, but are not limited to: pyridine, imidazole, thiophene, furan, thiazole, purine, indole, azaindole, etc.

[0040] "C6-12 heteroaralkyl" refers to a group in which an alkyl group is attached to a heteroaryl group containing 6 to 12 carbon atoms.

[0041] "C7-12 aralkyl" refers to a group in which an alkyl group is attached to an aryl group containing 7 to 12 carbon atoms.

[0042] "C1-3 alkyl" refers to an alkyl group having 1 to 3 carbon atoms;

[0043] "C2-6 unsaturated aliphatic hydrocarbon group" refers to a straight-chain or branched alkenyl or alkynyl group containing 2 to 6 carbon atoms with a double bond or a triple bond. Specific examples that may be mentioned include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, ethynyl, etc.

[0044] The compounds of the present invention or their pharmaceutically acceptable salts or deuterated compounds have the same efficacy, wherein the pharmaceutically acceptable salts refer to salts of formula (I), (II) or (III). Specific examples of the types of salts that may be mentioned include, but are not limited to: alkali metal salts, alkaline earth metal salts, other metal salts, inorganic base salts, organic base salts, inorganic acid salts, organic acid salts, lower alkanesulfonates, arylsulfonates, amino acid salts. Optical isomers refer to substances with the same molecular structure, similar physical and chemical properties, but different optical rotation properties. Deuterated compounds refer to compounds in which hydrogen in an organic compound molecule is replaced by its isotope deuterium (D).

[0045] The "pharmaceutical carrier" mentioned refers to a conventional pharmaceutical carrier in the pharmaceutical field, including conventional diluents, excipients (such as water, etc.), fillers (such as starch, etc.), binders (such as cellulose derivatives, gelatin, etc.), wetting agents (such as glycerol, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption promoters (such as quaternary ammonium compounds, etc.), surfactants (such as cetyl alcohol, etc.), adsorption carriers (such as kaolin and saponite clay, etc.), lubricants (such as talc powder, etc.). When necessary, flavoring agents, sweetening agents, etc. may also be added.

[0046] The "therapeutic agent" mentioned above refers to a therapeutic agent that can be formulated with the HDAC inhibitor, including but not limited to mitotic inhibitors (such as vinblastine, vindesine), microtubule depolymerizing inhibitors (such as paclitaxel), biologic alkylating agents (such as cyclophosphamide), antimetabolites (such as 5-fluorouracil, tegafur, methotrexate), antitumor antibiotics (such as doxorubicin, mitomycin), enzymes (such as asparaginase), topoisomerase inhibitors (such as etoposide and camptothecin), biologic response modifiers (such as interferon), proteasome inhibitors (such as bortezomib), etc.

[0047] The "any pharmaceutically acceptable dosage form" mentioned above is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal, nasal, topical (including buccal, sublingual, transdermal or inhalation administration), vaginal or parenteral (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection) routes. These preparations can be prepared by any method known in the pharmaceutical field. For example, by mixing the active ingredient with a carrier or excipient.

[0048] The tumors include but are not limited to breast cancer, sarcoma, lung cancer, prostate cancer, colon cancer, rectal cancer, kidney cancer, pancreatic cancer, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, liver cancer, ovarian cancer, uterine cancer, endometrial cancer, gastric cancer, bladder cancer, gastrointestinal stromal tumor, nasopharyngeal cancer, leukemia, lymphoma, multiple myeloma.

[0049] The autoimmune diseases mentioned above are a class of diseases caused by the body's immune system malfunctioning and reacting against its own antigens, resulting in damage to its own tissues, organs, and systems, including autoimmune liver diseases, autoimmune thyroid diseases, autoimmune hemolytic anemia, systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, multiple sclerosis, myasthenia gravis, scleroderma, mixed connective tissue disease, and many other diseases.

[0050] The inflammations include but are not limited to rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, pneumonia, gastroenteritis, hepatitis, tonsillitis, heart inflammation, ulcerative colitis, etc.

[0051] The present invention has the following beneficial effects:

[0052] The HDAC inhibitor of the present invention is a compound based on a flavone or flavone mimetic skeleton or a pharmaceutically acceptable salt, deuterated compound or optical isomer thereof. Flavones and their analogs have various pharmacological activities such as antioxidant, antibacterial and antitumor activities, and also have potential application value in the treatment of tumors, autoimmune diseases and neurodegenerative diseases. The present invention develops an HDAC inhibitor based on a flavone or its mimetic skeleton starting from this point.

[0053] Multiple activity tests have shown that the compounds based on flavone or flavone-like skeletons provided by the present invention all have HDAC inhibitory activity, and most of the compounds can strongly inhibit HDAC1 and / or HDAC6. Compounds with a specific structural formula, while strongly inhibiting HDAC6, exhibit significant anti-tumor cell proliferation activity, show excellent selectivity for HDAC6, and have tumor immunotherapy activity; there are also compounds with a specific structural formula that, while strongly inhibiting HDAC1, exhibit significant anti-tumor cell proliferation activity and have excellent selectivity for HDAC1 / 2 / 3. Compounds with HDAC6 selectivity or HDAC1 / 2 / 3 selectivity also have excellent safety. Pharmacodynamic experiments have shown that the HDAC inhibitors involved in the present invention can be used to prepare drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome, or Alzheimer's disease, thus providing new therapies for tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome, or Alzheimer's disease. Description of the Drawings

[0054] Figure 1 Immunoblot analysis of the regulatory effects of Compound 15 on P-STAT3 (Y705), Ac-tubulin (Lys40), and PD-L1 in MDA-MB-231 cells in the examples of the present invention;

[0055] Figure 2 Quantitative analysis of the regulatory levels of Ac-tubulin (Lys40), P-STAT3 (Y705), and PD-L1 in MDA-MB-231 cells by Compound 15 in the examples of the present invention. Detailed Description of the Embodiments

[0056] The embodiments of the present invention are described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0057] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. Additionally, unless otherwise specified, the methods without specific conditions or steps recorded are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0058] There are three different routes for the preparation of the HDAC inhibitors in the present invention. Taking Compound 1 (Route 1), Compound 23 (Route 2), and Compound 25 (Route 3) as examples below:

[0059] Route 1:

[0060]

[0061] In Route 1, the reactants and reaction conditions involved are as follows:

[0062] a is sodium hydroxide (NaOH), methanol (MeOH), 75 °C; NaOH (0.5 N), 30% hydrogen peroxide (30% H2O2), from 0 °C to room temperature;

[0063] b is methyl 4-(chloromethyl)benzoate, potassium carbonate (K2CO3), acetonitrile (CH3CN), under N2 protection, 80 °C;

[0064] c is lithium hydroxide (LiOH), tetrahydrofuran (THF), MeOH, water (H2O), room temperature; dilute hydrochloric acid (1 N);

[0065] d is hydroxylamine protected with THP group (NH2OTHP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N-methylmorpholine (NMM), N,N-dimethylformamide (DMF), room temperature;

[0066] e is trifluoroacetic acid (TFA), dichloromethane (DCM), from 0 °C to room temperature.

[0067] Route 2:

[0068]

[0069] In Route 2, the reactants and reaction conditions involved are as follows:

[0070] a is K2CO3, CH3CN, under N2 protection, 80 °C;

[0071] b is LiOH, THF, MeOH, H2O, room temperature; dilute hydrochloric acid (1 N);

[0072] c is methyl 4-(aminomethyl)benzoate hydrochloride, EDCI, HOBT, triethylamine (TEA), DMF, room temperature;

[0073] d is NH2OTHP, EDCI, HOBT, NMM, DMF, room temperature;

[0074] e is TFA, DCM, from 0 °C to room temperature.

[0075] Route 3:

[0076]

[0077] In Route 3, the reactants and reaction conditions involved are as follows:

[0078] a is N-BOC-4-(((methylsulfonyl)oxy)methyl)piperidine, K2CO3, DMF, 60 °C;

[0079] b is TFA, DCM, 0 °C to room temperature;

[0080] c is ethyl 2-chloropyrimidine-5-carboxylate, cesium carbonate (Cs2CO3), potassium iodide (KI), DMF, under N2 protection, 80 °C;

[0081] d is LiOH, THF, MeOH, H2O, room temperature; dilute hydrochloric acid (1N);

[0082] e is NH2OTHP, EDCI, HOBT, NMM, DMF, room temperature.

[0083] The preparation routes of other compounds are similar to the above, and all can be prepared with reference to the above routes. Among them, compounds 1-22 are prepared according to Route 1; compound 24 is prepared according to Route 2; compounds 26-28 are prepared according to Route 3.

[0084] Example 1 Synthesis of N-hydroxy-4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)benzamide (Compound 1)

[0085] (1) Synthesis of 3-hydroxy-2-phenyl-chromen-4-one (31)

[0086]

[0087] In a round-bottom flask, 2-hydroxyacetophenone (5.00 g, 36.7 mmol, 1.0 eq), benzaldehyde (3.90 g, 36.7 mmol, 1.0 eq), methanol (50.0 mL), and NaOH (4.40 g, 110 mmol, 3.0 eq) were added successively, and the reaction was carried out at 75 °C for 5 h. After the reaction solution was cooled, NaOH (0.5 N, 150 mL) and 30% H2O2 (3.75 g, 184 mmol, 5.0 eq) were added dropwise successively under an ice bath. After the addition was complete, the reaction was carried out at room temperature for 2 h. The reaction solution was poured into ice water, and a large amount of yellow solid was precipitated. The solid was filtered by suction, and the filter cake was recrystallized from ethanol to obtain 3.0 g of yellow solid.

[0088] (2) Synthesis of methyl 4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)benzoate (32)

[0089]

[0090] In a round-bottom flask, intermediate 31 (200 mg, 0.840 mmol, 1.0 eq), methyl 4-(chloromethyl)benzoate (187 mg, 1.01 mmol, 1.2 eq), K2CO3 (243 mg, 1.76 mmol, 2.1 eq) and CH3CN (4.0 mL) were successively added, and the reaction was carried out at 80 °C under N2 protection. After the reaction was completed, water was added to the reaction flask, and the mixture was allowed to stand to precipitate a solid, which was then filtered by suction. The filter cake was separated by silica gel column chromatography to obtain 150 mg of an off-white solid.

[0091] (3) Synthesis of 4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)benzoic acid (33)

[0092]

[0093] Intermediate 32 (140 mg, 0.362 mmol, 1.0 eq) and a mixed solvent of THF / MeOH / H2O (V / V / V = 3:1:1, 5.0 mL) were added to a round-bottom flask. Under an ice bath, LiOH (43.3 mg, 1.81 mmol, 5.0 eq) was added, and the mixture was transferred to room temperature for reaction for 3 h. The organic solvent was removed by distillation under reduced pressure. Under an ice bath, the pH was adjusted to 4 - 5 with freshly prepared dilute hydrochloric acid (1 N), and 120 mg of an off-white solid was obtained by suction filtration and directly used for the next step.

[0094] (4) Synthesis of 4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (34)

[0095]

[0096] Intermediate 33 (100 mg, 0.269 mmol, 1.0 eq), EDCI (56.7 mg, 0.296 mmol, 1.1 eq), HOBT (40.0 mg, 0.296 mmol, 1.1 eq) and DMF (2.0 mL) were successively added to a round-bottom flask, and the mixture was stirred at room temperature for 0.5 h. Subsequently, NMM (90.7 μL, 0.807 mmol, 3.0 eq) and NH2OTHP (34.7 mg, 0.296 mmol, 1.1 eq) were added, and the mixture was continuously stirred at room temperature for 3 h. The mixture was washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography to obtain 80 mg of an off-white solid.

[0097] (5) Synthesis of N-hydroxy-4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)benzamide (1)

[0098]

[0099] In a round-bottom flask, intermediate 34 (70 mg, 0.149 mmol, 1.0 eq), DCM (2.0 mL) were successively added. TFA (0.5 mL) was added dropwise under an ice bath, and the reaction was carried out at room temperature. After the reaction was completed, the organic solvent was removed by distillation under reduced pressure. Saturated sodium bicarbonate solution was added dropwise under an ice bath to adjust the pH to 7 - 8. Filtration was performed by suction, and the filter cake was purified by silica gel column chromatography to obtain 40 mg of a white solid.

[0100] The results of nuclear magnetic resonance were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.01 (s, 1H), 8.15 (d, J = 7.6 Hz, 1H), 8.02–7.96 (m, 2H), 7.89–7.81 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 7.6 Hz, 2H), 7.58–7.54 (m, 3H), 7.53–7.51 (m, 1H), 7.39 (d, J = 8.0 Hz, 2H), 5.14 (s, 2H); The results of electrospray ionization high-resolution mass spectrometry (ESI-HRMS) were: 388.1173 [M + H] + , which was compound 1.

[0101] Example 2 Synthesis of N-Hydroxy-4-((2-((oxo-2-phenyl-4H-chromen-3-yl)oxy)acetamido)methyl)benzamide (Compound 23)

[0102] (1) Synthesis of Ethyl 2-((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)acetate (35)

[0103]

[0104] In a round-bottom flask, intermediate 31 (1.00 g, 4.20 mmol, 1.0 eq), ethyl bromoacetate (557 μL, 5.04 mmol, 1.2 eq), K2CO3 (1.22 g, 8.82 mmol, 2.1 eq), CH3CN (10.0 mL) were successively added. The reaction was carried out at 80 °C under N2 protection. Water was added to the reaction flask, and after standing, filtration was performed by suction. The filter cake was purified by silica gel column chromatography to obtain 1.1 g of a white solid. Based on Route 2

[0105] (2) Synthesis of 2-((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)acetic Acid (36)

[0106]

[0107] In a round-bottom flask, intermediate 35 (1.00 g, 3.09 mmol, 1.0 eq) and a mixed solvent of THF / MeOH / H2O (V / V / V = 3:1:1, 10.0 mL) were successively added. LiOH (371 mg, 15.5 mmol, 5.0 eq) was added under an ice bath, and the reaction was carried out at room temperature for 3 h. The organic solvent was removed by distillation under reduced pressure. The pH was adjusted to 4 - 5 with freshly prepared dilute hydrochloric acid (1N) under an ice bath, and 800 mg of off-white solid was obtained by suction filtration and directly used for the next step of the reaction.

[0108] (3) Synthesis of methyl 4 - ((2 - ((oxo - 2 - phenyl - 4H - chromen - 3 - yl)oxy)acetamido)methyl)benzoate (37)

[0109]

[0110] In a round-bottom flask, intermediate 36 (200 mg, 0.675 mmol, 1.0 eq), EDCI (142 mg, 0.743 mmol, 1.1 eq), HOBT (100 mg, 0.743 mmol, 1.1 eq) and DMF (4.0 mL) were successively added. After stirring at room temperature for 0.5 h, methyl 4 - (aminomethyl)benzoate hydrochloride (163 mg, 0.810 mmol, 1.2 eq) and TEA (282 μL, 2.03 mmol, 3.0 eq) were successively added, and the reaction was continued at room temperature for 2 h. The mixture was washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 180 mg of off-white solid was obtained by silica gel column chromatography.

[0111] (4) Synthesis of 4 - ((2 - ((oxo - 2 - phenyl - 4H - chromen - 3 - yl)oxy)acetamido)methyl)benzoic acid (38)

[0112]

[0113] In a round-bottom flask, intermediate 37 (150 mg, 0.338 mmol, 1.0 eq) and a mixed solvent of THF / MeOH / H2O (V / V / V = 3:1:1, 5.0 mL) were successively added. LiOH (40.5 mg, 1.69 mmol, 5.0 eq) was added under an ice bath, and the reaction mixture was transferred to room temperature and reacted for 3 h. The organic solvent was removed by distillation under reduced pressure. The pH was adjusted to 4 - 5 with dilute hydrochloric acid (1N) under an ice bath, and 120 mg of off-white solid was obtained by suction filtration and directly used for the next step.

[0114] (5) Synthesis of 4 - ((2 - ((oxo - 2 - phenyl - 4H - chromen - 3 - yl)oxy)acetamido)methyl)-N - ((tetrahydro - 2H - pyran - 2 - yl)oxy)benzamide (39)

[0115]

[0116] In a round-bottom flask, intermediate 38 (100 mg, 0.233 mmol, 1.0 eq), EDCI (49.1 mg, 0.256 mmol, 1.1 eq), HOBT (34.6 mg, 0.256 mmol, 1.1 eq) and DMF (2.0 mL) were successively added, and the mixture was stirred at room temperature for 0.5 h. Subsequently, NMM (78.6 μL, 0.699 mmol, 3.0 eq) and NH2OTHP (30.0 mg, 0.256 mmol, 1.1 eq) were added, and stirring was continued at room temperature for 2 h. The mixture was washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Column chromatography on silica gel gave 80 mg of an off-white solid.

[0117] (6) Synthesis of N-hydroxy-4-((2-((oxo-2-phenyl-4H-chromen-3-yl)oxy)acetamido)methyl)benzamide (23)

[0118]

[0119] The intermediate 34 in step (5) of Example 1 was replaced with intermediate 39 to prepare an off-white solid compound 23.

[0120] The nuclear magnetic resonance results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.00 (s, 1H), 8.79 (t, J = 6.0 Hz, 1H), 8.21–8.05 (m, 3H), 7.92–7.82 (m, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.64–7.49 (m, 4H), 7.34 (d, J = 8.0 Hz, 2H), 4.55 (s, 2H), 4.39 (d, J = 6.0 Hz, 2H); ESI-HRMS: 445.1400 [M+H] + , which was compound 23.

[0121] Example 3 Synthesis of N-hydroxy-2-(4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidin-1-yl)pyrimidine-5-carboxamide (Compound 25)

[0122] (1) Synthesis of tert-butyl 4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidine-1-carboxylate (40)

[0123]

[0124] Add intermediate 31 (370 mg, 1.55 mmol, 1.0 eq), N-BOC-4-(((methylsulfonyl)oxy)methyl)piperidine (502 mg, 1.71 mmol, 1.1 eq), K2CO3 (428 mg, 3.10 mmol, 2.0 eq) and DMF (8.0 mL) to a round-bottom flask and react at 60 °C for 2 h. Add water to the reaction flask, filter by suction, and the filter cake is subjected to silica gel column chromatography to obtain 410 mg of an off-white solid.

[0125] (2) 2-Phenyl-3-(piperidin-4-yloxy)-4H-chromen-4-one (41)

[0126]

[0127] Add intermediate 40 (400 mg, 0.920 mmol, 1.0 eq) and DCM (8.0 mL) to a round-bottom flask, add TFA (2.0 mL) dropwise under an ice bath, transfer to room temperature and react for 5 h. Concentrate under reduced pressure to remove the organic solvent, wash successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extract with DCM, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 220 mg of a yellow oil, which is directly used for the next step.

[0128] (3) Synthesis of ethyl 2-(4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (42)

[0129]

[0130] Add intermediate 41 (200 mg, 0.596 mmol, 1.0 eq), ethyl 2-chloropyrimidine-5-carboxylate (122 mg, 0.656 mmol, 1.1 eq), Cs2CO3 (407 mg, 1.25 mmol, 2.1 eq), KI (119 mg, 0.715 mmol, 1.2 eq) and DMF (4.0 mL) to a round-bottom flask in sequence, react at 80 °C for 4 h under N2 protection. Wash successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extract with DCM, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and subject it to silica gel column chromatography to obtain 180 mg of an off-white solid.

[0131] (4) Synthesis of 2-(4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (43)

[0132]

[0133] In a round-bottom flask, intermediate 42 (150 mg, 0.309 mmol, 1.0 eq) and a mixed solvent of THF / MeOH / H2O (V / V / V = 3:1:1, 5.0 mL) were successively added. Under an ice bath, LiOH (37.1 mg, 1.55 mmol, 5.0 eq) was added, and the mixture was transferred to room temperature and reacted for 3 h. The organic solvent was removed by distillation under reduced pressure. Under an ice bath, the pH was adjusted to 4 - 5 with dilute hydrochloric acid (1 N), and a off-white solid (124 mg) was obtained by filtration and directly used for the next step.

[0134] (5) Synthesis of 2-4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidin-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (44)

[0135]

[0136] In a round-bottom flask, intermediate 43 (100 mg, 0.219 mmol, 1.0 eq), EDCI (46.2 mg, 0.241 mmol, 1.1 eq), HOBT (32.6 mg, 0.241 mmol, 1.1 eq) and DMF (2.0 mL) were successively added, and the mixture was stirred at room temperature for 0.5 h. Subsequently, NMM (73.9 μL, 0.657 mmol, 3.0 eq) and NH2OTHP (28.2 mg, 0.241 mmol, 1.1 eq) were added, and the stirring was continued at room temperature for 3 h. After the reaction was completed, the mixture was washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. A off-white solid (80 mg) was obtained by silica gel column chromatography.

[0137] (6) Synthesis of N-hydroxy-2-4-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)piperidin-1-yl)pyrimidine-5-carboxamide (25)

[0138]

[0139] The intermediate 34 in step (5) of Example 1 was replaced with intermediate 44 to prepare a off-white solid compound 25.

[0140] The nuclear magnetic resonance results were 11H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.97 (s, 1H), 8.64 (s, 2H), 8.10 (dd, J = 8.0, 1.6 Hz, 1H), 8.01 (d, J = 3.6 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.88–7.79 (m, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.60–7.54 (m, 3H), 7.53–7.47 (m, 1H), 4.69–4.63 (m, 1H), 4.62–4.59 (m, 1H), 3.88 (d, J = 6.4 Hz, 2H), 2.99–2.81 (m, 2H), 2.06–1.89 (m, 1H), 1.74–1.66 (m, 2H), 1.20–1.05 (m, 2H); ESI-HRMS: 473.1816 [M+H] + , namely, compound 25 was obtained.

[0141] Example 4 Synthesis of N-Hydroxy-5-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)thiophene-2-carboxamide (Compound 2)

[0142]

[0143] Compound 2 was prepared according to Example 1, except that: in step (2), methyl 4-(chloromethyl)benzoate was replaced with methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a off-white solid was obtained by the same method.

[0144] The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.11 (s, 1H), 8.15 (dd, J = 8.0, 1.6 Hz, 1H), 8.04–7.93 (m, 2H), 7.90–7.81 (m, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.61–7.49 (m, 4H), 7.42–7.35 (m, 1H), 7.02 (d, J = 4.0 Hz, 1H), 5.32 (s, 2H); ESI-HRMS: 394.0746 [M+H] + , which was compound 2.

[0145] Example 5 Synthesis of N-Hydroxy-5-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)isoxazole-3-carboxamide (Compound 3)

[0146]

[0147] Compound 3 was prepared according to Example 1, except that: in step (2), methyl 4-(chloromethyl)benzoate was replaced with ethyl 5-(bromomethyl)isoxazole-3-carboxylate, and then a white solid was obtained by the same method.

[0148] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.49(s,1H),9.37(s,1H),8.15(dd,J=8.0,1.6Hz,1H),7.96(d,J=1.6Hz,1H),7.94(d,J=2.4Hz,1H),7.91–7.82(m,1H),7.78(d,J=8.4Hz,1H),7.57–7.49(m,4H),6.72(s,1H),5.34(s,2H);ESI-HRMS:379.0922[M+H] + , which was Compound 3.

[0149] Example 6 Synthesis of 4-(((2-(4-chlorophenyl)-4-oxo-4H-chromen-3-yl)oxy)methyl)-N-hydroxybenzamide (Compound 4)

[0150]

[0151] Compound 4 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with 4-chlorobenzaldehyde, and then a white solid was obtained by the same method.

[0152] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.20(s,1H),9.03(s,1H),8.14(dd,J=8.0,1.6Hz,1H),8.02(d,J=8.4Hz,2H),7.89–7.82(m,1H),7.76(d,J=8.4Hz,1H),7.68(d,J=8.0Hz,2H),7.62(d,J=8.4Hz,2H),7.56–7.49(m,1H),7.40(d,J=8.0Hz,2H),5.15(s,2H);ESI-HRMS:422.0786[M+H] + , which was Compound 4.

[0153] Example 7 Synthesis of 5-(((2-(4-chlorophenyl)-4-oxo-4H-chromen-3-yl)oxy)methyl)-N-hydroxythiophene-2-carboxamide (Compound 5)

[0154]

[0155] Compound 5 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with 4-chlorobenzaldehyde, and in step (2), methyl 4-(chloromethyl)benzoate was replaced with methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a white solid was obtained by the same method.

[0156] The results of nuclear magnetic resonance were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 9.10 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.86 (t, J = 7.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.47–7.38 (m, 1H), 7.03 (d, J = 3.6 Hz, 1H), 5.34 (s, 2H); ESI-HRMS: 428.0534 [M+H] + , which was Compound 5.

[0157] Synthesis of N-hydroxy-4-(((4-oxo-2-(thiophen-2-yl)-4H-chromen-3-yl)oxy)methyl)benzamide (Compound 6) in Example 8

[0158]

[0159] Compound 6 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with thiophene-2-carbaldehyde, and then a white solid was obtained by the same method.

[0160] The results of nuclear magnetic resonance were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 9.21 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 3.6 Hz, 1H), 8.14 (d, J = 4.8 Hz, 1H), 8.01 (t, J = 7.6 Hz, 1H), 7.96–7.92 (m, 3H), 7.80 (d, J = 8.0 Hz, 2H), 7.68 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 4.4 Hz, 1H), 5.47 (s, 2H); ESI-HRMS: 394.0736 [M+H] + , which was Compound 6.

[0161] Synthesis of 4-(((2-(5-chlorothiophen-2-yl)-4-oxo-4H-chromen-3-yl)oxy)methyl)-N-hydroxybenzamide (Compound 7) in Example 9

[0162]

[0163] Compound 7 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with 5-chlorothiophene-2-carboxaldehyde, and then a white solid was obtained by the same method.

[0164] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.23(s,1H),9.04(s,1H),8.11(dd,J=8.0,1.6Hz,1H),7.88(d,J=4.4Hz,1H),7.87–7.81(m,1H),7.80–7.74(m,3H),7.61(d,J=8.0Hz,2H),7.55–7.47(m,1H),7.36(d,J=4.4Hz,1H),5.33(s,2H); ESI-HRMS:428.0356[M+H] + , which was Compound 7.

[0165] Synthesis of 5-(((2-(5-chlorothiophen-2-yl)-4-oxo-4H-chromen-3-yl)oxy)methyl)-N-hydroxythiophene-2-carboxamide (Compound 8) in Example 10

[0166]

[0167] Compound 8 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with 5-chlorothiophene-2-carboxaldehyde, and in step (2), methyl 4-(chloromethyl)benzoate was replaced with methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a white solid was obtained by the same method.

[0168] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.23(s,1H),9.12(s,1H),8.11(dd,J=8.0,1.6Hz,1H),7.88(d,J=4.4Hz,1H),7.87–7.80(m,1H),7.75(d,J=8.4Hz,1H),7.51(t,J=7.6Hz,1H),7.49–7.41(m,1H),7.36(d,J=4.0Hz,1H),7.18(d,J=3.6Hz,1H),5.56(s,2H); ESI-HRMS:433.9921[M+H] + , which was Compound 8.

[0169] Synthesis of Example 11 N-Hydroxy-4-(((4-oxo-2-(pyridin-2-yl)-4H-chromen-3-yl)oxy)methyl)benzamide (Compound 9)

[0170]

[0171] Compound 9 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with pyridine-2-carbaldehyde, and during the work-up, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method.

[0172] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.04(s,1H),8.81–8.75(m,1H),8.17(dd,J=8.0,1.6Hz,1H),8.03–7.94(m,2H),7.92–7.80(m,1H),7.74(d,J=8.8Hz,1H),7.66(d,J=8.0Hz,2H),7.60–7.51(m,2H),7.37(d,J=8.0Hz,2H),5.18(s,2H);ESI-HRMS:389.1126[M+H] + , which was Compound 9.

[0173] Synthesis of Example 12 N-Hydroxy-4-(((4-oxo-2-(pyridin-3-yl)-4H-chromen-3-yl)oxy)methyl)benzamide (Compound 10)

[0174]

[0175] Compound 10 was prepared according to Example 1, except that: in step (1), benzaldehyde was replaced with pyridine-3-carbaldehyde, and during the work-up, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method.

[0176] The results of nuclear magnetic resonance were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.14 (d, J = 2.4 Hz, 1H), 8.71 (dd, J = 4.8, 1.6 Hz, 1H), 8.33 (dd, J = 8.0, 2.0 Hz, 1H), 8.26–8.07 (m, 1H), 7.90–7.83 (m, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.59–7.56 (m, 1H), 7.55–7.51 (m, 1H), 7.37 (d, J = 8.0 Hz, 2H), 5.19 (s, 2H); ESI-HRMS: 389.1131 [M+H] + , which is Compound 10.

[0177] Example 13 Synthesis of N-Hydroxy-4-(((4-oxo-2-(pyridin-4-yl)-4H-chromen-3-yl)oxy)methyl)benzamide (Compound 11)

[0178]

[0179] Compound 11 was prepared according to Example 1, with the difference that: in step (1), benzaldehyde was replaced by pyridine-4-carbaldehyde. After the post-treatment, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method.

[0180] The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.76 (d, J = 1.6 Hz, 1H), 8.75 (d, J = 1.6 Hz, 1H), 8.16 (dd, J = 8.0, 1.6 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.95 (d, J = 2.0 Hz, 1H), 7.93–7.83 (m, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.60–7.49 (m, 1H), 7.42 (d, J = 8.0 Hz, 2H), 5.22 (s, 2H); ESI-HRMS: 389.1128 [M+H] + , which is Compound 11.

[0181] Example 14 Synthesis of 4-(((6-chloro-4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)-N-hydroxybenzamide (Compound 12)

[0182]

[0183] The synthesis of Compound 12 was prepared with reference to Example 1, except that: in Step (1), 2-hydroxyacetophenone was replaced with 5-chloro-2-hydroxyacetophenone, and then a white solid was obtained by the same method.

[0184] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.15(s,1H),8.99(s,1H),8.03(d,J=2.4Hz,1H),7.96(d,J=1.6Hz,1H),7.94(d,J=2.0Hz,1H),7.90–7.81(m,1H),7.80(d,J=9.2Hz,1H),7.63(d,J=8.4Hz,2H),7.58–7.45(m,3H),7.34(d,J=8.4Hz,2H),5.09(s,2H);ESI-HRMS:422.0786[M+H] + , which was Compound 12.

[0185] Example 15 Synthesis of 5-(((6-chloro-4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)-N-hydroxythiophene-2-carboxamide (Compound 13)

[0186]

[0187] Compound 13 was prepared according to Example 1, except that: in Step (1), 2-hydroxyacetophenone was replaced with 5-chloro-2-hydroxyacetophenone, and in Step (2), methyl 4-(chloromethyl)benzoate was replaced with methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a white solid was obtained by the same method.

[0188] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.11(s,1H),8.08(d,J=2.4Hz,1H),8.00(d,J=2.0Hz,1H),7.98(d,J=2.4Hz,1H),7.94–7.85(m,1H),7.84(d,J=9.2Hz,1H),7.60–7.49(m,3H),7.42–7.35(m,1H),7.02(d,J=4.0Hz,1H),5.31(s,2H);ESI-HRMS:428.0345[M+H] + , which was Compound 13.

[0189] Synthesis of Example 16 4-(((6-Chloro-2-(5-chlorothiophen-2-yl)-4-oxo-4H-chromen-3-yl)oxy)methyl)-N-hydroxybenzamide (Compound 14)

[0190]

[0191] The synthesis of Compound 14 was prepared with reference to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 5-chloro-2-hydroxyacetophenone, and benzaldehyde was replaced with 5-chlorothiophene-2-carbaldehyde, and then a white solid was obtained by the same method.

[0192] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.23(s,1H),9.04(s,1H),8.02(d,J=2.4Hz,1H),7.92–7.85(m,2H),7.82(d,J=9.2Hz,1H),7.78(d,J=8.0Hz,2H),7.60(d,J=8.0Hz,2H),7.36(d,J=4.0Hz,1H),5.33(s,2H);ESI-HRMS:461.9968[M+H] + which was Compound 14.

[0193] Synthesis of Example 17 N-Hydroxy-6-(((4-oxo-2-phenyl-4H-pyrano[3,2-b]pyridin-3-yl)oxy)methyl)benzamide (Compound 15)

[0194]

[0195] Compound 15 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 1-(3-hydroxypyridin-2-yl)ethanone, and when post-treated, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method.

[0196] The results of nuclear magnetic resonance were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.82 (d, J = 4.4 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 8.8, 4.4 Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.60–7.57 (m, 1H), 7.56–7.55 (m, 1H), 7.54–7.51 (m, 1H), 7.39 (d, J = 8.0 Hz, 2H), 5.15 (s, 2H); ESI-HRMS: 389.1133 [M+H] + , which is Compound 15.

[0197] Synthesis of N-Hydroxy-6-(((4-oxo-2-phenyl-4H-pyrano[3,2-b]pyridin-3-yl)oxy)methyl)nicotinamide (Compound 16), Example 18

[0198]

[0199] Compound 16 was prepared according to Example 1, with the differences that: in step (1), 2-hydroxyacetophenone was replaced by 1-(3-hydroxypyridin-2-yl)ethanone, and the pH was adjusted to 7 with dilute hydrochloric acid (1N) during the post-treatment; in step (2), methyl 4-(chloromethyl)benzoate was replaced by methyl 6-(bromomethyl)nicotinate, and then a white solid was obtained by the same method.

[0200] The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 9.17 (s, 1H), 8.83 (dd, J = 4.4, 1.6 Hz, 1H), 8.80 (d, J = 2.0 Hz, 1H), 8.29 (dd, J = 8.4, 1.2 Hz, 1H), 8.07 (dd, J = 8.4, 2.4 Hz, 1H), 8.05–7.98 (m, 2H), 7.88 (dd, J = 8.8, 4.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.57–7.49 (m, 3H), 5.23 (s, 2H); ESI-HRMS: 390.1084 [M+H] + , which is Compound 16.

[0201] Synthesis of N-Hydroxy-5-(((4-oxo-2-phenyl-4H-pyrano[3,2-b]pyridin-3-yl)oxy)methyl)thiophene-2-carboxamide (Compound 17), Example 19

[0202]

[0203] Compound 17 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 1-(3-hydroxypyridin-2-yl)ethanone, and during the post-treatment, the pH was adjusted to 7 with dilute hydrochloric acid (1N). In step (2), methyl 4-(chloromethyl)benzoate was replaced with methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a white solid was obtained by the same method.

[0204] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.11(s,1H),8.83(dd,J=4.4,1.6Hz,1H),8.28(dd,J=8.4,1.2Hz,1H),8.01(d,J=1.6Hz,1H),7.99(d,J=2.4Hz,1H),7.87(dd,J=8.8,4.4Hz,1H),7.61–7.50(m,3H),7.44–7.37(m,1H),7.04(d,J=4.0Hz,1H),5.33(s,2H);ESI-HRMS:395.0691[M+H] + , which was Compound 17.

[0205] Example 20 Synthesis of N-Hydroxy-6-(((4-oxo-2-(pyridin-3-yl)-4H-pyrano[3,2-b]pyridin-3-yl)oxy)methyl)benzamide (Compound 18)

[0206]

[0207] Compound 18 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 1-(3-hydroxypyridin-2-yl)ethanone, and benzaldehyde was replaced with pyridine-3-carbaldehyde. During the post-treatment, the pH was adjusted to 7 with dilute hydrochloric acid (1N), and then a white solid was obtained by the same method.

[0208] The results of nuclear magnetic resonance were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.14(s,1H),9.02(s,1H),8.84(d,J=4.4Hz,1H),8.71(d,J=4.0Hz,1H),8.37–8.27(m,2H),7.89(dd,J=8.8,4.4Hz,1H),7.67(d,J=8.0Hz,2H),7.60–7.53(m,1H),7.38(d,J=8.0Hz,2H),5.21(s,2H);ESI-HRMS:390.1071[M+H]+ , which is Compound 18.

[0209] Synthesis of Example 21 N-Hydroxy-4-(((4-oxo-2-phenyl-4H-pyrano[3,2-c]pyridin-3-yl)oxy)methyl)benzamide (Compound 19)

[0210]

[0211] Compound 19 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 1-(4-hydroxypyridin-3-yl)ethanone, and after the post-treatment, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method. ESI-HRMS: 389.1146 [M+H] + , which is Compound 19.

[0212] Synthesis of Example 22 N-Hydroxy-4-(((4-oxo-2-phenyl-4H-pyrano[3,2-c]pyridin-3-yl)oxy)methyl)benzamide (Compound 20)

[0213]

[0214] Compound 20 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 1-(3-hydroxypyridin-4-yl)ethanone, and after the post-treatment, the pH was adjusted to 7 with dilute hydrochloric acid (1N) first, and then a white solid was obtained by the same method. ESI-HRMS: 389.1141 [M+H] + , which is Compound 20.

[0215] Synthesis of Example 23 N-Hydroxy-4-(((7-oxo-5-phenyl-7H-thieno[3,2-c]pyran-6-yl)oxy)methyl)benzamide (Compound 21)

[0216]

[0217] Compound 21 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced with 3-hydroxythiophene-2-ethanone, and then a white solid was obtained by the same method. ESI-HRMS: 394.0740 [M+H] + , which is Compound 21.

[0218] Synthesis of Example 24 N-Hydroxy-5-(((7-oxo-5-phenyl-7H-thieno[3,2-b]pyran-6-yl)oxy)methyl)thiophene-2-carboxamide (Compound 22)

[0219]

[0220] Compound 22 was prepared according to Example 1, except that: in step (1), 2-hydroxyacetophenone was replaced by 3-hydroxythiophene-2-ethanone, and in step (2), methyl 4-(chloromethyl)benzoate was replaced by methyl 5-(bromomethyl)thiophene-2-carboxylate, and then a off-white solid was obtained by the same method. ESI-HRMS: 400.0327 [M+H] + , which is Compound 22.

[0221] Synthesis of Example 25 N-hydroxy-4-((N-methyl-2-((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)acetamido)methyl)benzamide (Compound 24)

[0222]

[0223] Compound 24 was prepared according to Example 2, except that in step (3), methyl 4-(aminomethyl)benzoate hydrochloride was replaced by methyl 4-((methylamino)methyl)benzoate hydrochloride, and then a off-white solid was obtained by the same method.

[0224] The results of nuclear magnetic resonance were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 0.35H), 11.02 (s, 0.6H), 8.84 (s, 1H), 8.02 (dd, J = 6.0, 2.4 Hz, 1H), 8.01–7.88 (m, 2H), 7.73–7.63 (m, 1H), 7.61–7.55 (m, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.45–7.35 (m, 3H), 7.34–7.29 (m, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 4.91 (s, 1H), 4.80 (s, 1H), 4.45 (s, 1H), 4.33 (s, 1H), 2.70 (s, 2H), 2.58 (s, 1H); ESI-HRMS: 459.1551 [M+H] + , which is Compound 24.

[0225] Synthesis of Example 26 N-hydroxy-2-(3-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)acridin-1-yl)pyrimidine-5-carboxamide (Compound 26)

[0226]

[0227] Compound 26 was prepared according to Example 3, except that in step (1), N-BOC-4-(((methylsulfonyl)oxy)methyl)piperidine was replaced with tert-butyl 3-(((methylsulfonyl)oxy)methyl)azetidine-1-carboxylate, and then a white solid was obtained by the same method. ESI-MS: 445.1529 [M+H] + , which is Compound 26.

[0228] Synthesis of N-hydroxy-2-(6-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)-2-azaspiro[3.3]heptan-2-yl)pyrimidine-5-carboxamide (Compound 27) in Example 27

[0229]

[0230] Compound 27 was prepared according to Example 3, except that in step (1), N-BOC-4-(((methylsulfonyl)oxy)methyl)piperidine was replaced with tert-butyl 6-(((methylsulfonyl)oxy)methyl)-2-azaspiro[3.3]heptane-2-carboxylate, and then a white solid was obtained by the same method. ESI-HRMS: 485.1832 [M+H] + , which is Compound 27.

[0231] Synthesis of N-hydroxy-2-[7-(((4-oxo-2-phenyl-4H-chromen-3-yl)oxy)methyl)-2-azaspiro[3.5]nonan-2-yl)pyrimidine-5-carboxamide (Compound 28) in Example 28

[0232]

[0233] Compound 28 was prepared according to Example 3, except that in step (1), N-BOC-4-((methylsulfonyl)oxy)methyl)piperidine was replaced with tert-butyl 7-(((methylsulfonyl)oxy)methyl)-2-azaspiro[3.5]nonane-2-carboxylate, and then a white solid was obtained by the same method. ESI-HRMS: 513.2143 [M+H] + , which is Compound 28.

[0234] Test Example 1 HDAC6 and HDAC1 Enzyme Inhibitory Activities of Compounds of the Present Invention

[0235] Tubastatin A is a representative highly selective HDAC6 inhibitor reported in the literature. In this example, Tubastatin A, the clinically investigated HDAC6 inhibitor Ricolinostat, and the marketed broad-spectrum HDAC inhibitor SAHA were used as positive controls, and the HDAC6 and HDAC1 inhibitory activities of the compounds of the present invention were evaluated using the Fluorescent-based HDAC Activity Assay. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as the compounds of the present invention having only the following beneficial effects.

[0236] The test procedure for the HDAC6 enzyme inhibitory activity was as follows: Using dimethyl sulfoxide (DMSO) as a solvent, a stock solution of the test compound was prepared; according to the kit instructions, a buffer solution was prepared, and an HDAC6 protein solution and a corresponding Substrate / Trypsin mixed solution were prepared using the buffer solution; the compound solution, the enzyme solution, and the Substrate / Trypsin mixed solution were added to a 384-well plate respectively to prepare a catalytic reaction system; after incubating at room temperature for a certain period of time, the fluorescence signal values were continuously read using a Synergy microplate reader, and the slope was obtained for the linear reaction segment, and then the inhibition rate at each concentration was calculated, and the half-inhibitory concentration (IC 50 ) was fitted.

[0237] The test method for the inhibitory activity of the compound on HDAC1 referred to the test method for the HDAC6 inhibitory activity, and only the substrate of the catalytic reaction system was changed when testing the corresponding enzyme inhibitory activity. The results are shown in Table 1.

[0238] Table 1 Enzyme inhibitory activities of the compounds on HDAC6 and HDAC1

[0239]

[0240]

[0241] In Table 1: “++++” represents 0 - 20 nM, “+++” represents 20 - 100 nM, “++” represents 100 - 1000 nM, and “+” represents 1000 - 10000 nM.

[0242] The literature shows that the IC 50 of the compound inhibiting HDAC1 50The difference between them can reflect the selectivity of the compound (Eugene Guorong Yang et.al. Design and Synthesis of Janus Kinase 2 (JAK2) and Histone Deacetlyase (HDAC) Bispecific Inhibitors Based on Pacritinib and Evidence of Dual Pathway Inhibition in Hematological Cell Lines. J. Med. Chem. 2016, 59, 8233–8262) — when the IC 50 for inhibiting HDAC1 is 50 greater than the IC for inhibiting HDAC6, and the greater the difference, the better the HDAC6 selectivity of the compound. From the data in Table 1, it can be seen that SAHA has comparable inhibitory activities against HDAC1 and HDAC6 and lacks selectivity for HDAC subtypes; while Tubastatin A has a significant difference in inhibitory activities against HDAC6 and HDAC1 and has excellent HDAC6 selectivity.

[0243] Compounds 1, 2, 6, 8 - 18 conforming to general formula (I) have better inhibitory activities against HDAC6 than HDAC1 and have HDAC6 selectivity; among them, Compounds 1, 6, 8, 12, 15 are similar to Tubastatin A and have significant HDAC6 inhibitory activities, and the IC 50 for enzyme inhibition is lower than 20 nM and shows high subtype selectivity; the IC 50 for Compounds 2, 9, 18 to inhibit HDAC6 is lower than 20 or 100 nM and has good HDAC6 selectivity. Compounds 23, 24 conforming to general formula (II) have excellent HDAC1 inhibitory activities, and the IC 50 for enzyme inhibition is lower than 20 or 100 nM. Compounds 25 - 28 conforming to general formula (III) have significant HDAC1 enzyme inhibitory activities and are significantly better than their inhibitory activities against HDAC6.

[0244] Test Example 2 Further evaluation of the subtype selectivity of the compounds in the present invention

[0245] The literature shows that the inhibitory activities of HDAC1, 2, 3, 6, 8, 10, and 11 can further reflect the subtype selectivity of the compound (Chao-Wu Yu et.al. Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone Deacetylase-6 Inhibitors for Treatment of Non-Small Cell Lung Cancer. J. Med. Chem. 2019, 62, 857–874).

[0246] The test method for the inhibitory activities of the compound against other HDAC isozymes refers to the test method for the inhibitory activity of HDAC6, and only the substrate of the catalytic reaction system is replaced when testing the inhibitory activity of the corresponding enzyme.

[0247] The selectivity of the compound is further elaborated below through the inhibitory activity data of some compounds of the present invention against HDAC1, 2, 3, 6, 8, 10, and 11. It should not be understood that only the following compounds of the present invention have HDAC6 or HDAC1 and / or HDAC2 and / or HDAC3 selectivity. The results are shown in Table 2.

[0248] Table 2 Selectivity of the compound against HDAC6 or HDAC1 and / or HDAC2 and / or HDAC3

[0249] Cpd HDAC1 HDAC2 HDAC3 HDAC6 HDAC8 HDAC10 HDAC11 1 + + + ++++ + + + 2 ++ + + ++++ + + + 6 + + + ++++ + + + 8 + + + ++++ + + + 12 + + + ++++ + + + 15 + + + ++++ + + + 25 ++++ +++ +++ ++ + + + 27 ++++ ++++ +++ ++ + + + SAHA ++++ +++ +++ ++++ +++ ++++ +++ Ricolinostat +++ + + ++++ ++ ++ + Tubastatin A + + + ++++ + + +

[0250] In Table 2: "++++" represents 0 - 20 nM, "+++" represents 20 - 100 nM, "++" represents 100 - 1000 nM, and "+" represents 1000 - 10000 nM.

[0251] As shown in Table 2, similar to Tubastatin A, Compounds 1, 2, 6, 8, 12, and 15 that conform to General Formula (I) have significant inhibitory activity against HDAC6, while their inhibitory activities against HDAC1, 2, 3, 8, 10, and 11 are significantly weaker than their inhibitory activity against HDAC6. Therefore, they are highly selective HDAC6 inhibitors, and their selectivity is significantly better than that of the HDAC6 inhibitor Ricolinostat under clinical investigation. Compounds 25 and 27 that conform to General Formula (III) have significant HDAC1 / 2 / 3 inhibitory activity, while their inhibitory activities against HDAC6, 8, 10, and 11 are relatively weak. Therefore, they are selective HDAC1 / 2 / 3 inhibitors. SAHA is a broad-spectrum HDACs inhibitor, and there is no obvious difference in its inhibitory activities against HDAC1, 2, 3, 6, 8, 10, and 11. While significantly inhibiting the corresponding HDAC subtypes, Compounds 1, 2, 6, 8, 12, 15, 25, and 27 are beneficial for reducing the toxicity caused by SAHA's inhibition of all HDAC subtypes.

[0252] Experimental Example 3 Antitumor Cell Proliferation Activity of the Compounds of the Invention

[0253] In this experimental example, SAHA and Ricolinostat were used as positive controls, and the CCK-8 method was adopted to evaluate the anti-proliferation activities of the compounds of the invention against human breast cancer cell line MDA-MB-231, human mammary ductal carcinoma cell line T47D, and colon cancer cell line HCT116. Other compounds of the invention have similar beneficial effects to the compounds listed below, but this should not be construed as the compounds of the invention only having the following beneficial effects.

[0254] The test steps were as follows: Digest and collect tumor cells, inoculate them in a 96-well culture plate at a certain density, and place them in an incubator (37°C, 5% CO2) overnight. Treat the cells with compound solutions at different concentrations. After the compounds acted for 72 h, discard the culture medium, and then gently wash the cells 3 times with PBS. Subsequently, add a certain volume of culture medium and CCK-8 to each well of the culture plate and continue culturing for a certain time. Finally, use a multifunctional microplate reader to measure the absorbance OD value at a wavelength of 570 nm, calculate the inhibition rate, and the IC 50 value was obtained by fitting with GraphPad Prism 5 software. The results are shown in Table 3.

[0255] Table 3 Antitumor Cell Proliferation Activity of the Compounds

[0256] Cpd. <![CDATA[MDA-MB-231(IC 50 )]]> <![CDATA[T47D(IC 50 )]]> <![CDATA[HCT-116(IC 50 )]]> 1 +++ +++ ++++ 2 +++ +++ +++ 6 +++ +++ +++ 8 +++ +++ +++ 13 +++ +++ +++ 15 ++++ +++ ++++ 17 +++ ++++ ++++ 23 ++++ ++++ ++++ 25 ++++ ++++ ++++ 27 ++++ ++++ ++++ SAHA ++++ +++ ++++ Ricolinostat +++ +++ ++

[0257] In Table 3: “++++” represents 0 - 0.5 μM, “+++” represents 0.5 - 1.0 μM, and “++” represents 1.0 - 5.0 μM.

[0258] As can be seen from the data in Table 3, the listed compounds (including Compounds 1, 2, 6, 8, 13, 15, 17 conforming to General Formula (I), Compound 23 conforming to General Formula (II), and Compounds 25, 27 conforming to General Formula (III)) all showed significant anti-proliferative activity against solid tumor cell lines, and the IC 50 of anti-proliferation was in the submicromolar range, and the activity was equivalent to or better than that of Ricolinostat.

[0259] Test Example 4 Toxicity of the Compounds of the Present Invention to Normal Cells

[0260] In this example, SAHA, Ricolinostat, and Tubastatin A were used as controls, and the CCK-8 method was used to evaluate the toxicity of the compounds of the present invention to human normal cells HUVEC. Other compounds of the present invention have similar beneficial effects to the following listed compounds, but this should not be construed as the compounds of the present invention only having the following beneficial effects. The test procedure was similar to the test for tumor cell proliferation inhibitory activity. The results are shown in Table 4.

[0261] Table 4 Toxicity of Compounds to Normal Cells HUVEC

[0262] Cpd. <![CDATA[HUVEC(IC 50 )]]> Cpd. <![CDATA[HUVEC(IC 50 )]]> 1 ++ 15 ++ 2 ++ 23 ++ 4 ++ 24 ++ 5 ++ 25 ++ 6 ++ 27 ++ 8 ++ SAHA ++++ 9 ++ Ricolinostat +++ 12 ++ Tubastatin A +++

[0263] In Table 4: "++++" represents 5 - 10 μM, "+++" represents 10 - 30 μM, "++" represents 30 - 100 μM, and "+" represents 100 - 1000 μM.

[0264] As can be seen from the data in Table 4, the listed compounds (including Compounds 1, 2, 4, 5, 6, 8, 9, 12, 15 conforming to General Formula (I), Compounds 23, 24 conforming to General Formula (II), and Compounds 25, 27 conforming to General Formula (III)) had relatively low cytotoxicity to normal cells and showed better safety than Tubastatin A, the clinically investigated HDAC6 inhibitor Ricolinostat, and the marketed broad-spectrum HDAC inhibitor SAHA.

[0265] Test Example 5 Tumor Immunomodulatory Effect of the Compounds of the Present Invention

[0266] In this example, the Western blot method was used to evaluate the regulatory effect of the compounds of the present invention on the STAT3 signaling pathway related to tumor cell immunity and the expression of the immune checkpoint PD-L1. Other compounds of the present invention have similar beneficial effects to the following listed compounds, but this should not be construed as the compounds of the present invention only having the following beneficial effects.

[0267] Test procedure: After digesting MDA-MB-231 cells in the logarithmic growth phase, pipette them into a single-cell suspension, inoculate them in a 6-well plate at a certain density, add the culture medium, and place them in an incubator overnight. Add different concentrations of the test compound solution to each well for drug administration, and set up control wells. After incubating for a certain period of time, add IL-6 and continue incubating. Wash the cells with PBS solution, lyse them with NP-40 lysis buffer, centrifuge, and collect the supernatant. Separate the proteins by SDS-PAGE, transfer them to a polyvinylidene difluoride membrane (PVDF membrane), and incubate them with PD-L1, STAT3, P-STAT3 (Y705), Acetyl-α-tubulin (Lys40), GAPDH antibodies and secondary antibodies respectively, and then expose them. The results are shown in Figure 1 and Figure 2 , where Figure 1 is the western blot analysis of the expression levels of STAT3, P-STAT3 (Y705), Acetyl-α-tubulin (Lys40), and PD-L1 by Compound 15, Figure 2 in which A is the quantitative analysis of the regulatory level of Acetyl-α-tubulin (Lys40) in MDA-MB-231 cells by Compound 15, Figure 2 in which B is the quantitative analysis of the regulatory level of P-STAT3 (Y705) in MDA-MB-231 cells by Compound 15, Figure 2 in which C is the quantitative analysis of the regulatory level of PD-L1 in MDA-MB-231 cells by Compound 15.

[0268] Activation of the STAT3 signaling pathway can mediate tumor immune escape. P-STAT3 (Y705) is an important biomarker of this signaling pathway, and the upregulation of its level reflects the activation of this pathway; PD-L1 is an immune checkpoint on the surface of tumor cells, which can escape immune system surveillance through interaction with PD-1 on the surface of immune cells. Figure 1It is shown that treating tumor cells with IL-6 can lead to upregulation of the levels of P-STAT3 (Y705) and PD-L1; the highly selective HDAC6 inhibitor (Compound 15) conforming to formula (I) can significantly resist the upregulation of the levels of P-STAT3 (Y705) and PD-L1 caused by IL-6 at a concentration as low as 0.1 μM, and shows a dose-dependence as a whole, suggesting that the compound can play a role in tumor immune regulation by intervening in the activation of the STAT3 signaling pathway and the expression of PD-L1, thereby effectively reversing the immune escape of tumor cells. Tubulin is a substrate of HDAC6. After treatment with Compound 15, an increase in the level of Ac-tubulin in tumor cells can be seen, indicating that the compound regulates the levels of P-STAT3 (Y705) and PD-L1 by inhibiting HDAC6 in cells. Therefore, while highly inhibiting HDAC6, this compound shows significant anti-tumor cell proliferation activity, excellent selectivity for HDAC6, and at the same time has tumor immunotherapy activity. Due to its dual anti-tumor cell proliferation and immunotherapy activities, the compound can intervene in tumors in a "multi-pathway, multi-factor" manner, which is beneficial to enhancing the drug effect and delaying drug resistance.

[0269] Literature has shown that HDAC6 can achieve anti-inflammatory efficacy by regulating the expression levels of intracellular inflammatory factors such as IL-6 (Park, Jin Kyun et al. Inhibition of histone deacetylase 6 suppresses inflammatory responses and invasiveness of fibroblast-like-synoviocytes in inflammatory arthritis. Arthritis Res. Ther. 2021, 23, 177); exert anti-Alzheimer's disease efficacy through mechanisms such as inhibiting zinc ion-mediated amyloid-β aggregation and promoting Tau protein clearance (Yu, Chao-Wu et al. Quinazolin-4-one derivatives as selective histone deacetylase-6 inhibitors for the treatment of Alzheimer's disease. J. Med. Chem. 2013, 56, 6775-6791; Rabal, Obdulia et al. Design, synthesis, biological evaluation and in vivo testing of dual phosphodiesterase 5 (PDE5) and histone deacetylase 6 (HDAC6)-selective inhibitors for the treatment of Alzheimer's disease. Eur. J. Med. Chem. 2018, 150, 506); slow down or reverse the pathogenesis of autoimmune diseases by enhancing the immunosuppressive ability of Foxp3+ regulatory T cells and maintaining immune homeostasis (Akimova, Tatiana et al. “Histone / protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs. Clin. Immunol. 2010, 136, 348-363; Kalin, Jay H et al. Second-generation histone deacetylase 6 inhibitors enhance the immunosuppressive effects of Foxp3+ T-regulatory cell.(J.Med.Chem. 2012, 55, 639 - 651). In addition, HDAC6 also has therapeutic effects on Parkinson's syndrome and Rett syndrome (LoPresti P. HDAC6 in Diseases of Cognition and of Neurons. Cells. 2020, 10, 12.). Given the significant HDAC6 inhibitory effect and excellent subtype selectivity of some compounds of the present invention, it can be inferred that the compounds proposed in the present invention have application prospects for treating autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

Claims

1. A class of HDAC inhibitors, characterized in that, The HDAC inhibitor is a compound based on a flavone or flavone-mimicking skeleton represented by any one of the general formulas (II)-(III) or a pharmaceutically acceptable salt thereof: Wherein, X is a benzene ring; Y is , , , , , or ; Ring A is a benzene ring substituted with at least one R5; R3 is selected from a benzene ring; R4 is selected from hydrogen or methyl; R5 is selected from hydrogen, methyl or halogen; n2=0-3。 2. A class of HDAC inhibitors according to claim 1, characterized in that, The HDAC inhibitor is selected from any one of the following compounds or a pharmaceutically acceptable salt or deuterated product thereof: 。 3. A class of selective HDAC1 inhibitors, characterized in that, The selective HDAC1 inhibitor is a compound based on a flavone or flavone-mimicking skeleton represented by formula (III) in claim 1 or a pharmaceutically acceptable salt thereof.

4. Use of the HDAC inhibitor according to claim 1 in the preparation of HDAC inhibitor drugs.

5. Use of the HDAC inhibitor according to claim 1 in the preparation of drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

6. An HDAC inhibitor composition, characterized in that, It comprises the HDAC inhibitor according to claim 1 or 2, and further comprises at least one pharmaceutical carrier or excipient.

7. The HDAC inhibitor composition according to claim 6, wherein It further comprises at least one therapeutic agent.

8. The HDAC inhibitor composition according to claim 6 or 7, characterized in that, The dosage form of the HDAC inhibitor composition is any clinically or pharmaceutically acceptable dosage form.

9. Use of the HDAC inhibitor composition according to claim 6 in the preparation of drugs for preventing and / or treating tumors, autoimmune diseases, inflammation, Parkinson's syndrome, Rett syndrome or Alzheimer's disease.

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