Coumarin HDAC6 inhibitors and their preparation method and application

Abstract

The present invention discloses a coumarin HDAC6 inhibitor and its preparation method and application, belonging to the field of chemical medicine technology. The present invention provides coumarin compounds shown in Formula I, Formula II and Formula III. Based on the structure of coumarin, the characteristic group of HDAC6 inhibitor hydroxamic acid is introduced to achieve good homologous selectivity for HDAC1 / 6; at the enzyme level, cell level and animal experiment level, it is verified that the compound has excellent target inhibition effect; by studying the example of HDAC6-NF-κB participating in the inflammatory response of colonic tissue, a new practical basis is provided for the treatment of ulcerative colitis. The compound provided by the present invention can be used as a new HDAC6 inhibitor to prevent and treat HDAC6-related diseases, especially it is expected to solve the shortcomings of some current small molecule drugs for the treatment of ulcerative colitis, such as poor relief effect and large side effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and specifically relates to a coumarin-based HDAC6 inhibitor and a preparation method and application thereof. Background Art

[0002] Inflammation is a common and important physiological process that occurs in mammals following infections, such as those caused by bacteria and viruses, on the surface of the body and its organs, as well as in the organs themselves. Many common lesions and recurring diseases, such as pneumonia, enteritis, and arthritis, are associated with inflammatory responses. Currently, the molecular pathways associated with inflammation are being extensively studied, among which histone deacetylase 6 (HDAC6) is one of the emerging therapeutic targets for the treatment of inflammatory diseases.

[0003] HDAC6, a member of the class IIb HDAC family, is a zinc-dependent protease located in the cytoplasm. HDAC6 contains two functional catalytic domains. HDAC6 deacetylates various substrates, including α-tubulin and HSP90α, and is involved in protein trafficking and degradation, cell shape, and migration. Consequently, deactivation of HDAC6 has been associated with a variety of diseases, including cancer, neurodegenerative diseases, and pathological autoimmune responses. In recent years, research on the mechanisms involved in HDAC6 in inflammatory diseases has become increasingly prominent. The acetylation and ubiquitination domains of HDAC6 play a key role in regulating the NF-κB pathway and the NLRP3 inflammasome, further influencing the production of inflammatory cytokines, including proinflammatory cytokines (IL-6, IL-1β, TNF-α, IL-17) and anti-inflammatory cytokines (IL-10). Given the direct and indirect regulatory effects of HDAC6 on inflammatory responses, inhibition of HDAC6 may be an effective treatment for a variety of inflammatory diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and chronic obstructive pulmonary disease (COPD). Unlike other HDAC inhibitors, selective HDAC6 inhibitors do not have serious side effects.

[0004] In recent years, HDAC6 has become a particularly attractive target for the development of drugs to treat various diseases. For example, the HDAC6-selective inhibitor Tubastatin A has been studied for the treatment of neurodegenerative diseases, cancer, and COPD without significant adverse effects. Another HDAC6-selective inhibitor, CKD-506, blocks the NF-κB signaling pathway in IEC macrophages by affecting HDAC6 acetylation and improves inflammation in a mouse colitis model.

[0005] However, most current HDAC6 inhibitors lack selectivity for class I HDAC enzyme subtypes. Therefore, further research is urgently needed to develop new and effective HDAC6 inhibitors with higher selectivity and provide research value and application significance for the treatment of inflammatory diseases. Summary of the Invention

[0006] To solve the above problems, the first object of the present invention is to provide a novel coumarin-based HDAC6 inhibitor, which has a significant improvement effect on HDAC6-related diseases such as ulcerative colitis.

[0007] The second purpose of the present invention is to provide specific applications of the above-mentioned coumarin HDAC6 inhibitors.

[0008] The third purpose of the present invention is to provide a method for preparing a novel coumarin-based HDAC6 inhibitor.

[0009] In order to achieve this goal, the present invention adopts the following technical solutions.

[0010] First, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in Formula I, Formula II or Formula III:

[0011]

[0012] in,

[0013] X is selected from C1 to C10 alkyl chains,

[0014] (The left end bond of these X structural units is connected to O or Y, and the right end bond is connected to the hydroxylamine acyl group);

[0015] X1 is selected from a bond or a C1-C6 alkyl chain;

[0016] X2 is selected from a bond or a C1-C4 alkyl chain;

[0017] Y is selected from O, NH or NCH3.

[0018] In some embodiments of the present invention, in the above compounds, X is selected from C1 to C6 alkyl chains,

[0019]

[0020] (The left-end bond of these X structural units is connected to O or Y, and the right-end bond is connected to the hydroxylamine group).

[0021] In some embodiments of the present invention, in the above compounds, X1 is selected from a bond or a C1-C4 alkyl chain.

[0022] In some preferred embodiments of the present invention, in the above compounds, X1 is selected from a bond or -CH2-.

[0023] In some embodiments of the present invention, in the above compounds, X2 is selected from a bond or -CH2-.

[0024] In some preferred embodiments of the present invention, in the above compounds, X1 is selected from a bond or -CH2-.

[0025] In some preferred embodiments of the present invention, in the above compounds, Y is O.

[0026] In some more preferred embodiments of the present invention, in the above compounds, X is selected from -CH2-,

[0027] (The left-end bond of these X structural units is connected to O or Y, and the right-end bond is connected to the hydroxylamine group).

[0028] The present invention also provides some specific compounds of the above formula I, formula II or formula III, wherein the compound of formula I is selected from:

[0029]

[0030] The compound of formula II is selected from:

[0031]

[0032] The compound of formula III is selected from:

[0033]

[0034] The present invention also provides a class of pharmaceutical compositions, which contain the above-mentioned compound or a pharmaceutically acceptable salt thereof as an active ingredient, supplemented with pharmaceutically acceptable auxiliary ingredients.

[0035] The present invention also provides a method for preparing the pharmaceutical composition of the present invention, which comprises combining any compound of Formula I, Formula II or Formula III or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable auxiliary ingredients.

[0036] The pharmaceutically acceptable carrier that can be used in the pharmaceutical composition of the present invention is a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0037] Second, based on improving the inhibitory effect of the compound on the HDAC6 enzyme, the present invention provides the use of the above-mentioned Formula I, Formula II or Formula III, and related specific compounds or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of the present invention in the preparation of HDAC6 small molecule inhibitors.

[0038] The present invention also provides a method for preventing or treating HDAC6 enzyme-related diseases, comprising administering a compound of Formula I, Formula II or Formula III or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention to an individual in need thereof.

[0039] The present invention also provides the use of the above-mentioned compounds of Formula I, Formula II or Formula III or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of the present invention, in the preparation of a method for preventing and treating HDAC6-related inflammatory diseases; wherein the HDAC6-related inflammatory diseases are pneumonia, enteritis, arthritis, cancer or neurodegenerative diseases.

[0040] In some preferred embodiments of the present invention, in the above use, the pneumonia is asthma and chronic obstructive pulmonary disease (COPD).

[0041] In some preferred embodiments of the present invention, in the above use, the enteritis is ulcerative colitis (UC) or Crohn's disease (CD).

[0042] In some preferred embodiments of the present invention, in the above use, the arthritis is rheumatoid arthritis (RA).

[0043] Overexpression of HDAC6 is associated with advanced tumor stage and increased tumor aggressiveness, resulting in lower survival rates. In some preferred embodiments of the present invention, in the above use, the cancer is oral squamous cell carcinoma, acute myeloid leukemia (AML), ovarian cancer or hepatocellular carcinoma.

[0044] HDAC6 is also associated with neurodegenerative diseases, and inhibition of HDAC6 can affect the transport of mitochondria and neurotransmitters. In some preferred embodiments of the present invention, in the above use, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or Huntington's disease.

[0045] The present invention also provides a method for preventing or treating HDAC6-related diseases in combination with the above-mentioned compound of Formula I, Formula II or Formula III or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, wherein the additional treatment method includes but is not limited to: radiotherapy, chemotherapy, immunotherapy, or a combination thereof.

[0046] Third, the present invention also provides a method for preparing the compound of Formula I or Formula II. The method for preparing the compound of Formula I and Formula II is a synthetic route 1, comprising the following steps:

[0047] Synthesis route 1:

[0048]

[0049] a. Dissolve raw materials A-1 / A-2 in solvent a, add alkaline reagent a, and then add chloromethyl methyl ether to react to form intermediates B-1 / B-2;

[0050] b. Reducing the raw materials B-1 / B-2, catalyst b, and solvent b under a hydrogen atmosphere to generate intermediates C-1 / C-2;

[0051] c. Dissolve intermediate C-1 / C-2 and paraformaldehyde in solvent c, add alkaline reagent c, and then add reducing agent c to react to form intermediate D-1 / D-2;

[0052] d. Dissolve the raw material E in the solvent d, add the brominating agent d, add phosphorus pentoxide, and react to form the intermediate F;

[0053] e. Dissolve intermediate D-1 / D-2 and intermediate F in solvent e, add alkaline reagent e, and undergo substitution reaction to generate intermediate G-1 / G-2;

[0054] f. Dissolve the intermediate G-1 / G-2 in solvent f, add alkaline reagent f, and undergo hydrolysis reaction to generate intermediate H-1 / H-2;

[0055] g. Dissolve the intermediate H-1 / H-2 and the alkaline reagent g in the solvent g, add the bromine-substituted ester I (the structure of the bromine-substituted ester I depends on the structure of the compounds of Formula I and Formula II, and R is generally methyl or ethyl), and react to form the intermediate J-1 / J-2;

[0056] h. Dissolve the intermediate J-1 / J-2 in solvent h, add alkaline reagent h, and then add an aqueous solution of hydroxylamine to undergo hydrolysis reaction to generate compounds of formula I / formula II.

[0057] For the purpose of improving the solubility of raw materials and the yield of products, in the preparation method of the compound of formula I or formula II:

[0058] In step a, the solvent a is N,N-dimethylformamide.

[0059] In step a, the alkaline reagent a is 60% sodium hydride.

[0060] In step a, the molar ratio of raw material A-1 / A-2: alkaline reagent a: chloromethyl methyl ether is 1:1-3:1-3.

[0061] In step b, the catalyst b is 5% or 10% palladium carbon.

[0062] In step b, the solvent b is methanol.

[0063] In step c, the solvent c is methanol.

[0064] In step c, the alkaline reagent c is sodium methoxide or potassium methoxide.

[0065] In step c, the reducing agent c is sodium borohydride or potassium borohydride.

[0066] In step c, the molar ratio of the intermediate C-1 / C-2: paraformaldehyde: alkaline reagent c: reducing agent c is 1:1.0-1.5:1-3:1-3.

[0067] In step d, the solvent d is toluene.

[0068] In step d, the brominating agent d is tetrabutylammonium bromide.

[0069] In step d, the molar ratio of raw material E: brominating agent d: phosphorus pentoxide is 1 to 2: 1 to 2: 1. In an embodiment of the present invention, the molar ratio of raw material E: brominating agent: phosphorus pentoxide is 2: 2: 1.

[0070] In step e, the solvent e is N,N-dimethylformamide.

[0071] In step e, the alkaline reagent e is DMAP.

[0072] In step e, the molar ratio of the intermediate D-1 / D-2:intermediate F:alkaline reagent e is 0.8-1.2:1:0.05-0.2.

[0073] In step f, the solvent f is a solution of hydrogen chloride in 1,4-dioxane.

[0074] In step f, the alkaline reagent f is sodium bicarbonate or potassium bicarbonate.

[0075] In step g, the solvent g is acetonitrile.

[0076] In step g, the alkaline reagent g is cesium carbonate.

[0077] In step g, the molar ratio of the intermediate H-1 / H-2: alkaline reagent g: bromo-substituted ester I is 1:1-3:1-3.

[0078] In step h, the solvent h is methanol.

[0079] In step h, the alkaline reagent h is potassium carbonate or sodium carbonate.

[0080] The present invention also provides a method for preparing the compound of formula III. The method for preparing the compound of formula III is a synthetic route 2, comprising the following steps:

[0081] Synthesis route 2:

[0082]

[0083] i. Dissolve raw material E, raw material K (the structure of raw material K depends on the structure of the compound of formula III, and R is generally methyl or ethyl), alkaline reagent i, and catalyst i in solvent i, and perform substitution reaction to generate intermediate L;

[0084] 1. Dissolve the intermediate L in solvent 1, add alkaline reagent 1, and then add an aqueous solution of hydroxylamine to produce a compound of formula III through hydrolysis reaction;

[0085] Alternatively, j. reacting the raw material E with phosphorus oxychloride to form an intermediate M;

[0086] k. Dissolve the intermediate M, the raw material HO-X-COOR (the structure of HO-X-COOR depends on the structure of the compound of formula III, and R is generally methyl or ethyl), the alkaline reagent k, and the catalyst k in the solvent k, and perform a substitution reaction to generate the intermediate N;

[0087] 1. Dissolve intermediate N in solvent 1, add alkaline reagent 1, and then add an aqueous solution of hydroxylamine to undergo hydrolysis reaction to generate a compound of formula III.

[0088] For the purpose of improving the solubility of raw materials and the yield of products, in the preparation method of the compound of formula III:

[0089] In step i, the alkaline reagent ii is potassium carbonate or sodium carbonate.

[0090] In step i, the catalyst i is potassium iodide.

[0091] In step i, the solvent i is N,N-dimethylformamide.

[0092] In step i, the molar ratio of raw material E: raw material K: alkaline reagent i: catalyst i is 1:1-3:1-3:0.05-0.2.

[0093] In step k, the alkaline reagent k is potassium carbonate or sodium carbonate.

[0094] In step k, the catalyst k is potassium iodide.

[0095] In step k, the solvent k is N,N-dimethylformamide.

[0096] In step k, the molar ratio of the raw material E: the raw material HO-X-COOR: the alkaline reagent k: the catalyst k is 1:1-3:1-3:0.05-0.2.

[0097] In step 1, the solvent 1 is methanol.

[0098] In step 1, the alkaline reagent 1 is potassium carbonate or sodium carbonate.

[0099] Definition of terms:

[0100] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. The terms "include," "comprising," "having," "containing," or "involving," and their variations herein, are inclusive or open-ended and do not exclude other unrecited elements or method steps. It should be understood by those skilled in the art that the above terms, such as "comprising," encompass the meaning of "consisting of."

[0101] In the present invention, unless otherwise specified, "alkyl chain" includes a linear or branched monovalent saturated hydrocarbon chain. For example, alkyl chains include methylene, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, etc. Similarly, the C1-10 in "C1-10 alkyl chain" refers to an alkyl chain containing 1, 2, 3, 4, ... 10 carbon atoms in a linear or branched arrangement.

[0102] On the basis of not violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0103] Beneficial effects of the present invention:

[0104] Based on the structure of coumarin, this invention introduces the characteristic hydroxamic acid group of HDAC6 inhibitors for the first time, designing novel derivatives. By adopting a design strategy that selectively inhibits the HDAC6 enzyme, the compounds achieve excellent homologous selectivity for HDAC1 / 6. The invention verifies the compounds' excellent target inhibition efficacy at the enzyme, cellular, and animal levels. By studying the involvement of HDAC6-NF-κB in colonic tissue inflammatory responses, the invention provides new practical evidence for the treatment of ulcerative colitis. The compounds provided by this invention can be used as novel HDAC6 inhibitors, potentially addressing the shortcomings of some current small molecule drugs for the treatment of ulcerative colitis, such as poor relief and significant side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1The figure shows the effect of compound B22 in treating ulcerative colitis; A shows the changes in mouse body weight, B shows the changes in mouse colon length, C shows the changes in mouse disease activity index, D shows the results of in vitro mouse colon tissue staining, E shows the results of RT-PCR experiments, and F shows the results of ELISA experiments.

[0106] Figure 2 These are the results of the effects of compound B22 on the levels of HDAC, NF-κB and inflammation-related proteins in the colon tissues of mice with ulcerative colitis. DETAILED DESCRIPTION

[0107] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications shall be followed.

[0108] The reagents and raw materials used in the examples of the present invention are all commercially available.

[0109] Example 1

[0110] N-hydroxy-2-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)acetamide (B1), the structural formula of which is shown below:

[0111]

[0112] The synthesis steps are as follows:

[0113] (1) 3-Nitro-5-methoxyphenol A-1 (200 mmol) was dissolved in 500 mL of N,N-dimethylformamide, and the mixture was cooled to 0°C. 60% NaH (400 mmol) was added in three batches. After half an hour, MOMCl (400 mmol) was slowly added to the system. After the addition was complete, the reaction was transferred to room temperature and monitored by TLC. After the reaction was complete, the reaction solution was slowly poured into 2.5 L of water to quench the reaction. A large amount of yellow solid was observed. The filter cake was filtered and recrystallized from ethanol to obtain intermediate B-1.

[0114] (2) Intermediate B-1, 10% palladium on carbon, and 500 mL of methanol were added sequentially to a three-necked flask. The reaction system was purged with nitrogen, and hydrogen at a pressure of 0.4 MPa was introduced, followed by stirring at room temperature for 4 hours. After the reaction was complete, the mixture was filtered through celite and washed with ethyl acetate. The filtrate was collected and concentrated to obtain Intermediate C-1.

[0115] (3) Dissolve intermediate C-1 (100 mmol) and paraformaldehyde (120 mmol) in 300 mL of methanol, place at 0°C, and then add sodium methoxide (200 mmol) to the system in batches. Stir at room temperature overnight. Add sodium borohydride (200 mmol) to the reaction flask and stir at room temperature for 2 hours. After the reaction is complete, quench with 300 mL of water and extract with methyl tert-butyl ether three times, 100 mL each time. Combine the organic phases, concentrate under reduced pressure, and cool to obtain intermediate D-1.

[0116] (4) Intermediate D-1 (18 mmol) and 4-bromocoumarin F (20 mmol) were mixed and added to a 250 mL eggplant-shaped flask. The mixture was dissolved in 100 mL of N,N-dimethylformamide, and a catalytic amount of DMAP (2 mmol) was added. The mixture was reacted at 110°C for 12 hours. After the reaction was complete, 200 mL of water was added, and the mixture was extracted with ethyl acetate three times, each time with 100 mL. The organic phase was concentrated, and the concentrate was separated by column chromatography (PE / EA = 80 / 20 v / v) to obtain intermediate G-1.

[0117] (5) Intermediate G-1 was placed in a 250 mL eggplant-shaped flask, 100 mL of a solution of hydrogen chloride in 1,4-dioxane was added, the system was sealed, and the reaction was carried out at room temperature for 2 hours. 150 mL of saturated sodium bicarbonate was added to precipitate a large amount of yellow solid, which was filtered, washed with water, and dried to obtain intermediate H-1.

[0118] (6) Intermediate H-1 (1 mmol) and cesium carbonate (2 mmol) were added to a 50 mL flask, dissolved in 15 mL of acetonitrile, and heated under reflux for 0.5 h. Ethyl bromoacetate I (2 mmol) was then slowly added. After 2 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure and separated by TLC (DCM / MeOH = 10 / 1) to obtain intermediate J-1.

[0119] (7) Intermediate J-1 (2 mmol) was dissolved in 15 mL of methanol, and anhydrous potassium carbonate (1 mmol) was added. The mixture was stirred at 0°C for 15 min, and an aqueous solution of hydroxylamine (2 mL) was added dropwise. The reaction was monitored by TLC until completion. 0.5 N hydrochloric acid was added to adjust the pH to 7-8, and the mixture was concentrated under reduced pressure. The mixture was separated and purified by preparative thin-layer chromatography (DCM / MeOH = 7 / 1), and dried in vacuo to obtain the target compound B1.

[0120] Light yellow solid powder, yield: 58%, mp: 107.8-108.6. 1H NMR (400MHz, DMSO) δ10.74(s,1H),9.01(s,1H),7.47–7.39(m,1H),7.30(d,J=8.2Hz,1H),6.99(dd,J=16.8,3.3 Hz,3H),6.89(d,J=8.6Hz,1H),6.63(dd,J=8.6,2.4Hz,1H),5.81(s,1H),4.41(s,2H),3.75(s,3H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d6)δ161.67,156.84,154.08,148.60,147.90,141.57,131.59,126.8 3,123.34,118.84,117.57,116.19,112.99,112.22,95.86,67.46,56.17,49.07,44.30. HRMS(ESI):C 19 H 18 N2O6[M+Na] + The theoretical value is 393.1063, and the measured value is 393.1060.

[0121] Example 2

[0122] N-hydroxy-4-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)butanamide (B2), whose structure is shown below:

[0123]

[0124] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by ethyl 4-bromobutyrate.

[0125] Light brown solid powder, yield: 66%, mp: 83.1-84.5. 1 H NMR (400MHz, DMSO-d6) δ7.42(s,1H),7.30(d,J=7.8Hz,1H),6.96(s,3H),6.87(d,J=8.1Hz,1H),6.59( d,J=7.5Hz,1H),5.80(s,1H),3.92(s,2H),3.74(s,3H),3.34(s,3H),2.10(s,2H),1.91–1.86(m,2H). 13C NMR (101MHz, DMSO-d6) δ169.02,161.68,156.81,154.06,149.20,147.79,141.71,131.56,126. 81,123.30,117.86,116.21,112.75,111.20,102.98,95.55,68.30,56.15,44.34,29.14,25.14. HRMS(ESI)C 21 H 22 N2O6[M+Na] + The theoretical value is 421.1376, and the measured value is 421.1372.

[0126] Example 3

[0127] N-hydroxy-5-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)-pentanamide (B3), whose structure is shown below:

[0128]

[0129] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by ethyl 5-bromovalerate.

[0130] Brown oily liquid, yield: 48%. 1 H NMR (400MHz, DMSO) δ10.38(s,1H),7.42(d,J=2.9Hz,1H),7.30(d,J=8.2Hz,1H),6.96(d,J=13.5Hz,3H),6.85(t,J=10.0Hz, 1H), 6.59 (d, J = 6.9 Hz, 1H), 5.80 (s, 1H), 3.92 (s, 2H), 3.74 (s, 3H), 3.47 (dd, J = 18.9, 13.0Hz, 2H), 1.99 (s, 3H), 1.63 (s, 4H). 13 C NMR(101MHz,DMSO-d6)δ169.50,161.74,156.83,154.04,149.29,147.72,141.68,131.57, 126.83,123.29,117.57,116.19,112.68,95.46,56.15,56.07,44.32,32.38,28.53,22.28. HRMS(ESI)C 22 H 24 N2O6[M+H] +The theoretical value is 413.1713, and the measured value is 413.1706.

[0131] Example 4

[0132] N-hydroxy-6-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)-hexanamide (B4), whose structure is shown below:

[0133]

[0134] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by ethyl 6-bromohexanoate.

[0135] Brown oily liquid, yield: 59%. 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),7.68(d,J=7.6Hz,1H),7.30(d,J=25.6Hz,1H ),7.05(d,J=22.7Hz,1H),6.90(d,J=7.2Hz,1H),6.71(d,J=8.4Hz,1H),6.39(s,1H) ,6.21(s,1H),5.99(d,J=8.0Hz,1H),3.85(t,J=6.1Hz,2H),3.62(s,3H),2.61(s,3 H),1.97(t,J=5.8Hz,2H),1.74–1.63(m,2H),1.58–1.50(m,2H),1.41–1.32(m,2H). 13 C NMR(101MHz,DMSO-d6)δ171.50,169.58,165.97,155.15,149.85,145.78,140.77,131.55,126.54,119 .68,116.88,115.47,114.91,102.77,99.81,95.10,68.28,57.32,32.71,30.87,29.06,25.68,25.37. HRMS(ESI)C 23 H 26 N2O6[M+Na] + The theoretical value is 449.1689, and the measured value is 449.1683.

[0136] Example 5

[0137] N-hydroxy-4-((2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)methyl)benzamid e(B5), whose structure is shown below:

[0138]

[0139] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by methyl 4-bromomethylbenzoate.

[0140] Light brown solid powder, yield: 77%, mp: 104.2-105.7. 1 H NMR (400MHz, DMSO-d6) δ7.86(s,1H),7.74(d,J=6.8Hz,1H),7.43(d,J=7.2Hz,2H),7.29(s,2H),7.06(d,J=13.1Hz,1H),6. 90 (d, J = 9.5 Hz, 3H), 6.59 (dd, J = 18.0, 7.7 Hz, 1H), 5.79 (s, 1H), 5.11 (d, J = 21.3 Hz, 2H), 3.75 (d, J = 4.4 Hz, 3H), 3.29 (s, 3H). 13 C NMR(101MHz,DMSO-d6)δ161.68,156.87,154.04,148.89,148.65,148.00,141.65,131.55,130.25,12 9.58,127.90,127.38,127.25,126.77,123.31,117.53,116.18,113.96,112.01,95.64,49.07,44.31. HRMS(ESI)C 25 H 22 N2O6[M+Na] + The theoretical value is 469.1376, and the measured value is 469.1370.

[0141] Example 6

[0142] N-hydroxy-3-((2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)methyl)benzamid e(B6), whose structure is shown below:

[0143]

[0144] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by methyl 3-bromomethylbenzoate.

[0145] Light brown solid powder, yield: 81%, mp: 113.4-114.7. 1 H NMR (400MHz, DMSO) δ11.28(s,1H),9.11(s,1H),7.85(d,J=5.5Hz,1H),7.70(d,J=7.4Hz,1H),7.59–7.51(m,1H),7.49–7.38(m,2H),7. 30(d,J=7.9Hz,1H),7.10(s,1H),7.00–6.82(m,3H),6.59(t,J=12.9Hz,1H),5.81(s,1H),5.14(s,2H),3.84–3.68(m,3H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d6)δ161.80,156.89,154.01,148.69,147.97,141.61,137.54,131.61,130.93,12 8.95,126.98,126.73,123.30,118.27,117.55,116.13,112.78,112.02,95.52,70.19,56.15,44.29. HRMS(ESI)C 25 H 22 N2O6[M+Na] + The theoretical value is 469.1376, and the measured value is 469.1378.

[0146] Example 7

[0147] (E)-N-hydroxy-3-(4-((2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)-methyl)phenyl)acrylamide (B7), whose structure is shown below:

[0148]

[0149] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by methyl 4-bromomethylcinnamate.

[0150] Light yellow solid powder, yield: 69%, mp: 142.7-148.9. 1H NMR (400MHz, DMSO) δ7.50(d,J=7.8Hz,2H),7.44–7.38(m,2H),7.35(t,J=9.7Hz,2H),7.27(d,J=8.3Hz,1H),7.03(d,J=2.6Hz,1 H),6.94–6.84(m,3H),6.62(dd,J=8.5,2.5Hz,1H),6.45(d,J=15.8Hz,1H),5.79(s,1H),5.10(s,2H),3.75(s,3H),3.30(s,3H). 13 C NMR(101MHz,Chloroform-d)δ167.12,162.50,156.75,156.41,154.00,144.01,141.58,138.63,134.01,130.62, 128.12,128.03,127.61,127.53,126.48,126.19,122.51,117.94,117.36,115.87,115.76,95.79,69.56,51.55. HRMS(ESI)C 27 H 24 N2O6[M+Na] + The theoretical value is 495.1532, and the measured value is 495.1537.

[0151] Example 8

[0152] N-hydroxy-6-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)-nicotinamide (B8), whose structure is shown below:

[0153]

[0154] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by methyl 4-chloronicotinate.

[0155] Light brown solid powder, yield: 71%, mp: 114.7-115.2. 1 H NMR (400MHz, DMSO) δ8.41(s,1H),8.08(s,1H),7.46(s,1H),7.31(d,J=7.7Hz,1H),7.10( d,J=11.1Hz,3H),7.04(s,2H),6.96(s,1H),5.84(s,1H),3.68(s,3H),3.32–3.21s,3H). 13C NMR (101MHz, DMSO) δ173.57,162.78,161.45,152.15,148.75,147.94,141.37,132. 32,127.94,126.12,124.57,119.79,114.71,114.51,113.51,75.57,56.25,42.72. HRMS(ESI)C 23 H 19 N3O6[M+Na] + The theoretical value is 456.1172, and the measured value is 456.1165.

[0156] Example 9

[0157] N-hydroxy-4-(2-methoxy-5-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)benzamide (B9), whose structure is shown below:

[0158]

[0159] The synthesis steps are the same as those in Example 1, except that ethyl bromoacetate in step (6) is replaced by methyl 4-bromobenzoate.

[0160] Brown solid powder, yield: 78%, mp: 142.1-143.5. 1 H NMR (400MHz, DMSO) δ7.65(d,J=8.4Hz,2H),7.48(t,J=7.8Hz,1H),7.31(d,J=8.0Hz,1H),7.19(q,J=9.1Hz,2H),7 .09(t,J=7.6Hz,1H),7.00(dd,J=11.0,5.1Hz,2H),6.75(d,J=8.3Hz,2H),5.85(s,1H),3.73(s,3H),3.35(s,3H). 13 C NMR (101MHz, DMSO) δ166.89,162.79,161.44,152.13,148.76,148.30,141.39,132.32, 127.92,126.17,124.57,119.56,114.72,113.34,113.10,70.56,68.01,56.24,42.75. HRMS(ESI)C 24 H 20 N2O6[M+Na] + The theoretical value is 455.1219, and the measured value is 455.1218.

[0161] Example 10

[0162] N-hydroxy-2-(4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)acetamide (B10), whose structure is shown below:

[0163]

[0164] The synthesis steps are as follows:

[0165] (1) 4-Nitrophenol A-2 (200 mmol) was dissolved in 500 mL of N,N-dimethylformamide, and the mixture was cooled to 0°C. 60% NaH (400 mmol) was added in three batches. After half an hour, MOMCl (400 mmol) was slowly added to the system. After the addition was complete, the reaction was transferred to room temperature and monitored by TLC. After the reaction was complete, the reaction solution was slowly poured into 2.5 L of water to quench. A large amount of yellow solid was observed. The filter cake was filtered and recrystallized from ethanol to obtain intermediate B-2.

[0166] (2) Intermediate B-2, 10% palladium on carbon, and 500 mL of methanol were added sequentially to a three-necked flask. The reaction system was purged with nitrogen, and hydrogen at a pressure of 0.4 MPa was introduced, followed by stirring at room temperature for 4 hours. After the reaction was complete, the mixture was filtered through celite and washed with ethyl acetate. The filtrate was collected and concentrated to obtain Intermediate C-2.

[0167] (3) Dissolve intermediate C-2 (100 mmol) and paraformaldehyde (120 mmol) in 300 mL of methanol, place at 0°C, and then add sodium methoxide (200 mmol) to the system in batches. Stir at room temperature overnight. Add sodium borohydride (200 mmol) to the reaction flask and stir at room temperature for 2 hours. After the reaction is complete, quench with 300 mL of water and extract with methyl tert-butyl ether three times, 100 mL each time. Combine the organic phases, concentrate under reduced pressure, and cool to obtain intermediate D-2.

[0168] (4) Intermediate D-2 (18 mmol) and 4-bromocoumarin F (20 mmol) were mixed and added to a 250 mL eggplant-shaped flask. The mixture was dissolved in 100 mL of N,N-dimethylformamide, and a catalytic amount of DMAP (2 mmol) was added. The mixture was reacted at 110°C for 12 hours. After the reaction was complete, 200 mL of water was added, and the mixture was extracted with ethyl acetate three times, each time with 100 mL. The organic phase was concentrated, and the concentrate was separated by column chromatography (PE / EA = 80 / 20 v / v) to obtain intermediate G-2.

[0169] (5) Intermediate G-2 was placed in a 250 mL eggplant-shaped flask, 100 mL of a solution of hydrogen chloride in 1,4-dioxane was added, the system was sealed, and the reaction was carried out at room temperature for 2 hours. 150 mL of saturated sodium bicarbonate was added to precipitate a large amount of yellow solid, which was filtered, washed with water, and dried to obtain intermediate H-2.

[0170] (6) Intermediate H-2 (1 mmol) and cesium carbonate (2 mmol) were added to a 50 mL flask, dissolved in 15 mL of acetonitrile, and heated under reflux for 0.5 h. Ethyl bromoacetate I (2 mmol) was then slowly added. After 2 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure and separated by TLC (DCM / MeOH = 10 / 1) to obtain intermediate J-2.

[0171] (7) Intermediate J-2 (2 mmol) was dissolved in 15 mL of methanol, and anhydrous potassium carbonate (1 mmol) was added. The mixture was stirred at 0°C for 15 min, and an aqueous solution of hydroxylamine (2 mL) was added dropwise. The reaction was monitored by TLC until completion. 0.5 N hydrochloric acid was added to adjust the pH to 7-8, and the mixture was concentrated under reduced pressure. The mixture was separated and purified by preparative thin-layer chromatography (DCM / MeOH = 7 / 1), and dried in vacuo to obtain the target compound B10.

[0172] Brown oily liquid, yield: 51%. 1 H NMR (400MHz, DMSO) δ7.42 (d, J = 5.9 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.1 Hz, 2H), 6.94 (d, J = 5.5 Hz, 4H), 5.83 (s, 1H), 4.45 (s, 2H), 3.31 (s, 3H). 13 C NMR(101MHz,DMSO-d6)δ164.56,161.64,156.90,156.09,154.14,142.24,131.6 1,126.84,126.74,123.32,117.65,116.26,116.11,96.00,66.47,49.07,44.40. HRMS(ESI)C 18 H 16 N2O5[M+Na] + The theoretical value is 363.0957, and the measured value is 363.0952.

[0173] Example 11

[0174] N-hydroxy-4-(4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)butanamide (B11), whose structure is shown below:

[0175]

[0176] The synthesis steps were the same as those in Example 10, except that ethyl bromoacetate in step (6) was replaced with ethyl 4-bromobutyrate.

[0177] Brown oily liquid, yield: 61%. 1 H NMR (400MHz, DMSO) δ7.44–7.38(m,1H),7.30(d,J=8.2Hz,1H),7.11(d,J=8.7Hz,2H),6.92(dd,J=10.7, 7.3Hz, 4H), 5.81 (s, 1H), 3.93 (t, J = 6.0Hz, 2H), 3.30 (s, 3H), 2.12 (q, J = 7.7Hz, 2H), 1.98–1.89 (m, 2H). 13 C NMR (101MHz, DMSO) δ167.03,162.79,161.47,152.14,148.92,148.10,141.33,132.34,127 .92,126.15,124.59,119.86,114.68,114.68,113.69,77.67,42.72,26.60,18.16,14.01. HRMS(ESI)C 20 H 20 N2O5[M+Na] + The theoretical value is 391.1270 and the measured value is 391.1266.

[0178] Example 12

[0179] N-hydroxy-5-(4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)pentanamide (B12), whose structure is shown below:

[0180]

[0181] The synthesis steps are the same as those in Example 10, except that ethyl bromoacetate in step (6) is replaced by ethyl 5-bromovalerate.

[0182] Brown oily liquid, yield: 58%. 1H NMR (400MHz, DMSO) δ10.39(s,1H),8.73(s,1H),7.49–7.38(m,1H),7.30(d,J=8.0Hz,1H),7.12(d,J=8.1Hz,2H) ,7.04–6.75(m,4H),5.81(s,1H),3.93(t,J=5.8Hz,2H),3.35(s,3H),2.01(t,J=6.7Hz,2H),1.71–1.59(m,4H). 13 CNMR(101MHz,DMSO-d6)δ161.67,157.04,156.89,154.13,141.55,131.60,1 26.84,123.31,117.63,116.11,115.99,95.62,44.42,32.35,28.59,22.24. HRMS(ESI)C 21 H 22 N2O5[M+Na] + The theoretical value is 405.1427, and the measured value is 405.1436.

[0183] Example 13

[0184] N-hydroxy-6-(4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)hexanamide (B13), whose structure is shown below:

[0185]

[0186] The synthesis steps are the same as those in Example 10, except that ethyl bromoacetate in step (6) is replaced by ethyl 6-bromohexanoate.

[0187] Brown oily liquid, yield: 65%. 1 H NMR (400MHz, DMSO) δ10.36(s,1H),8.69(s,1H),7.41(d,J=5.7Hz,1H),7.30(d,J=8.0Hz,1H),7.12(d,J=8.3Hz,2H),6.93(dd,J=12.1,7.2H z,4H),5.81(s,1H),3.92(t,J=6.0Hz,2H),3.30(s,3H),1.96(t,J=7.1Hz,2H),1.73–1.62(m,2H),1.58–1.49(m,2H),1.38(d,J=6.7Hz,2H). 13C NMR (101MHz, DMSO-d6) δ161.68,157.07,156.90,154.13,141.53,131.62,126. 86,123.32,117.64,116.11,115.98,68.06,44.43,32.66,28.84,25.61,25.34. HRMS(ESI)C 22 H 24 N2O5[M+Na] + The theoretical value is 419.1583, and the measured value is 419.1583.

[0188] Example 14

[0189] N-hydroxy-4-((4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)methyl)benzamide (B14), whose structure is shown below:

[0190]

[0191] The synthesis steps were the same as those in Example 10, except that ethyl bromoacetate in step (6) was replaced by methyl 4-bromomethylbenzoate.

[0192] Light brown solid powder, yield: 79%, mp: 110.3-112.0. 1 H NMR (400MHz, DMSO) δ7.76(s,2H),7.52–7.40(m,3H),7.31(d,J=7.6Hz,1H),7.15(d,J=7.3Hz ,2H),7.02(d,J=7.2Hz,2H),6.93(s,2H),5.83(s,1H),5.17(d,J=20.8Hz,2H),3.31(s,3H). 13 C NMR(101MHz,DMSO-d6)δ161.62,156.87,156.53,154.15,149.82,145.12,141.95,131.59,127.89,127.67,12 7.46,127.36,126.86,123.29,117.65,116.45,116.34,116.11,114.03,112.96,95.87,69.76,69.39,44.38. HRMS(ESI)C 24 H 20 N2O5[M+Na] + The theoretical value is 439.1270, and the measured value is 439.1270.

[0193] Example 15

[0194] N-hydroxy-3-((4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)methyl)benzamide (B15), whose structure is shown below:

[0195]

[0196] The synthesis steps were the same as those in Example 10, except that ethyl bromoacetate in step (6) was replaced by methyl 3-bromomethylbenzoate.

[0197] Light brown solid powder, yield: 80%, mp: 166.5-167.2. 1 H NMR (400MHz, DMSO) δ11.27(s,1H),9.09(s,1H),7.84(d,J=11.5Hz,1H),7.70(t,J=9.2Hz,1H),7.57(t,J=10.0Hz,1H),7.52–7.38(m,2H), 7.32(d,J=7.9Hz,1H),7.14(t,J=11.6Hz,2H),7.03(d,J=8.0Hz,2H),6.98–6.84(m,2H),5.82(d,J=11.5Hz,1H),5.15(s,2H),3.32(s,3H). 13 C NMR(101MHz,DMSO-d6)δ161.70,156.90,156.57,154.13,141.92,137.68,13 1.63,129.04,126.87,123.31,117.64,116.44,116.08,95.76,69.58,44.37. HRMS(ESI)C 24 H 20 N2O5[M+Na] + The theoretical value is 439.1270 and the measured value is 439.1267.

[0198] Example 16

[0199] (E)-N-hydroxy-3-(4-((4-(methyl(2-oxo-2H-chromen-4-yl)amino)phenoxy)methyl)phenyl)acryla mide(B16), whose structure is shown below:

[0200]

[0201] The synthesis steps are the same as those in Example 10, except that ethyl bromoacetate in step (6) is replaced by methyl 4-bromomethylcinnamate.

[0202] Light brown solid powder, yield: 75%, mp: 186.3-188.1. 1 H NMR (400MHz, DMSO) δ7.58(d,J=7.6Hz,2H),7.50–7.39(m,4H),7.32(d,J=8.1Hz,1H),7.15(d,J=8.4Hz,2H ),7.02(d,J=8.1Hz,2H),6.97–6.87(m,2H),6.47(d,J=8.5Hz,1H),5.83(s,1H),5.13(s,2H),3.32(s,3H). 13 C NMR(101MHz,DMSO-d6)δ161.62,156.86,156.59,154.16,141.90,131.57,12 8.62,128.05,126.85,123.27,117.64,116.46,116.11,95.85,69.55,44.38. HRMS(ESI)C 26 H 22 N2O5[M+Na] + The theoretical value is 465.1427, and the measured value is 465.1425.

[0203] Example 17

[0204] N-hydroxy-4-((2-oxo-2H-chromen-4-yl)oxy)butanamide (B17), whose structure is shown below:

[0205]

[0206] The synthesis steps are as follows:

[0207] (1) A mixture of 4-hydroxycoumarin E (1.5 mmol), potassium carbonate (3 mmol), and a catalytic amount of potassium iodide (0.15 mmol) was dissolved in 10 mL of N,N-dimethylformamide. Ethyl 4-bromobutyrate K (3 mmol) was added and the mixture was reacted at 60°C for 2 hours. The reaction was monitored by TLC. After the reaction was complete, 20 mL of water was added to precipitate a white solid, which was filtered to obtain intermediate L.

[0208] (2) Intermediate L (2 mmol) was dissolved in 15 mL of methanol, and anhydrous potassium carbonate (1 mmol) was added. The mixture was stirred at 0°C for 15 min, and an aqueous solution of hydroxylamine (2 mL) was added dropwise. The reaction was monitored by TLC until completion. 0.5 N hydrochloric acid was added to adjust the pH to 7-8, and the mixture was concentrated under reduced pressure. The mixture was separated and purified by preparative thin-layer chromatography (DCM / MeOH = 7 / 1), and dried in vacuo to obtain the target compound B17.

[0209] White solid powder, yield: 76%, mp: 172.6-173.6. 1 H NMR (400MHz, DMSO) δ10.47(s,1H),8.75(s,1H),7.83(dd,J=14.3,7.9Hz,1H),7.66(t,J=7.7Hz,1H),7.38(dd,J= 14.8,7.8Hz,2H),5.88(d,J=4.7Hz,1H),4.23(dt,J=12.1,6.1Hz,2H),2.21(t,J=7.1Hz,2H),2.14–1.96(m,2H). 13 C NMR (101MHz, DMSO-d6) δ168.94,165.44,162.16,153.22,133.24,124.65,116.87,115.70,90.92. HRMS(ESI)C 13 H 13 NO5[M+Na] + The theoretical value is 286.0692, and the measured value is 286.0696.

[0210] Example 18

[0211] N-hydroxy-6-((2-oxo-2H-chromen-4-yl)oxy)hexanamide (B18), the structure of which is shown below:

[0212]

[0213] The synthesis steps are the same as those in Example 17, except that ethyl 4-bromobutyrate in step (1) is replaced by ethyl 6-bromohexanoate.

[0214] White solid powder, yield: 79%, mp: 144.3-145.6. 1H NMR (400MHz, DMSO) δ10.37(s,1H),8.70(s,1H),7.79(dd,J=7.8,1.6Hz,1H),7.71–7.60(m,1H),7.36(dd,J=14.4,7.7Hz,2H),5.8 7 (s, 1H), 4.19 (t, J = 6.3Hz, 2H), 1.99 (t, J = 7.3Hz, 2H), 1.81 (p, J = 6.6Hz, 2H), 1.59 (p, J = 7.3Hz, 2H), 1.46 (dd, J = 9.1, 6.2Hz, 2H). 13 C NMR (101MHz, DMSO-d6) δ169.54,165.45,162.20,153.20,133.20,124.67,123.27,116.90,115.70,90.86,69.85,32.61,28.13,25.48,25.23. HRMS(ESI)C 15 H 17 NO5[M+Na] + The theoretical value is 314.1005, and the measured value is 314.1000.

[0215] Example 19

[0216] N-hydroxy-4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)benzamide (B19), whose structure is shown below:

[0217]

[0218] The synthesis steps are the same as those in Example 17, except that ethyl 4-bromobutyrate in step (1) is replaced by methyl 4-bromomethylbenzoate.

[0219] White solid powder, yield: 44%, mp: 190.9-192.2. 1 H NMR (400MHz, DMSO) δ7.92–7.76(m,3H),7.70–7.64(m,1H),7.60(d,J=8.0Hz,2H),7.44–7.29(m,2H),6.01(s,1H),5.40(s,2H). 13 C NMR (101MHz, DMSO-d6) δ165.07,162.11,153.21,138.64,133.36,133.22,128.15,127.72,124.82,123.40,116.94,115.60,91.78,70.71. HRMS(ESI)C 17H 13 NO5[M+H] + The theoretical value is 312.0873, and the measured value is 312.0870.

[0220] Example 20

[0221] N-hydroxy-3-(((2-oxo-2H-chromen-4-yl)oxy)methyl)benzamide (B20), whose structure is shown below:

[0222]

[0223] The synthesis steps are the same as those in Example 17, except that ethyl 4-bromobutyrate in step (1) is replaced by methyl 3-bromomethylbenzoate.

[0224] White solid powder, yield: 50%, mp: 233.0-234.6. 1 H NMR (400MHz, DMSO) δ7.91(s,1H),7.86(d,J=7.8Hz,1H),7.77(d,J=7.7Hz,1H),7.68(t,J=7.8Hz,1H),7.62(d,J= 7.6Hz, 1H), 7.49 (t, J = 7.6Hz, 1H), 7.43 (d, J = 8.3Hz, 1H), 7.37 (dd, J = 14.4, 6.9Hz, 1H), 6.05 (s, 1H), 5.38 (s, 2H). 13 C NMR(101MHz,Chloroform-d)δ166.30,164.89,162.39,153.15,139.04,132. 40,130.38,129.96,127.09,123.82,122.83,116.63,115.30,91.18,70.13. HRMS(ESI)C 17 H 13 NO5[M+Na] + The theoretical value is 334.0692, and the measured value is 334.0687.

[0225] Example 21

[0226] (E)-N-hydroxy-3-(4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)phenyl)acrylamide (B21), whose structure is shown below:

[0227]

[0228] The synthesis steps are the same as those in Example 17, except that ethyl 4-bromobutyrate in step (1) is replaced by methyl 4-bromomethylcinnamate.

[0229] Light brown solid powder, yield: 46%, mp: 198.2-199.1. 1 H NMR (400MHz, DMSO-d6) δ7.83 (d, J = 7.2 Hz, 1H), 7.65 (q, J = 10.4, 8.8 Hz, 3H), 7.56 (s, 2H), 7.46–7.17 (m, 3H), 6.57 (s, 1H), 6.03 (s, 1H), 5.37 (s, 2H). 13 C NMR (101MHz, DMSO-d6) δ163.20,162.69,152.51,140.30,139.24,138.19,133.96,128. 86,127.66,119.21,115.72,104.34,61.20,60.14,59.97,50.09,35.02,27.89,25.62. HRMS(ESI)C 19 H 15 NO5[M+Na] + The theoretical value is 360.0848, and the measured value is 360.0846.

[0230] Example 22

[0231] (E)-N-hydroxy-3-(4-((2-oxo-2H-chromen-4-yl)oxy)phenyl)acrylamide (B22), whose structure is shown below:

[0232]

[0233] The synthesis steps are as follows:

[0234] (1) 4-Hydroxycoumarin E (8 mmol) and 20 mL of phosphorus oxychloride were added to a flask, and triethylamine (12 mmol) was slowly added dropwise. The mixture was refluxed and stirred for 2 hours. After the reaction was completed, it was cooled to room temperature and quenched by slowly adding 30 mL of ice water. Saturated sodium bicarbonate solution was added to adjust the pH to 7, and DCM was added for extraction. The organic phase was concentrated and purified by column chromatography (DCM / MeOH, 95 / 5 v / v) to obtain intermediate M.

[0235] (2) A mixture of intermediate M (3 mmol), potassium carbonate (6 mmol), and a catalytic amount of potassium iodide (0.3 mmol) was dissolved in 20 mL of acetonitrile. Methyl 4-hydroxycinnamate (4.6 mmol) was added and reacted at 80°C for 6 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure and purified by preparative thin layer chromatography (PE / EA = 2 / 1) to obtain intermediate N.

[0236] (3) Intermediate N (2 mmol) was dissolved in 15 mL of methanol, anhydrous potassium carbonate (1 mmol) was added, and the mixture was stirred at 0°C for 15 min. An aqueous solution of hydroxylamine (2 mL) was added dropwise. The reaction was monitored by TLC until completion. 0.5 N hydrochloric acid was added to adjust the pH to 7-8, and the mixture was concentrated under reduced pressure. The mixture was separated and purified by preparative thin-layer chromatography (DCM / MeOH = 7 / 1), and dried in vacuo to obtain the target compound B22.

[0237] Pale pink solid powder, yield: 66%, mp: 162.9-164.0. 1 H NMR (400MHz, DMSO) δ10.86 (s, 1H), 9.08 (s, 1H), 8.04 (d, J = 7.5Hz, 1H), 7.83–7.68 (m, 3H), 7.59–7. 52(m,1H),7.51–7.45(m,2H),7.42(d,J=8.0Hz,2H),6.50(d,J=15.7Hz,1H),5.29(d,J=6.9Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ166.87,165.71,162.17,153.45,153.39,142.85,132.87,1 32.77,129.85,129.64,123.99,122.79,121.62,118.60,116.65,115.74,114.93,93.58. HRMS(ESI)C 18 H 13 NO5[M+Na] + The theoretical value is 346.0692, and the measured value is 346.0688.

[0238] Biological activity test:

[0239] 1. In vitro enzyme activity assay

[0240] The compounds (B1-B22) having the structures represented by Formula I, Formula II, and Formula III were subjected to in vitro enzyme activity assays as follows:

[0241] The AlphaLISA method was used to test the inhibition percentage of the derivatives on HDAC1 and HDAC6 at single concentrations (50 nM and 500 nM); the IC 50 In the value test, the inhibition rate of 10 concentrations of gradient dilution was used as IC 50 The curve is calculated and the results are obtained. The specific steps are:

[0242] (1) Prepare 1x detection buffer (modified Tris buffer).

[0243] (2) Compound serial dilution: Compounds were transferred to the assay plate by Echo in 100% DMSO. The final DMSO concentration was 1%.

[0244] (3) Prepare enzyme solution: Prepare enzyme solution in 1x assay buffer.

[0245] (4) Prepare substrate solution: Add trypsin and AC peptide substrate to 1x detection buffer to prepare substrate solution.

[0246] (5) Transfer 15 μL of enzyme solution to the assay plate or transfer 15 μL of 1x assay buffer to the low control plate.

[0247] (6) Incubate at room temperature for 15 minutes.

[0248] (7) Add 10 μL of substrate solution to each well to start the reaction, with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

[0249] (8) Curve fitting: The inhibition value was obtained by fitting the data in Excel using Formula 1. Formula 1: Inh% = ((maximum signal) / (minimum signal)*100).

[0250] Fitting the data in XL-Fit yields the half-inhibitory concentration value of Formula 2. Formula 2: Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X)*HillSlope), where Y is the inhibition rate and X is the concentration of the compound.

[0251] The results of the inhibitory activity test are shown in Table 1.

[0252] Table 1 Selective inhibitory activity of the compounds of the present invention on HDAC6

[0253]

[0254]

[0255] As shown in Table 1, all compounds synthesized from Formulas I-III, except B1 and B17, exhibited strong inhibitory activity against HDAC6 at a concentration of 500 nM (inhibition rate > 50%). Among them, compounds B2-B5, B9, B10, B14, and B22 exhibited high HDAC6 inhibitory activity (inhibition rates > 50% at a concentration of 50 nM) while avoiding HDAC1 activity (inhibition rates < 50% at a concentration of 50 nM), suggesting that compounds B2, B10, B14, and B22 have good HDAC1 / 6 selectivity.

[0256] 2. In vitro anti-inflammatory activity assay

[0257] The compounds (B1-B22) having the structures represented by Formula I, Formula II, and Formula III were subjected to in vitro anti-inflammatory activity assays as follows:

[0258] (1) The Griess method was used to screen the in vitro anti-nitric oxide activity. RAW264.7 cells were first incubated in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / ml streptomycin, and then transferred to a humidified environment at 37°C containing 5% CO2 for culture.

[0259] (2) Secondly, collect the logarithmically growing RAW264.7 cells in a 15 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, dilute with fresh culture medium to an appropriate density, and set aside. Place a special coverslip in the center of the cell counting plate, aspirate 10 μL of suspension from the centrifuge tube, add it between the counting plate and the special coverslip, and fill the coverslip with liquid through siphoning. Place the counting plate under a microscope for counting. Counting formula: (number of cells per well × 100 × number of 96-well plate) / (number of cells in 4 large grids × 104) = suspension volume (mL).

[0260] (3) Next, calculate the volume of the cell suspension. By calculating the cell suspension density, plate 8,000-12,000 cells per well, i.e., plate one 96-well plate. Aspirate the cell suspension volume (mL) = (number of cells per well × 96 × 1) / cell suspension density. Mix the required cell suspension and culture medium, and add the suspension to the 96-well plate using a dispenser, 100 μL per well. Mix the cell suspension once every three additions. After the cell suspension is added, cover the plate and observe the cell density and uniformity under an inverted microscope.

[0261] (4) Finally, in a 96-well plate, the experimental group was treated with 1 μg / mL LPS for 1 h, followed by treatment with different derivatives for 48 h. Three wells served as blank groups, with an equal volume of DMSO added, and three wells served as LPS groups, with 100 μL of culture medium added to each well. Side wells were left untreated. After 48 h, the cell suspensions from each group were transferred to a new cell counting plate, and Solution A and Solution B from the NO assay kit were added to each well. After the solution changed from colorless to pink, the culture medium was collected and the nitrite level (an indicator of NO production) was determined using the Griess method. The absorbance (OD540) of the samples was measured at a wavelength of 540 nm using a microplate reader.

[0262]

[0263] Control: DMSO solution treated with LPS only;

[0264] Compound: solution after treatment with LPS and derivatives;

[0265] Blank: DMSO solution without LPS.

[0266] Table 2 In vitro anti-inflammatory activity test results of the compounds of the present invention

[0267]

[0268] As shown in Table 2, among the above 22 compounds, B1, B3, B5, B7, B10-B14, B16, B21 and B22 all showed good inhibitory activity on NO production in RAW264.7 mouse macrophages at a concentration of 5 μM (inhibition rates were all greater than 50%). Among them, the inhibition rates of B21 and B22 were greater than 90%, which was comparable to the positive drug S-methylisothiourea sulfate (SMT).

[0269] 3. In vivo animal experiments in ulcerative colitis mouse models

[0270] In vivo animal experimental study on the therapeutic effect of compound B22 on DSS-induced ulcerative colitis mouse model.

[0271] The results are as follows Figure 1 The results showed that the compound B22 had a good therapeutic effect on DSS-induced ulcerative colitis, and administration of 15 mg / kg or 30 mg / kg could significantly reduce the effect of the disease on the body weight of mice ( Figure 1 Middle A), the disease activity index can also be significantly reduced ( Figure 1 C), and compared with the DSS model group, the colon length of the B22-treated mice was longer ( Figure 1 Middle B), no obvious ulcers and inflammatory cell infiltration were observed in the colon ( Figure 1Compound B22 showed a good therapeutic effect on DSS-induced ulcerative colitis in mice.

[0272] The results showed that the 15 mg / kg and 30 mg / kg doses of compound B22 could effectively prevent the weight loss of UC mice ( Figure 1 A), growth of disease activity index (DAI) ( Figure 1 Middle C) and shortening of colon length ( Figure 1 B) and other disease indicators. Histopathological analysis showed that ( Figure 1 In D), the high-dose group of compound B22 can effectively maintain the integrity of the structure of each layer of colon tissue, reduce the degree of lesions, and reduce tissue damage. The therapeutic effect is comparable to that of the positive drug tofacitinib. By RT-PCR and ELISA experiments ( Figure 1 Zhong E and Figure 1 (F) Compound B22 was found to significantly reduce the levels of inflammatory factors such as IL-1β, IL-6, and TNF-α in the colon tissue of UC mice. Combined with the results of other animal experiments, this suggests that compound B22 has a good therapeutic effect on DSS-induced ulcerative colitis in mice.

[0273] 1. Animal Model Establishment and Evaluation Methods

[0274] C57BL / 6J mice were randomly divided into normal group, DSS-induced group, tofacitinib (40 mg / kg), and B22 (15 mg / kg, 30 mg / kg) according to body weight. On the first day of the experiment, except for the normal group, all groups were given free drinking of 2.5% DSS aqueous solution (this time marked as the first day). The normal control group was given the same sterile water without DSS. After drinking it for 6 consecutive days, on the 7th day, all mice in all groups were switched to sterile drinking water without DSS. At this time, drug administration began. The DSS-induced group was given the same solvent for the preparation of the drug. The dosage volume was approximately 0.1 ml / 10 g, and the drug was administered for 6 consecutive days. The specific treatment plan is as follows:

[0275] Table 3 Treatment options

[0276] Group <![CDATA[Dose (mg·kg -1 )]]> Dosage Normal group / / Model Group / / Tofacitinib control group 40 Oral gavage, once a day B22 low-dose group 15 Intraperitoneal, once a day B22 high-dose group 30 Intraperitoneal, once a day

[0277] From the date of modeling, mice were observed daily for food intake, activity, and coat condition. Animals were weighed, and fecal characteristics, fecal occult blood, and bleeding were observed to assess colitis severity. Disease activity was scored using the Disease Activity Index (DAI) which is a combination of weight loss, stool characteristics, fecal occult blood, and coat color. The total score for each mouse is 16.

[0278] Table 4 Disease Activity Index (DAI) evaluation criteria

[0279]

[0280]

[0281] 2. Pathological Tissue Section Analysis

[0282] Sampling: Fix fresh tissue in fixative for at least 24 hours. Remove the tissue from the fixative and trim the desired area with a scalpel in a fume hood. Place the trimmed tissue and corresponding label in a dehydration box.

[0283] Dehydration: Place the dehydration box in the hanging basket and dehydrate in a dehydrator using a gradient of alcohols: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, absolute ethanol I for 30 minutes, absolute ethanol II for 30 minutes, alcohol / benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, wax III for 1 hour.

[0284] Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the embedding surface requirements. Label the frame accordingly. Cool in a -20°C freezer. Once the wax solidifies, remove the wax block from the embedding frame and trim it.

[0285] Sectioning: Place the trimmed wax block on a paraffin microtome and slice to 3 μm thickness. Float the slices on a 40°C warm water slide to flatten the tissue. Remove the tissue slides with glass slides and bake in a 60°C oven. Once the wax is melted, remove the slices and store at room temperature until ready for use.

[0286] Dewax the paraffin sections to water: place the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and wash with tap water.

[0287] Hematoxylin staining: Hematoxylin staining for 3-5 minutes, differentiated with hydrochloric acid aqueous solution, blued with ammonia aqueous solution, and washed with water; Eosin staining: sections were dehydrated in 85% and 95% graded alcohol in sequence, and then stained in eosin solution for 5 minutes.

[0288] Dehydration and sealing: slices were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min to make them transparent, and then sealed with neutral gum.

[0289] Photography: optical microscope, image acquisition (microscope: NIKON Eclipse ci, imaging system: NIKON digital sight DS-FI2, MADE IN JAPAN, photography magnification: 100x and 200x).

[0290] 3.ELISA test

[0291] IL-1β levels in colonic tissue samples were measured using a commercially available ELISA kit according to the manufacturer's instructions (Beijing 4A Biotechnology Co., Ltd., Beijing, China). Colonic homogenates were first prepared. The entire homogenate preparation process was performed on ice. Colonic tissue, frozen in liquid nitrogen, was dissected and accurately weighed on an analytical balance. Five times the sample weight of pre-chilled PBS was added, along with protease inhibitors (protease inhibitors, PBS = 1:100). Homogenization was performed using a tissue grinder at 4°C. The homogenate was then transferred to a 1.5 mL EP tube and centrifuged at 3000 rpm for 20 min at 4°C. The supernatant was collected (carefully avoiding the pellet at the bottom of the tube), aliquoted, and stored at -80°C. Prior to measurement, the sample was removed and thawed on ice. The IL-1β content of each sample was determined using an ELISA kit. Specific steps and final data processing were performed strictly according to the manufacturer's instructions. GraphPad Prism 7.0 software was used for analysis.

[0292] 4. PCR testing

[0293] Sample preparation:

[0294] The tissue was removed from liquid nitrogen, quickly crushed with a heavy hammer, quickly ground into powder in an RNase-free mortar (liquid nitrogen was continuously added during the grinding process), and transferred to an RNase-free centrifuge tube.

[0295] Extraction of total RNA:

[0296] (1) Add 1 mL of Trizol per 30 mg of tissue, vortex vigorously, and lyse the cells at room temperature for 5 minutes.

[0297] (2) Add an appropriate amount of chloroform at a ratio of 1 mL of Trizol to 0.2 mL of chloroform, shake vigorously up and down for 15 seconds, let it stand at room temperature for 2-3 minutes, and then centrifuge at 12000 rpm at 4°C for 15 minutes;

[0298] (3) Place the upper aqueous phase (chloroform layer) in a new centrifuge tube and add an appropriate amount of isopropanol at a ratio of 1 mL Trizol to 0.5 mL isopropanol. Gently invert the tube to mix thoroughly. After standing at room temperature for 10-15 minutes, centrifuge at 12,000 rpm and 4°C for 10 minutes.

[0299] (4) Discard the supernatant and wash the RNA pellet by adding 1 mL of 75% ethanol per 1 mL of Trizol.

[0300] (5) Centrifuge at 7500 rpm, 2-8°C for 5 min, discard the supernatant, and repeat washing 2-3 times;

[0301] (6) Discard the supernatant and allow the RNA pellet to dry naturally at room temperature;

[0302] (7) Dissolve in 20 μL of DEPC water and store at -80°C until use.

[0303] RNA quantification:

[0304] Aspirate 2 μL of RNA sample to determine the RNA concentration and OD260 / OD280 ratio. Use DEPC water to adjust the value to zero. If the OD260 / OD280 value of the sample is between 1.8-2.0, it indicates that the extracted RNA is pure and qualified, and the next step of the experiment can be continued.

[0305] Reverse transcription of cDNA:

[0306] The experiment was performed according to the following reaction system, and the volume of RNA added was determined based on the quantitative RNA concentration.

[0307] Table 5 Reverse transcription reaction system

[0308] Reagents Dosage Final concentration Reverse transcription reaction reagents 2μL 1× Total RNA * / DEPC treated water A total of 10 μL /

[0309] In Table 5, 1× means 1 times the sample volume, and * indicates that the volume of RNA added was determined based on the quantified RNA concentration.

[0310] Reverse transcriptase chain reaction (RT-PCR):

[0311] (1) The reaction system was carried out according to the instructions of the Takara kit:

[0312] Table 6 Reverse transcription reaction system

[0313] Reagents Dosage / μL Final concentration Reverse transcription mixture (2×) 12.5 1× PCR forward primer 1.0 0.4 μM PCR reverse primer 1.0 0.4μM RT reagents 2.0 / <![CDATA[dH2O]]> 8.5 / Total 25 /

[0314] (2) Primer sequence:

[0315] Table 7 Primer sequences used in RT-PCR

[0316]

[0317] (3) Reaction conditions:

[0318] Table 8 RT-PCR process and conditions

[0319]

[0320] 5. Western blot experiment

[0321] The results of Western blot experiments on compound B22 and the statistics of relative protein levels are shown in Figure 2. Figure 2 The results showed that compared with the model group, treatment with 30 mg kg-1 of B22 significantly increased the level of acetylated α-tubulin (#1, P < 0.001, #2, P < 0.01), suggesting that B22 can affect the levels of HDAC6-related proteins in colon tissue.

[0322] Regarding the expression of NF-κB-related proteins, B22 significantly reduced the levels of phosphorylated IκB-α and phosphorylated IKK-α / β (compared with the model group, P < 0.001), suggesting that B22 can affect IκB kinase and IKK, leading to their phosphorylation and proteasomal degradation, thereby affecting the NF-κB signaling pathway. Furthermore, the derivative B22 effectively inhibited the expression of COX-2 and iNOS proteins (statistically significant compared with the model group, P values less than 0.001 and 0.01, respectively), suggesting that B22 can also affect the levels of proteins downstream of the NF-κB pathway.

[0323] The specific experimental methods are as follows:

[0324] (1) Sample preparation: The colon tissue was removed from liquid nitrogen and quickly ground into powder in a mortar (liquid nitrogen was continuously added during the grinding process), and then RIPA lysis buffer was added at a ratio of 100 mg tissue to 150 μL RIPA lysis buffer. Vortex once every 5 minutes. After lysis for about 30 minutes, ultrasonicate 5 times, each ultrasonicate for 3 seconds, with an interval of 9 seconds. Then centrifuge at 13000 rpm and 4°C for 15 minutes, take the supernatant and add it to a new EP tube. The protein concentration was determined by Bradford method, and 6× loading buffer was added and placed in a water bath above 95°C for 5 minutes. Cool and set aside.

[0325] (2) Electrophoresis: Prepare an SDS-PAGE gel of appropriate concentration based on the size of the target protein to be separated; load approximately 30 μg of sample based on the protein sample concentration; start electrophoresis at 80 V. When the sample passes through the stacking gel, switch to 120 V and continue electrophoresis. Stop electrophoresis when the loading buffer moves to the bottom of the gel.

[0326] (3) Transfer: Prepare transfer buffer in advance and pre-cool it, and place the gel after electrophoresis separation in the buffer; cut off the separation gel part and place it on the transfer clamp, cut a PVDF membrane of appropriate size, place it in methanol for soaking and activation, place the activated PVDF membrane on the gel, remove the bubbles between the gel and the membrane, and prepare a sandwich transfer device; determine the transfer current conditions according to the size of the protein, and after the transfer is completed, remove the PVDF membrane and briefly wash it in TBS for 5 minutes.

[0327] (4) Blocking, antibody incubation, and development: The transferred PVDF membrane was blocked in 5% skim milk for 2 h, then incubated with the primary antibody at 4°C overnight. The next day, the membrane was washed three times with TBS / T, each for 10 min. The membrane was then incubated with the secondary antibody at room temperature for 45 min, and then washed three times with TBS / T, each for 10 min. After washing, the developing solution was added and the membrane was developed.

[0328] (5) Imaging analysis: The protein bands were scanned in grayscale using Image J software using a multifunctional imaging analysis system. β-action was used as an internal reference to calculate and analyze the optical density of each target band.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is as follows ,Mode or As shown: Formula I Formula II Formula III in, Mode The compound is selected from: or ; Mode The compound is selected from: or ; Mode The compound is selected from: 。 2. A pharmaceutical composition, characterized in that: The active ingredient is the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and is supplemented with pharmaceutically acceptable auxiliary ingredients.

3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 2 in the preparation of a small molecule HDAC6 inhibitor.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and the pharmaceutical composition according to claim 2 in the preparation of a drug for preventing and treating HDAC6-related inflammatory diseases; wherein, The HDAC6-related inflammatory disease is enteritis.

5. The use according to claim 4, characterized in that: The enteritis is ulcerative colitis.

6. The method for preparing the compound according to claim 1, characterized in that: Synthesis route 1: ； Synthesis route 2: ； The preparation method of the compounds of Formula I and Formula II is Synthesis Route 1, comprising the following steps: a. Dissolve raw materials A-1 / A-2 in solvent a, add alkaline reagent a, and then add chloromethyl methyl ether to react to form intermediates B-1 / B-2; b. Reducing the raw materials B-1 / B-2, catalyst b, and solvent b under a hydrogen atmosphere to generate intermediates C-1 / C-2; c. Dissolve intermediate C-1 / C-2 and paraformaldehyde in solvent c, add alkaline reagent c, and then add reducing agent c to react to form intermediate D-1 / D-2; d. Dissolve the raw material E in the solvent d, add the brominating agent d, add phosphorus pentoxide, and react to form the intermediate F; e. Dissolve intermediate D-1 / D-2 and intermediate F in solvent e, add alkaline reagent e, and undergo substitution reaction to generate intermediate G-1 / G-2; f. Dissolve the intermediate G-1 / G-2 in solvent f, add alkaline reagent f, and undergo hydrolysis reaction to generate intermediate H-1 / H-2; g. Dissolve intermediate H-1 / H-2 and alkaline reagent g in solvent g, add bromine-substituted ester I, and react to form intermediate J-1 / J-2; h. Dissolve the intermediate J-1 / J-2 in solvent h, add alkaline reagent h, and then add an aqueous solution of hydroxylamine to produce a compound of formula I / formula II through hydrolysis reaction; Mode The preparation method of the compound is a synthetic route 2, comprising the following steps: i. Dissolve raw material E, raw material K, alkaline reagent i and catalyst i in solvent i, and generate intermediate L through substitution reaction; 1. Dissolve the intermediate L in solvent 1, add alkaline reagent 1, and then add an aqueous solution of hydroxylamine to produce a compound of formula III through hydrolysis reaction; Alternatively, j. reacting the raw material E with phosphorus oxychloride to form an intermediate M; k. Dissolve the intermediate M, the raw material HO-X-COOR, the alkaline reagent k and the catalyst k in the solvent k, and generate the intermediate N through substitution reaction; 1. Dissolve intermediate N in solvent 1, add alkaline reagent 1, and then add an aqueous solution of hydroxylamine to undergo hydrolysis reaction to generate a compound of formula III.

7. The preparation method according to claim 6, characterized in that: At least one of the following must be met: In step a, the solvent a is N,N-dimethylformamide; In step a, the alkaline reagent a is 60% sodium hydride; In step a, the molar ratio of raw material A-1 / A-2: alkaline reagent a: chloromethyl methyl ether is 1:1~3:1~3; In step b, the catalyst b is 5% or 10% palladium on carbon; In step b, the solvent b is methanol; In step c, the solvent c is methanol; In step c, the alkaline reagent c is sodium methoxide or potassium methoxide; In step c, the reducing agent c is sodium borohydride or potassium borohydride; In step c, the molar ratio of the intermediate C-1 / C-2: paraformaldehyde: alkaline reagent c: reducing agent c is 1:1.0-1.5:1-3:1-3; In step d, the solvent d is toluene; In step d, the brominating agent d is tetrabutylammonium bromide; In step d, the molar ratio of the raw material E: the brominating agent d: phosphorus pentoxide is 1-2:1-2:1; In step e, the solvent e is N,N-dimethylformamide; In step e, the alkaline reagent e is DMAP; In step e, the molar ratio of intermediate D-1 / D-2: intermediate F: alkaline reagent e is 0.8~1.2:1:0.05~0.2; In step f, the solvent f is a 1,4-dioxane solution of hydrogen chloride; In step f, the alkaline reagent f is sodium bicarbonate or potassium bicarbonate; In step g, the solvent g is acetonitrile; In step g, the alkaline reagent g is cesium carbonate; In step g, the molar ratio of the intermediate H-1 / H-2: alkaline reagent g: bromo-substituted ester I is 1:1~3:1~3; In step h, the solvent h is methanol; In step h, the alkaline reagent h is potassium carbonate or sodium carbonate; In step i, the alkaline reagent i is potassium carbonate or sodium carbonate; In step i, the catalyst i is potassium iodide; In step i, the solvent i is N,N-dimethylformamide; In step i, the molar ratio of raw material E: raw material K: alkaline reagent i: catalyst i is 1:1-3:1-3:0.05-0.2; In step k, the alkaline reagent k is potassium carbonate or sodium carbonate; In step k, the catalyst k is potassium iodide; In step k, the solvent k is acetonitrile; In step k, the molar ratio of the intermediate M: the raw material HO-X-COOR: the alkaline reagent k: the catalyst k is 1:1-3:1-3:0.05-0.2; In step 1, the solvent 1 is methanol; In step 1, the alkaline reagent 1 is potassium carbonate or sodium carbonate.

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