Targeting histone deacetylase 8 proteolysis body, methods of making and uses thereof

Abstract

The application discloses a targeted histone deacetylase 8 proteolysis chimera, a preparation method and application thereof, and relates to the technical field of drug design and synthesis. The targeted histone deacetylase 8 proteolysis chimera provided by the application is an HDAC8-PROTACs targeted proteolysis chimera designed based on the structure of PCI-34051 and VHL as an E3 ligase. Experiments prove that the targeted proteolysis chimera can significantly degrade deacetylase 8 in tumor cells, and indicates the value of the targeted proteolysis chimera in preparation of drugs for preventing or treating diseases related to abnormal activity or expression of HDAC8, especially anti-tumor active drugs.

Description

Technical Field

[0001] This invention relates to the field of drug design and synthesis technology, and in particular to a target histone deacetylase 8 protein hydrolysis chimera, its preparation method and its application. Background Technology

[0002] Protein degradation-targeting chimeras (PROTACs) technology is a novel strategy for chemically inducing target protein degradation via the ubiquitin-proteasome pathway. The mechanism of action of PROTACs inducing protein degradation is as follows: After entering the cell, the target protein ligand (POI) at the PROTAC's structure specifically binds to the target protein (POI), while the E3 ligand at the other end binds to the E3 ligase, forming a ternary complex of POI-PROTAC-E3 ligase. Then, the E3 ligase mediates the ubiquitin-binding enzyme E2, which ubiquitinates the target protein POI in the ternary complex. After the ternary complex dissociates, the multiubiquitinated target protein is transported to the proteasome for degradation, thereby selectively reducing the target protein level.

[0003] As of December 2021, the number of protein targets that can be degraded by PROTACs has increased to more than 130, covering multiple disease areas such as cancer, immune disorders, viral infections, and neurodegenerative diseases, with cancer being the primary application. Among them, Arvinas' ARV-110 and ARV-471 have entered Phase II clinical trials, making them the most advanced PROTAC drugs in clinical trials. Some R&D startups have already attracted the attention of major global pharmaceutical companies such as Roche, Sanofi, Merck, Pfizer, and Gilead.

[0004] Histone deacetylases (HDACs), as important functional proteins in epigenetic regulation, have attracted widespread attention from scientists in recent years. HDAC8 is a class I HDAC, namely Zn... 2+HDAC8-dependent hematoxylin and eosinophils (HDACs), mainly distributed in the nucleus and cytoplasm, typically induce histone deacetylation and inhibit gene transcription. Dysregulation of HDAC8 is closely related to the development and progression of various diseases, especially tumors such as gastric cancer, colon cancer, lung cancer, and breast cancer. With the deepening research into the structure and function of the HDAC8 target, small-molecule selective inhibitors targeting HDAC8 have been reported, such as PCI-34051, NCC149, OJI-1, and A8B4. However, these inhibitors have poor efficacy and significant side effects. Given the unique mechanism of action of PROTACs, such as their ability to target the non-enzymatic functions of proteins, the development of PROTAC-related drugs targeting HDAC8 is of great significance for the treatment of tumors and other diseases. To date, only one HDAC8-PROTAC degrader based on the HDAC8 inhibitor NCC-149 has been reported in the literature (Chem. Commun. 2022, 58, 4635-4638.). The inventors' research group is currently developing an HDAC8-PROTAC degrader based on the structure of the HDAC8 inhibitor PCI-34051 and CRBN as an E3 ligase (CN202210122650.8). Building upon this foundation, developing more selective drugs targeting the degradation of histone deacetylase 8 is of great significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the deficiency of the prior art as described in the background art, and provides a targeted histone deacetylase 8 protein hydrolysis chimera, its preparation method and its application.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] In a first aspect, the present invention provides a chimeric target histone deacetylase 8 protein hydrolysis compound, said chimeric compound having at least one compound as shown in formulas 9X-1 and 9X-2, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] In Equations 9X-1 and 9X-2, the chiral sites can be either R or S, m is an integer from 1 to 4, and n is an integer from 1 to 4.

[0010] In a second aspect, the present invention provides a method for preparing a target histone deacetylase 8 protein hydrolysis chimera as described in the first aspect, the preparation method comprising the following steps:

[0011] S1. Compound 1 and Compound 2 are dissolved in organic solvents to obtain a first solution and a second solution, respectively. NaH is added to the first solution to react and obtain a reaction solution. Then, the second solution is added dropwise to the reaction solution to continue the reaction and obtain compound 3.

[0012] S2. Compound 3 was dissolved in methanol solution, and Pd / C was added to react and give compound 4.

[0013] S3. Dissolve compound 4, compound 5A or compound 5B, and potassium carbonate in anhydrous DMF, stir and heat to 90°C, react for 8 hours, and after the reaction is complete, obtain compound 6a-6h.

[0014] S4. Dissolve compound 6a-6h in anhydrous dichloromethane and react it with trifluoroacetic acid at room temperature to obtain a reaction solution. Add petroleum ether to the reaction solution, stir evenly, and remove the solvent under vacuum to obtain a concentrated residue. Dissolve compound 7, EDCI, and HOBt in DMF and add them to the above concentrated residue. Cool to 5°C, add DIPEA, restore the temperature to room temperature, and continue the reaction for 18 hours to obtain compound 8X-1 or 8X-2.

[0015] S5. Cool the NH2OH solution to 5℃, add an alkaline reagent, and stir to dissolve to obtain a hydroxylamine solution; dissolve compound 8X-1 or 8X-2 in MeOH / THF solution, then add the hydroxylamine solution, and after the reaction is complete, add an acid reagent to neutralize to obtain compound 9X-1 or 9X-2.

[0016] The synthetic route for steps S1-S2 is as follows:

[0017]

[0018] The synthetic route for step S3 is as follows:

[0019]

[0020] The synthetic route for step S4 is as follows:

[0021]

[0022] In this compound, the chiral site of compound 7 can be either R or S; m is an integer from 1 to 4, and n is an integer from 1 to 4.

[0023] Specifically, due to the different chiral structures, compound 7 of the present invention can arbitrarily take any of the following five structures:

[0024]

[0025] Specifically, when the structural formula of compound 7 is as shown in 7-1 below,

[0026]

[0027] For compound 5A, m=1, the synthesized compound is 9a:

[0028]

[0029] For compound 5A, m=2, the synthesized compound is 9b:

[0030]

[0031] For compound 5A, m=3, the synthesized compound is 9c:

[0032] For compound 5A, m=4, and the synthesized compound is 9d:

[0033] For compound 5B, n=1, the synthesized compound is 9e:

[0034]

[0035] For compound 5B, n=2, the synthesized compound is 9f:

[0036] For compound 5B, n=3, and the synthesized compound weighs 9g:

[0037]

[0038] For compound 5B, n=4, and the synthesized compound is 9h:

[0039]

[0040] Furthermore, steps S1-S5 also include steps of concentrating, separating, and purifying the products obtained in each step.

[0041] Specifically, in steps S1-S5, in order to improve the yield and purity of the corresponding products, after obtaining the preliminary reaction products, further steps are included to concentrate, separate and purify the products by means of extraction, washing, concentration and separation by silica gel column.

[0042] Further, in step S1, the organic solvent is N,N-dimethylacetylamine.

[0043] Furthermore, in step S4, the concentrated residue does not require purification.

[0044] Further, in step S5, the alkaline reagent is sodium hydroxide; the acidic reagent is acetic acid.

[0045] Furthermore, in step S5, the volume ratio of MeOH to THF in the MeOH / THF solution is 1:1.

[0046] Thirdly, the present invention provides a pharmaceutical composition comprising a target histone deacetylase 8 protein hydrolysis chimera as described in the first aspect, or a target histone deacetylase 8 protein hydrolysis chimera prepared by the preparation method described in the second aspect.

[0047] Furthermore, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0048] The pharmaceutical composition of the present invention uses the target histone deacetylase 8 protein hydrolysis chimera as described in the first aspect as the active ingredient. Variations in the formulation system and administration method, derivatives of the chimera after simple chemical modification, pharmaceutical salts, multiple compounds, and multiple degradation agents are not excluded.

[0049] Specifically, the pharmaceutically acceptable salts of the compounds provided by the present invention as described above can be: sodium salts, potassium salts, ammonium salts, amino acid salts, lactates, hydrochlorides, phosphates, acetates, malates, citrates, or aspartates, etc. The present invention does not specifically limit the pharmaceutical salts.

[0050] In this invention, the target histone deacetylase 8 protein hydrolysis chimera of this invention can be formulated as an active ingredient in a non-toxic, inert, and pharmaceutically acceptable carrier medium; the formulated drug can be administered via conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.

[0051] When the pharmaceutical composition of the present invention is in the form of a drug for oral administration, it contains a safe and effective amount of the target histone deacetylase 8 protein hydrolysis chimera of the present invention and a pharmaceutically acceptable carrier and / or excipient. The drug for oral administration can be formulated into commonly used dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, and suppositories. In the present invention, no specific limitation is made on the carrier and / or excipient.

[0052] The pharmaceutical composition of the present invention can also be formulated into an injection, which can be prepared in an aseptic environment with water for injection, physiological saline, or glucose solution. The above-mentioned injection can be prepared by conventional methods.

[0053] Fourthly, the present invention provides the use of the target histone deacetylase 8 protein hydrolysis chimera as described in the first aspect and the pharmaceutical composition as described in the third aspect in the preparation of medicaments for the prevention or treatment of diseases related to abnormal HDAC8 activity or expression.

[0054] Specifically, the aforementioned related diseases include hematologic malignancies, solid tumors and other tumors, viral infections, and parasitic diseases.

[0055] Compared with the prior art, the technical effects achieved by the present invention include:

[0056] The histone deacetylase 8-targeting proteolytic chimera provided by this invention is an HDAC8-PROTAC designed based on the PCI-34051 structure and VHL as an E3 ligase. It has the effect of selectively targeting the degradation of histone deacetylase 8. Experiments have shown that it can significantly degrade deacetylase 8 in tumor cells, suggesting its value in the preparation of drugs for the prevention or treatment of diseases related to abnormal HDAC8 activity or expression, especially anti-tumor drugs. Attached Figure Description

[0057] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0058] Figure 1 This is a schematic diagram of the test results of the protein degradation experiment of the present invention. Detailed Implementation

[0059] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0060] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0061] The term "pharmaceutical composition" means one or more of the compounds described herein or their physiologically / pharmaceutical acceptable salts or prodrugs or mixtures in combination with other active pharmaceutical ingredients.

[0062] Example 1

[0063] This embodiment provides a method for preparing the above-mentioned target histone deacetylase 8 protein hydrolysis chimera. Specifically, the preparation method includes the following steps:

[0064] Synthesis of Compound 3

[0065] The synthesis route is shown below:

[0066]

[0067] The specific steps are as follows:

[0068] Compound 1 (3.0 g, 17.1 mmol) was dissolved in 5 mL of N,N-dimethylacetylamine. The solution was cooled to 0 °C, and NaH (1.1 g, 42.8 mmol) was slowly added in portions. The reaction was continued for 1 h. Then, compound 2 (4-benzyloxybenzyl chloride) (4.8 g, 21.1 mmol) was dissolved in 5 mL of N,N-dimethylacetylamine and slowly added dropwise to the above reaction solution. The reaction was continued at 0 °C for 15 min. After that, the temperature was slowly restored to room temperature, and the reaction was continued for 2 h. After the reaction was completed by TLC, 5 mL of ice water was slowly added, and the mixture was stirred for another 5 min. The mixture was then extracted with ethyl acetate. The organic phases were combined and washed three times (30 mL) with saturated brine. After washing and concentrating the organic phase, it was purified by silica gel column chromatography (VEA:VPE = 1:4) to obtain compound 3 (4.8 g), a white solid, with a yield of 75%.

[0069] The structural characterization data of compound 3 are as follows:

[0070] 1 H NMR (600MHz, d6-DMSO): δ = 8.94 (brs, 1H), 8.09 (s, 1H), 7.73-7.75 (m, 1H), 7.60-7.62 (m, 1H), 7.13 (d, J=8.2Hz,1H),7.00(d,J=8.2Hz,2H),6.75-6.78(m,2H),6.53-6.54(m,1H),5.28(s,2H),3.90(s,3H). 13 C NMR (125MHz, d6-DMSO): δ=167.9,157.1,135.4,132.3,131.6,129.4,128.4,127.4,122.9,120.4,115.7,112.0,101.7,51.8,49.7.HRMS(EI)calcd.For[C 24 H 22 NO3](M+H) + :372.1594,found:372.1596.

[0071] Synthesis of Compound 4

[0072] The synthesis route is as follows:

[0073]

[0074] The specific steps are as follows:

[0075] Compound 3 (3.6 g, 9.7 mmol) was weighed and dissolved in 10 mL of methanol. Pd / C (0.4 g) was added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the solvent was weighed under reduced pressure and purified by silica gel column chromatography (VEA:VPE = 1:3) to obtain compound 4 (2.5 g), a white solid, with a yield of 92%.

[0076] The structural characterization data of compound 4 are as follows:

[0077] 1 H NMR (600MHz, d6-DMSO): δ = 8.09 (s, 1H), 7.62-7.67 (m, 3H), 7.40 (d, J = 8.4Hz, 2H), 7.36 (d, J = 8.4Hz, 2H), 7.13 (d, J =8.2Hz,2H),6.92-6.95(m,3H),6.53(d,J=10.0Hz,1H),6.56-6.58(m,1H),5.44(s,2H),5.04(s,2H),3.84(s,3H). 13 C NMR (125MHz, d6-DMSO): δ=167.5,158.2,155.4,137.3,135.4,132.1,132.4,130.4,128.7,127 .9,127.5,122.8,120.7,120.2,115.4,112.4,101.8,69.7,52.1,49.2.HRMS(EI)calcd.For[C 17 H 16 NO3](M+H) + :282.1125,found:282.1126.

[0078] Synthesis of Compound 6

[0079] The synthesis route is as follows:

[0080]

[0081] The specific steps are as follows:

[0082] Taking the synthesis of 6a as an example: Compound 4 (112 mg, 0.4 mmol), p-toluenesulfonate derivative 5A (m = 1) (289 mg, 1.2 mmol), and potassium carbonate (0.2 g, 1.6 mmol) were dissolved in 4 mL of anhydrous DMF, stirred, and heated to 90 °C for 8 h. After the reaction was complete, the reaction system was cooled to room temperature, the reaction solution was extracted three times with ethyl acetate, washed twice with saturated brine, the organic layer was separated and concentrated, and the residue was purified by silica gel column chromatography to obtain compound 6a (103 mg, 61%) as a colorless oil.

[0083] The chemical formula of compound 6a is as follows:

[0084]

[0085] The structural characterization data of compound 6a are as follows:

[0086] Methyl 1-(4-(4-(tert-butoxy)-4-oxobutoxy)benzyl)-1H-indole-6-carboxylate(6a).Colorless oil(103mg, 61%). 1 H NMR (600MHz, CDCl3): δ = 8.09 (s, 1H), 7.80 (d, J = 8.0Hz, 1H), 7.65 (d, J = 8.0Hz, 1H), 7.20 (d, J = 6.0Hz, 1H), 7.04 (d, J = 8.0Hz, 2H), 6.81 (d, J = 8.0Hz, 2H) ,6.54(d,J=6.0Hz,1H),5.26(s,2H),3.91(s,3H),3.90(t,J=6.0Hz,2H),2.21(t,J=6.0Hz,2H),1.67-1.70(m,2H),1.47(s,9H).HRMS(EI)calcd.For[C 25 H 30 NO5](M+H) + :424.2118,found:424.2123.

[0087] The synthesis method of compound 6b is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with p-toluenesulfonate derivative 5A (m=2). The resulting compound 6b is a colorless oil (91 mg) with a yield of 52%.

[0088] The chemical formula of compound 6b is as follows:

[0089]

[0090] The structural characterization data of compound 6b are as follows:

[0091] Methyl 1-(4-((5-(tert-butoxy)-5-oxopentyl)oxy)benzyl)-1H-indole-6-carboxylate(6b).Colorless oil(91mg, 52%). 1 H NMR (600MHz, CDCl3): δ = 8.10 (s, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.66 (d, J = 8.0Hz, 1 H),7.20(d,J=6.0Hz,1H),7.04(d,J=8.0Hz,2H),6.82(d,J=8.0Hz,2H),6.53(d, J=6.0Hz,1H),5.27(s,2H),3.91(s,3H),3.90(t,J=6.0Hz,2H),2.21(t,J=6.0Hz ,2H),1.68-1.71(m,2H),1.86-1.70(m,2H),1.47(s,9H).HRMS(EI)calcd.For[C 26 H 32 NO5](M+H) + :438.2275,found:438.2270.

[0092] The synthesis method of compound 6c is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with p-toluenesulfonate derivative 5A (m=3). The resulting compound 6c is a colorless oil (71 mg) with a yield of 39%.

[0093] The chemical formula of compound 6c is as follows:

[0094]

[0095] The structural characterization data of compound 6c are as follows:

[0096] Methyl 1-(4-((6-(tert-butoxy)-6-oxohexyl)oxy)benzyl)-1H-indole-6-carboxylate(6c).Colorless oil(71mg, 39%). 1H NMR (600MHz, CDCl3): δ = 8.09 (s, 1H), 7.80 (d, J = 8.0Hz, 1H), 7.65 (d, J = 8.0Hz, 1 H),7.19(d,J=6.0Hz,1H),7.04(d,J=8.0Hz,2H),6.82(d,J=8.0Hz,2H),6.54(d, J=6.0Hz,1H),5.26(s,2H),3.91(s,3H),3.90(t,J=6.0Hz,2H),2.20(t,J=6.0Hz ,2H),1.68-1.72(m,2H),1.85-1.69(m,4H),1.47(s,9H).HRMS(EI)calcd.For[C 27 H 34 NO5](M+H) + :452.2431,found:452.2430.

[0097] The synthesis method of compound 6d is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with p-toluenesulfonate derivative 5A (m=4). The resulting compound 6d is a colorless oil (70 mg) with a yield of 38%.

[0098] The chemical formula of compound 6d is as follows:

[0099]

[0100] The structural characterization data of compound 6d are as follows:

[0101] Methyl 1-(4-((7-(tert-butoxy)-7-oxoheptyl)oxy)benzyl)-1H-indole-6-carboxylate(6d).Colorless oil(70mg, 38%). 1 H NMR (600MHz, CDCl3): δ = 8.09 (s, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.65 (d, J = 8.0Hz, 1 H),7.20(d,J=6.0Hz,1H),7.04(d,J=8.0Hz,2H),6.82(d,J=8.0Hz,2H),6.53(d, J=6.0Hz,1H),5.26(s,2H),3.91(s,3H),3.90(t,J=6.0Hz,2H),2.21(t,J=6.0Hz ,2H),1.68-1.72(m,2H),1.86-1.70(m,6H),1.47(s,9H).HRMS(EI)calcd.For[C 28 H35 NO5](M+H) + :465.2510,found:465.2515.

[0102] The synthesis method of compound 6e is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with brominated compound 5B (n=1). The resulting compound 6e is a colorless oil (132 mg) with a yield of 75%.

[0103] The chemical formula of compound 6e is as follows:

[0104]

[0105] The structural characterization data of compound 6e are as follows:

[0106] Methyl 1-(4-(2-(2-(tert-butoxy)-2-oxoethoxy)ethoxy)benzyl)-1H-indole-6-carboxylate(6e).Colorless oil(132mg, 75%). 1 H NMR (600MHz, CDCl3): δ = 8.09 (s, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.63 (d, J = 8.0Hz, 1H), 7.21 (d, J = 6.0Hz, 1H), 7.03 (d, J = 8.0Hz, 2H), 6.83 (d, J = 8.0Hz, 2 H),6.54(d,J=6.0Hz,1H),5.26(s,2H),4.10(t,J=6.0Hz,2H),4.06(s,2H),3.90(s,3H),3.87(t,J=6.0Hz,2H),1.47(s,9H).HRMS(EI)calcd.For[C 25 H 30 NO6](M+H) + :440.2068,found:440.2070.

[0107] The synthesis method of compound 6f is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with brominated compound 5B (n=2). The resulting compound 6f is a colorless oil (116 mg) with a yield of 60%.

[0108] The chemical formula of compound 6f is as follows:

[0109]

[0110] The structural characterization data of compound 6f are as follows:

[0111] Methyl 1-(4-(2-(2-(2-(tert-butoxy)-2-oxoethoxy)ethoxy)ethoxy)benzyl)-1H-indole-6-carboxyl ate(6f).Colorless oil(116mg, 60%). 1 H NMR (600MHz, CDCl3): δ = 8.12 (s, 1H), 7.81 (d, J = 8.0Hz, 1H), 7.66 (d, J = 8.0Hz, 1H), 7. 25(d,J=6.0Hz,1H),7.07(d,J=8.0Hz,2H),6.87(d,J=8.0Hz,2H),6.58(d,J=6.0Hz,1 H),5.32(s,2H),4.11(t,J=6.0Hz,2H),4.04(s,2H),3.93(s,3H),3.86(t,J=6.0Hz,2 H),3.74(t,J=6.0Hz,2H),3.50(t,J=6.0Hz,2H),1.48(s,9H).HRMS(EI)calcd.For[C 27 H 34 NO7](M+H) + :484.2330,found:484.2332.

[0112] The synthesis method of compound 6g is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with brominated compound 5B (n=3). The resulting compound 6g is a colorless oil (148mg) with a yield of 70%.

[0113] The chemical formula of 6g of compound is as follows:

[0114]

[0115] The structural characterization data of compound 6g are as follows:

[0116] Methyl 1-(4-((13,13-dimethyl-11-oxo-3,6,9,12-tetraoxatetradecyl)oxy)benzyl)-1H-indole-6-ca rboxylate(6g).Colorless oil(148mg,70%). 1H NMR (600MHz, CDCl3): δ = 8.10 (s, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.65 (d, J = 8.0Hz, 1H),7.24(d,J=6.0Hz,1H),7.06(d,J=8.0Hz,2H),6.85(d,J=8.0Hz,2H),6.56( d,J=6.0Hz,1H),5.31(s,2H),4.09(t,J=6.0Hz,2H),4.01(s,2H),3.92(s,3H) ,3.84(t,J=6.0Hz,2H),3.67-3.72(m,8H),1.46(s,9H).HRMS(EI)calcd.For[C 29 H 38 NO8](M+H) + :528.2592,found:528.2594.

[0117] The synthesis method of compound 6h is the same as that of compound 6a. Specifically, it is synthesized by reacting compound 4 with brominated compound 5B (n=4). The resulting compound 6h is a colorless oil (128 mg) with a yield of 56%.

[0118] The chemical formula of compound 6h is as follows:

[0119]

[0120] The structural characterization data of compound 6h are as follows:

[0121] Methyl 1-(4-((16,16-dimethyl-14-oxo-3,6,9,12,15-pentaoxaheptadecyl)oxy)benzyl)-1H-indole-6-carboxylate(6h).Colorless oil(128mg, 56%). 1H NMR (600MHz, CDCl3): δ = 8.10 (s, 1H), 7.79 (d, J = 8.0Hz, 1H), 7.64 (d, J = 8.0Hz, 1H), 7.24(d,J=6.0Hz,1H),7.05(d,J=8.0Hz,2H),6.84(d,J=8.0Hz,2H),6.56(d,J=6.0H z,1H),5.31(s,2H),4.09(t,J=6.0Hz,2H),4.01(s,2H),3.92(s,3H),3.83(t,J=6. 0Hz,2H),3.69-3.72(m,6H),3.66-3.68(m,6H),1.47(s,9H).HRMS(EI)calcd.For[C 31 H 42 NO9](M+H) + :572.2854,found:572.2856.

[0122] Synthesis of compound 8 (8X-1 or 8X-2)

[0123] The synthesis route is as follows:

[0124]

[0125] Since the chiral site of compound 7 can be either R or S, this embodiment uses compound 7-1 as an example to introduce the specific method for synthesizing compound 8 (8X-1 or 8X-2).

[0126]

[0127] The specific steps are as follows:

[0128] Taking the synthesis of 8a as an example: 6a (70 mg, 0.2 mmol) was dissolved in 2 mL of anhydrous dichloromethane, and 0.35 mL of trifluoroacetic acid was slowly added dropwise over 3 minutes. After the addition was complete, the reaction was continued at room temperature for 3-5 hours. 10 mL of petroleum ether was added to the reaction solution, and the mixture was stirred for 10 minutes. The solvent was then removed under vacuum to obtain a concentrated residue, which was used for the next coupling reaction without purification. Compound 7-1 (89 mg, 0.2 mmol), EDCI (96 mg, 0.5 mmol), and HOBt (76 mg, 0.5 mmol) were weighed and dissolved in 3 mL of DMF. This solution was added to the concentrated residue, cooled to 5°C, and then DIPEA (0.1 mL, 0.6 mmol) was added. The temperature was restored to room temperature, and the reaction was continued for 18 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain compound 8a (87 mg, 56%) as a white solid.

[0129] The chemical formula of compound 8a is as follows:

[0130]

[0131] The structural characterization data of compound 8a are as follows:

[0132] Methyl 1-(4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)ca rbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutoxy)benzyl)-1H-indole-6-carboxylate(8a).Whitesolid(87mg,56%). 1 H NMR (600MHz, CDCl3): δ = 8.65 (s, 1H), 8.15 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.64 (d, J = 8.0Hz, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.3 4-7.40(m,5H),7.23(d,J=6.0Hz,1H),7.05(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,2H),6.58(d,J=6.0Hz,1H),5.31(s,2H),5.0 8-5.10(m,1H),4.76(t,J=6.0Hz,1H),4.55(d,J=6.0Hz,1H),4.50-4.51(m,1H),4.30-4.31(m,2H),3.92(s,3H),3.80(t,J =6.0Hz,3H),2.47(s,3H),2.35-2.45(m,2H),2.05-2.09(m,4H),1.49(d,J=6.0Hz,3H),1.05(s,9H).HRMS(EI)calcd.for[C 44 H 52 [N5O7S](M+H) + :794.3582,found:794.3588.

[0133] The synthesis method of compound 8b is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6b and reacted with compound 7-1 to obtain compound 8b as a white solid (81 mg) with a yield of 51%.

[0134] The chemical formula of compound 8b is as follows:

[0135]

[0136] The structural characterization data of compound 8b are as follows:

[0137] Methyl 1-(4-((5-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolid in-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)benzyl)-1H-indole-6-carboxylate(8b).White solid(81mg,51%). 1 H NMR (600MHz, CDCl3): δ = 8.66 (s, 1H), 8.15 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.66 (d, J = 8.0Hz, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.34-7. 40(m,5H),7.24(d,J=6.0Hz,1H),7.05(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,2H),6.59(d,J=6.0Hz,1H),5.31(s,2H),5.09-5.10(m ,1H),4.77(t,J=6.0Hz,1H),4.56(d,J=6.0Hz,1H),4.50-4.51(m,1H),4.31-4.32(m,2H),3.91(s,3H),3.79(t,J=6.0Hz,3H),2 .46(s,3H),2.36-2.45(m,2H),2.06-2.09(m,4H),1.51-1.68(m,2H),1.49(d,J=6.0Hz,3H),1.06(s,9H).HRMS(EI)calcd.for[C 45 H 54 [N5O7S](M+H) + :808.3738,found:808.3740.

[0138] The synthesis method of compound 8c is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6c and reacted with compound 7-1 to obtain compound 8c as a white solid (78 mg) with a yield of 48%.

[0139] The chemical formula of compound 8c is as follows:

[0140]

[0141] The structural characterization data of compound 8c are as follows:

[0142] Methyl 1-(4-((6-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl )-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)oxy)benzyl)-1H-indole-6-carboxylate(8c).Whitesolid(78mg, 48%). 1 H NMR (600MHz, CDCl3): δ = 8.67 (s, 1H), 8.16 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.64 (d, J = 8.0Hz, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.35-7.40 (m ,5H),7.25(d,J=6.0Hz,1H),7.06(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,2H),6.70(d,J=6.0Hz,1H),5.32(s,2H),5.09-5.10(m,1H),4.7 8(t,J=6.0Hz,1H),4.56(d,J=6.0Hz,1H),4.50-4.52(m,1H),4.30-4.31(m,2H),3.90(s,3H),3.79(t,J=6.0Hz,3H),2.47(s,3H),2. 38-2.44(m,2H),2.07-2.09(m,4H),1.51-1.69(m,2H),1.49(d,J=6.0Hz,3H),1.28-1.29(m,2H),1.06(s,9H).HRMS(EI)calcd.for[C 46 H 56 [N5O7S](M+H) + :822.3895,found:822.3900.

[0143] The synthesis method of compound 8d is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6d and reacted with compound 7-1 to obtain compound 8d as a white solid (82 mg) with a yield of 49%.

[0144] The chemical formula of compound 8d is as follows:

[0145]

[0146] The structural characterization data of compound 8d are as follows:

[0147] Methyl 1-(4-((7-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin -1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)oxy)benzyl)-1H-indole-6-carboxylate(8d).Colorless oil (82mg, 49%). 1 H NMR (600MHz, CDCl3): δ = 8.67 (s, 1H), 8.16 (s, 1H), 7.77 (d, J = 8.0Hz, 1H), 7.64 (d, J = 8.0Hz, 1H), 7.75 (d, J = 8.0Hz, 1H), 7.36-7.40 (m, 5H) ,7.26(d,J=6.0Hz,1H),7.06(d,J=8.0Hz,2H),6.97(d,J=8.0Hz,2H),6.71(d,J=6.0Hz,1H),5.31(s,2H),5.09-5.10(m,1H),4.78(t,J=6. 0Hz,1H),4.57(d,J=6.0Hz,1H),4.50-4.52(m,1H),4.31-4.32(m,2H),3.91(s,3H),3.79(t,J=6.0Hz,3H),2.47(s,3H),2.39-2.45(m,2H ),2.07-2.09(m,4H),1.52-1.69(m,2H),1.50(d,J=6.0Hz,3H),1.43-1.46(m,2H),1.33-1.35(m,2H),1.06(s,9H).HRMS(EI)calcd.for[C 47 H 58 [N5O7S](M+H) + :836.4051,found:836.4054.

[0148] The synthesis method of compound 8e is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6e and reacted with compound 7-1 to obtain compound 8e as a white solid (98 mg) with a yield of 62%.

[0149] The chemical formula of compound 8e is as follows:

[0150]

[0151] The structural characterization data of compound 8e are as follows:

[0152] Methyl 1-(4-(2-(2-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin -1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)benzyl)-1H-indole-6-carboxylate(8e).Colorless oil (98mg, 62%). 1 H NMR (600MHz, CDCl3): δ = 8.66 (s, 1H), 8.15 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.63 (d, J = 8.0Hz, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.3 3-7.39(m,5H),7.23(d,J=6.0Hz,1H),7.05(d,J=8.0Hz,2H),6.94(d,J=8.0Hz,2H),6.58(d,J=6.0Hz,1H),5.31(s,2H),5.0 8-5.10(m,1H),4.76(t,J=6.0Hz,1H),4.54(d,J=6.0Hz,1H),4.50-4.51(m,1H),4.07-4.08(m,4H),3.92(s,3H),3.81(t,J =6.0Hz,3H),3.25-3.30(m,2H),2.47(s,3H),2.35-2.46(m,2H),1.48(d,J=6.0Hz,3H),1.05(s,9H).HRMS(EI)calcd.for[C 44 H 52 [N5O8S](M+H) + :810.3531,found:810.3534.

[0153] The synthesis method of compound 8f is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6f and reacted with compound 7-1 to obtain compound 8f as a white solid (97 mg) with a yield of 57%.

[0154] The chemical formula of compound 8f is as follows:

[0155]

[0156] The structural characterization data of compound 8f are as follows:

[0157] Methyl 1-(4-(2-(2-(2-(((S)-1-((2S,4R))-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin -1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)benzyl)-1H-indole-6-carboxylate(8f).White solid(97mg,57%). 1 H NMR (600MHz, CDCl3): δ = 8.68 (s, 1H), 8.12 (s, 1H), 7.81 (d, J = 8.0Hz, 1H), 7.65 (d, J = 8.0Hz, 1H), 7.48 (d, J = 8.0Hz, 1H), 7.41 (d, J = 8.0Hz, 2 H),7.36(d,J=8.0Hz,2H),7.33(d,J=6.0Hz,1H),7.24(d,J=d,J=6.0Hz,1H),7.06(d,J=8.0Hz,2H),6.84(d,J=8.0Hz,2H),6.57(d,J=6.0H z,1H),5.31(s,2H),5.07-5.10(m,1H),4.75(t,J=6.0Hz,1H),4.51-4.55(m,2H),4.11-4.16(m,6H),3.92(s,3H),3.85-3.88(m,2H),3.71 -3.76(m,4H),3.60-3.62(m,1H),2.52-2.54(m,2H),2.47(s,3H),2.06(s,1H),1.46(d,J=6.0Hz,3H),1.06(s,9H).HRMS(EI)calcd.for[C 46 H 56 [N5O9S](M+H) + :854.3793,found:854.3795.

[0158] The synthesis method of compound 8g is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6g and reacted with compound 7-1 to obtain compound 8g as a white solid (90mg) with a yield of 50%.

[0159] The chemical formula of 8g of compound is as follows:

[0160]

[0161] The structural characterization data of compound 8g are as follows:

[0162] Methyl 1-(4-(((S)-13-((2S,4R)-4-hydroxy-2-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-car bonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)oxy)benzyl)-1H-indole-6-carboxylate(8g).Colorless oil(90mg,50%). 1 H NMR (600MHz, CDCl3): δ = 8.66 (s, 1H), 8.10 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.63 (d, J = 8.0Hz, 1H), 7.47 (d, J = 6.0Hz, 1H), 7.41 (d, J = 8.0Hz, 2H), 7.38 (d, J=8.0Hz,2H),7.33(d,J=6.0Hz,1H),7.24(d,J=6.0Hz,1H),7.05(d,J=8.0H z,2H),6.83(d,J=8.0Hz,2H),6.56(d,J=6.0Hz,1H),5.30(s,2H),5.06-5.0 9(m,1H),4.73(t,J=6.0Hz,1H),4.53(d,J=6.0Hz,1H),4.50-4.51(m,1H), 4.07-4.09(m,4H),3.91(s,3H),3.81(t,J=6.0Hz,3H),3.66-3.70(m,4H),3 .58-3.61(m,2H),3.28(t,J=6.0Hz,2H),3.23-3.25(m,2H),2.47(s,3H),2. 45-2.46(m,2H),1.48(d,J=6.0Hz,3H),1.05(s,9H).HRMS(EI)calcd.for[C 48 H60 N5O 10 S](M+H + :898.4055,found:898.4058.

[0163] The synthesis method of compound 8h is the same as that of compound 8a. Specifically, compound 6a is replaced by compound 6h and reacted with compound 7-1 to obtain compound 8h as a white solid (85 mg) with a yield of 45%.

[0164] The chemical formula of compound 8h is as follows:

[0165]

[0166] The structural characterization data of compound 8h are as follows:

[0167] Methyl 1-(4-(((S)-16-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)car bamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaocta decyl)oxy)benzyl)-1H-indole-6-carboxylate(8h).White solid(85mg,45%). 1H NMR (600MHz, CDCl3): δ = 8.66 (s, 1H), 8.09 (s, 1H), 7.78 (d, J = 8.0Hz, 1H), 7.63 (d, J = 8.0Hz, 1H), 7.52 (d, J = 6.0Hz, 1H), 7.35-7.39 (m, 5 H),7.23(d,J=6.0Hz,1H),7.04(d,J=8.0Hz,2H),6.83(d,J=8.0Hz,2H),6.55(d,J=6.0Hz,1H),5.29(s,2H),5.06-5.08(m,1H),4.71(t, J=6.0Hz,1H),4.56(t,J=6.0Hz,1H),4.47-4.59(m,1H),4.06(t,J=6.0Hz,2H),3.96-4.05(m,4H),3.90(s,3H),3.80(t,J=6.0Hz,2H),3 .70-3.71(m,2H),3.66-3.67(m,10H),3.60-3.62(m,1H),2.48(s,3H),2.41-2.43(m,2H),1.46(d,J=6.0Hz,3H).HRMS(EI)calcd.For[C 50 H 64 N5O 11 S](M+H + :942.4323,found:942.4323.

[0168] Synthesis of Compound 9 (9X-1, 9X-2)

[0169] Following the synthesis of compounds 8a-8h described above, the synthesis of compound 9 is now described using the synthesis of 9a as an example: 100 mg of sodium hydroxide was weighed and slowly added to a 0.5 mL solution of hydroxylamine (50 wt%) that had been cooled (5 °C), and stirring was continued for 10 minutes. 8a (79 mg, 0.1 mmol) was weighed and dissolved in 2 mL of a mixed solvent composed of THF / MeOH. This solution was then slowly added dropwise to the above mixed solvent, and the mixture was allowed to return to room temperature and the reaction continued for 2 hours. After the reaction was complete, acetic acid was added to adjust the pH of the reaction system to approximately 7. Ethyl acetate was added to the reaction system for organic phase extraction. The mixture was then washed three times with saturated brine, and the organic layer was concentrated and purified by column chromatography to obtain compound 9a (44 mg, yield 56%), which was a light white solid.

[0170] The chemical formula of compound 9a is as follows:

[0171]

[0172] The structural characterization data of compound 9a are as follows:

[0173] N-Hydroxy-1-(4-(4-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxo butoxy)benzyl)-1H-indole-6-carboxamide(9a).Off-white solid(44mg,56%). 1 HNMR(600MHz,d 6 -DMSO):δ=10.95(brs,2H),8.98(s,1H),8.37(d,J=8.0Hz,1H),7.94(s,1H),7.88(d,J=8.0Hz,1H),7.16(d,J=8.0Hz,1H),6.86(d,J=8.0Hz,2H),6.51(d,J=6.0Hz,1H),5.36(s,2H),5.09(s,1H),4.91(t,J=6.0Hz,1H),4.50(d,J=6.0Hz,1H),4.41(t,J=6.0Hz,1H),4.27(brs,1H),3.88-3.91(m,2H),3.59-3.60(m,2H),2.45(s,3H),2.34-2.38(m,1H),2.25-2.30(m,1H),1.98-2.02(m,1H),1.84-1.90(m,2H),1.75-1.80(m,3H),1.37(d,J=6.0Hz,3H),0.91(s,9H). 13 C NMR(150MHz,d 6 -DMSO):δ=176.7,175.8,174.7,163.1,156.8,156.6,152.9,149.8,145.9,140.8,140.2,136.3,135.4,135.1,134.8,134.0,133.6,132.6,131.5,131.4,125.2,119.7,106.3,73.9,72.1,70.1,63.7,61.6,52.9,42.3,40.4,36.4,31.6,30.2,27.6,21.2,20.4.HRMS(EI)calcd.for[C 43 H 51 N6O7S](M+H) +:795.3534,found:795.3593.

[0174] The synthesis method of compound 9b is the same as that of compound 9a, except that compound 8a is replaced by compound 8b in the reaction. The resulting compound 9b is a light white solid (40 mg) with a yield of 50%.

[0175] The chemical formula of compound 9b is as follows:

[0176]

[0177] The structural characterization data of compound 9b are as follows:

[0178] N-Hydroxy-1-(4-((5-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrro lidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)oxy)benzyl)-1H-indole-6-carboxamide(9b).Off-white solid(40mg,50%). 1 H NMR (600MHz, d) 6 -DMSO): δ=10.94(brs,2H),8.98(s,1H),8.38(d,J=8.0Hz,1H),7.95(s,1H),7.88(d,J=8.0Hz,1H),7.15(d,J=8.0H z,1H),6.87(d,J=8.0Hz,2H),6.50(d,J=6.0Hz,1H),5.36(s,2H),5.10(s,1H),4.91(t,J=6.0Hz,1H),4.51(d,J=6. 0Hz,1H),4.40(t,J=6.0Hz,1H),4.28(brs,1H),3.88-3.91(m,2H),3.58-3.59(m,2H),2.46(s,3H),2.35-2.38(m,1 H),2.26-2.29(m,1H),1.98-2.01(m,1H),1.84-1.91(m,2H),1.76-1.80(m,5H),1.38(d,J=6.0Hz,3H),0.90(s,9H). 13 C NMR (150MHz, d) 6-DMSO): δ=175.9,175.3,174.8,163.2,157.1,156.5,153.0,149.9,145 .9,141.0,140.1,136.3,135.6,135.3,134.9,134.1,133.8,132.4,131 .8,131.6,125.0,119.8,107.0,74.0,72.0,70.2,63.8,61.9,53.0,42.5,40.6,36.6,31.8,30.4,27.8,26.6,21.3,20.5.HRMS(EI)calcd.for[C 44 H 53 [N6O7S](M+H) + :809.3691,found:809.3693.

[0179] The synthesis method of compound 9c is the same as that of compound 9a, except that compound 8a is replaced by compound 8c in the reaction. The resulting compound 9c is a light white solid (41 mg) with a yield of 50%.

[0180] The chemical formula of compound 9c is as follows:

[0181]

[0182] The structural characterization data of compound 9c are as follows:

[0183] N-Hydroxy-1-(4-((6-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl) carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)oxy)benzyl)-1H-indol e-6-carboxamide(9c).Off-white solid(41mg,50%). 1 H NMR (600MHz, d) 6-DMSO): δ=11.02(brs,2H),8.99(s,1H),8.37(d,J=8.0Hz,1H),7.96(s,1H),7.81(d,J=8.0Hz,1H),7.62(s,1H),7.58(d,J=8.0Hz,1H),7.4 5-7.46(m,3H),7.39(d,J=8.0Hz,2H),7.17(d,J=8.0Hz,2H),6.86(d,J=8.0Hz,2H),6.51(d,J=6.0Hz,1H),5.37(s,2H),5.10(s,1H),4.92(t ,J=6.0Hz,1H),4.51(d,J=6.0Hz,1H),4.42(t,J=6.0Hz,1H),4.28(s,1H),3.89(t,J=6.0Hz,2H),3.58-3.63(m,2H),2.46(s,3H),2.24-2.28 (m,1H),2.12-2.14(m,1H),1.99-2.02(m,1H),1.77-1.81(m,2H),1.64-1.68(m,2H),1.48-1.56(m,3H),1.37(d,J=6.0Hz,3H),0.93(s,9H). 13 CNMR (150MHz, d) 6 -DMSO): δ=172.4,172.1,171.0,158.4,151.9,148.2,145.1,139.8,135. 4,131.8,131.5,130.7,130.2,130.1,129.2,128.8,126.8,120.5,118.2, 114.8,110.1,109.7,101.5,69.2,67.7,65.4,58.9,56.7,49.1,48.1,38.1,35.6,35.2,28.8,26.9,25.6,22.9,16.5,16.4.HRMS(EI)calcd.for[C 45 H 55 [N6O7S](M+H) + :823.3847,found:823.3850.

[0184] The synthesis method of compound 9d is the same as that of compound 9a, except that compound 8a is replaced by compound 8d in the reaction. The resulting compound 9d is a white solid (38 mg) with a yield of 45%.

[0185] The chemical formula of compound 9d is as follows:

[0186]

[0187] The structural characterization data of compound 9d are as follows:

[0188] N-Hydroxy-1-(4-((7-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyr rolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)oxy)benzyl)-1H-indole-6-carboxamide(9d).White solid(38mg,45%). 1 H NMR (600MHz, d) 6 -DMSO): δ=11.01(brs,2H),8.99(s,1H),8.38(d,J=8.0Hz,1H),7.96(s,1H),7.80(d,J=8.0Hz,1H),7.61(s,1H),7.58(d,J=8.0Hz,1H),7.4 6-7.47(m,3H),7.40(d,J=8.0Hz,2H),7.18(d,J=8.0Hz,2H),6.87(d,J=8.0Hz,2H),6.51(d,J=6.0Hz,1H),5.36(s,2H),5.10(s,1H),4.91(t ,J=6.0Hz,1H),4.50(d,J=6.0Hz,1H),4.41(t,J=6.0Hz,1H),4.28(s,1H),3.90(t,J=6.0Hz,2H),3.59-3.64(m,2H),2.47(s,3H),2.24-2.28 (m,1H),2.13-2.14(m,1H),1.99-2.01(m,1H),1.78-1.81(m,2H),1.65-1.69(m,2H),1.47-1.58(m,5H),1.38(d,J=6.0Hz,3H),0.93(s,9H). 13 C NMR (150MHz, d) 6-DMSO): δ=172.2,172.0,171.0,158.6,152.0,148.1,145.0,139.8,135.6 ,131.9,131.6,130.7,130.3,130.0,129.2,128.9,126.8,120.6,118.2,11 5.0,110.2,109.7,101.6,69.8,67.8,65.6,59.0,56.8,49.3,48.0,38.0,3 5.8,35.0,29.0,27.0,25.8,22.9,16.8,16.6,16.4.HRMS(EI)calcd.for[C 46 H 57 [N6O7S](M+H) + :837.4004,found:837.4010.

[0189] The synthesis method of compound 9e is the same as that of compound 9a, except that compound 8a is replaced by compound 8e in the reaction. The resulting compound 9e is a light white solid (48 mg) with a yield of 60%.

[0190] The chemical formula of compound 9e is as follows:

[0191]

[0192] The structural characterization data of compound 9e are as follows:

[0193] N-Hydroxy-1-(4-(2-(2-(((S)-1-((2S,4R))-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrro lidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)benzyl)-1H-indole-6-carboxamide(9e).Off-white solid(48mg,60%). 1 H NMR (600MHz, d) 6-DMSO): δ=11.09(brs,2H),9.0(s,1H),8.90(brs,1H),8.43(d,J=6.0Hz,1H),7.96(s,1H),7.63(s,1H),7.57(d,J=8.0Hz,1H) ,7.44-7.46(m,3H),7.34(d,J=8.0Hz,2H),7.17(d,J=8.0Hz,2H),6.97(d,J=8.0Hz,2H),6.52(d,J=6.0Hz,1H),5.39(s,2H),5. 13(s,1H),4.90-4.92(m,1H),4.56(d,J=4.0Hz,1H),4.43(t,J=6.0Hz,1H),4.29(s,1H),4.08-4.12(m,2H),4.01-3.96(m,2H) ,3.80-3.81(m,2H),3.55-3.62(m,2H),2.47(s,3H),2.02-2.06(m,1H),1.76-1.80(m,1H),1.33(d,J=6.0Hz,3H),0.93(s,9H). 13 C NMR (150MHz, d) 6 -DMSO): δ=170.9,169.4,168.8,158.2,151.9,148.2,145.1,135.5,131.8,131.5,130.7,130.1,129.3,128.7,126.8,127.7,126.0,120.5, 118.2,115.0,109.7,101.5,99.9,70.0,69.2,67.2,65.4,59.0,57.0,56.1,49.0,48.1,38.1,36.2,26.7,22.9,16.4.HRMS(EI)calcd.for[C 43 H 51 [N6O8S](M+H) + :811.3484,found:811.3533.

[0194] The synthesis method of compound 9f is the same as that of compound 9a, except that compound 8a is replaced by compound 8f in the reaction. The resulting compound 9f is a white solid (55 mg) with a yield of 64%.

[0195] The chemical formula of compound 9f is as follows:

[0196]

[0197] The structural characterization data of compound 9f are as follows:

[0198] N-Hydroxy-1-(4-(2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)benzyl)-1H-indole-6-carboxamide(9f).White solid(55mg,64%). 1 HNMR(600MHz,d 6 -DMSO):δ=11.11(brs,2H),8.98(s,1H),8.44(s,1H),7.94(s,1H),7.59(s,1H),7.56(d,J=8.0Hz,1H),7.35-7.47(m,5H),7.16(d,J=8.0Hz,2H),6.88(d,J=8.0Hz,2H),6.50(d,J=6.0Hz,1H),5.36(s,2H),5.16(s,1H),4.89(t,J=6.0Hz,1H),4.54(d,J=6.0Hz,1H),4.44(d,J=6.0Hz,1H),4.29(s,1H),4.05-4.07(m,2H),3.95-3.96(m,2H),3.73-3.75(m,2H),3.60-3.64(m,6H),2.46(s,3H),2.03-2.07(m,1H),1.75-1.77(m,1H),1.34(d,J=6.0Hz,3H),0.92(s,9H). 13 C NMR(150MHz,d 6 -DMSO):δ=170.9,169.5,169.0,168.1,158.2,151.9,148.2,145.1,135.5,131.5,130.4,130.1,130.0,129.3,128.8,126.7,126.0,120.4,118.1,114.9,109.7,101.5,99.8,70.8,70.1,69.4,69.2,59.0,56.9,56.1,49.0,48.2,38.1,36.2,31.7,26.7,25.6,22.9,16.4.HRMS(EI)calcd.For[C 45 H 55[N6O9S](M+H) + :855.3746,found:855.3804.

[0199] The synthesis method of compound 9g is the same as that of compound 9a, except that compound 8a is replaced by compound 8g in the reaction. The resulting compound 9g is a light white solid (52mg) with a yield of 58%.

[0200] The chemical formula of 9g of compound is as follows:

[0201]

[0202] The structural characterization data of compound 9g are as follows:

[0203] N-Hydroxy-1-(4-(((S)-13-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine- 1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)oxy)benzyl)-1H-indole-6-carboxamide(9g).Off-white solid(52mg,58%). 1 H NMR (600MHz, d) 6 -DMSO): δ=10.98(brs,2H),8.99(s,1H),8.44(d,J=6.0Hz,1H),7.96(s,1H),7.62(d,J=6.0Hz,1H),7.56(d,J=8.0Hz,1H),7 .44-7.45(m,3H),7.34-7.39(m,3H),7.17(d,J=8.0Hz,2H),6.89(d,J=8.0Hz,2H),6.51(d,J=6.0Hz,1H),5.37(s,2H),5.14 (brs,1H),4.89-4.91(m,1H),4.54(d,J=6.0Hz,1H),4.45(t,J=6.0Hz,1H),4.29(s,1H),4.03-4.04(m,2H),3.95(s,2H),3. 70-3.72(m,2H),3.55-3.62(m,10H),2.46(s,3H),2.04-2.07(m,1H),1.75-1.79(m,1H),1.37(d,J=6.0Hz,3H),0.93(s,9H). 13C NMR (150MHz, d) 6 -DMSO): δ=170.9,169.4,168.9,158.2,151.9,148.2,145.1,135.5,131.7, 131.5,130.4,130.1,129.3,129.2,128.8,126.7,120.5,118.1,115.0,114. 9,109.6,101.5,99.9,70.8,70.3,70.0,69.3,69.2,67.5,65.3,59.0,56.9, 56.1,49.0,48.2,38.2,36.2,26.7,25.6,22.9,16.4.HRMS(EI)calcd.for[C 47 H 59 N6O 10 S](M+H + :899.4008,found:899.4018.

[0204] The synthesis method of compound 9h is the same as that of compound 9a, except that compound 8a is replaced by compound 8h in the reaction. The resulting compound 9h is a white solid (46 mg) with a yield of 49%.

[0205] The chemical formula of compound 9h is as follows:

[0206]

[0207] The structural characterization data of compound 9h are as follows:

[0208] N-Hydroxy-1-(4-(((S)-16-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetr aoxa-15-azaoctadecyl)oxy)benzyl)-1H-indole-6-carboxamide (9h). White solid (46 mg, 49%). 1 H NMR (600MHz, d) 6-DMSO): δ=11.10(brs,2H),8.99(s,1H),8.44(d,J=6.0Hz,1H),7.96(s,1H),7.62(d,J=6.0Hz,1H),7.57(d,J=8.0Hz,1H),7 .43-7.45(m,3H),7.36-7.39(m,3H),7.18(d,J=8.0Hz,2H),5.88(d,J=8.0Hz,1H),6.51(d,J=6.0Hz,1H),5.37(s,2H),5.14 (brs,1H),4.89-4.92(m,1H),4.54(d,J=6.0Hz,1H),4.45(t,J=6.0Hz,1H),4.29(s,1H),4.02-4.04(m,2H),3.95(s,2H),3. 70-3.71(m,2H),3.52-3.60(m,14H),2.46(s,3H),2.04-2.07(m,1H),1.75-1.78(m,1H),1.37(d,J=6.0Hz,3H),0.93(s,9H). 13 C NMR (150MHz, d) 6 -DMSO): δ=170.9,169.4,168.9,168.1,158.2,151.9,148.2,145.1,135.4,13 1.8,131.5,130.8,130.4,130.1,129.3,129.2,128.8,126.7,120.5,118.1,1 14.9,109.6,101.5,70.8,70.3,70.2,70.0,69.3,69.2,67.5,65.4,59.0,56. 9,56.1,49.0,48.2,38.2,36.2,26.6,22.9,16.5,15.6.HRMS(EI)calcd.For[C 49 H 63 N6O 11 S](M+H + :943.4270,found:943.4275.

[0209] The following experiment uses compound 9a-9h as an example to verify the HDAC8 protein degradation experiment of the target histone deacetylase 8 protein hydrolysis chimera provided in this invention.

[0210] Experiment 1: Degradation efficiency of HDAC8 protein by compounds 9a-9h

[0211] A549 cells were seeded in 96-well plates, with 5 × 10⁶ cells per well. 4After 12 hours, add the appropriate dose of degradation agent or DMSO (control wells) to each well. Incubate for 20 hours, then add 75 μL of 8% formaldehyde-based TBS solution to each well and fix for 20 minutes at room temperature. Remove the solution, wash twice with 200 μL / well of TBST, add 50 μL / well of 0.1% Ttiton PBS solution, and incubate for 15 minutes. Remove the solution from the 96-well plate, wash twice with 150 μL / well of TBST. Then add 100 μL / well of 1% H2O2-TBS solution and incubate for 20 minutes at room temperature. Remove the solution, wash three times with an appropriate amount of TBST. Add 50 μL of blocking buffer to each well and incubate overnight at 4°C. Remove the blocking buffer, add 100 μL of blocking buffer containing 1:2000 diluted HDAC8 antibody to each well (control group added blocking buffer containing DMSO), and incubate for 2 hours at room temperature. The HDAC8 primary antibody was recovered and washed twice with an appropriate amount of TBST. 150 μL of HRP-labeled rabbit antibody was added to each well, and the mixture was incubated at room temperature for 1 h. The secondary antibody was removed, and the mixture was washed three times with TBST. 100 μL of pre-prepared TMB substrate solution was added to each well, and the mixture was incubated at room temperature in the dark for 20 min. 10 μL of stop solution containing 1N HCl was added to each well, and the mixture was shaken for 5 min. The absorbance values ​​at 450 and 570 nm were measured and recorded on a densitometer, and the relative content of HDAC8 protein was calculated.

[0212] The test results are shown in Table 1.

[0213] Table 1: HDAC8 degradation efficiency in A549 cells

[0214]

[0215] Experiment 2: Analysis of HDAC8 protein degradation of compound 9a-9h using Western blot analysis

[0216] A549 cells were treated with the aforementioned synthetic compounds 9a-9h (2 μM), then lysed for 30 minutes with RIPA at ice temperature. Following this, a protease degradation agent was added, and protein concentration was quantified using a BCA kit. Equal amounts of protein were subjected to electrophoresis with 10-12% polyacrylamide. Protein bands were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. The target band was blocked with primary antibody at 4°C for 12 hours, washed, and then incubated with secondary antibody at room temperature for 1 hour. Immunofluorescence substrate was added, followed by rapid chemifluorescence imaging. The test results are as follows: Figure 1 As shown.

[0217] according to Figure 1The test results showed that some compounds, such as 9b, 9d, 9e, and 9f, could significantly degrade deacetylase 8 in tumor cells; other compounds, such as 9a, 9c, 9g, and 9h, had a certain degrading effect on deacetylase 8 in tumor cells.

[0218] In summary, the target histone deacetylase 8 proteolytic chimera provided by this invention can significantly degrade deacetylase 8 in tumor cells, suggesting its value in the preparation of drugs for the prevention or treatment of diseases related to abnormal HDAC8 activity or expression, especially anti-tumor drugs.

[0219] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0220] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A chimeric protein hydrolysant targeting histone deacetylase 8, characterized in that, The chimera is at least one of compounds 9a, 9b, 9c, 9d, 9e, 9f, 9g, and 9h, or a pharmaceutically acceptable salt thereof. 、 、 、 、 、 、 、 。 2. A method for preparing the target histone deacetylase 8 protein hydrolysis chimera as described in claim 1, characterized in that, Includes the following steps: S1. Compound 1 and Compound 2 are dissolved in N,N-dimethylacetylamine to obtain a first solution and a second solution, respectively. NaH is added to the first solution to react and obtain a reaction solution. Then, the second solution is added dropwise to the reaction solution to continue the reaction and obtain Compound 3. S2. Compound 3 was dissolved in methanol solution, and Pd / C was added to react and give compound 4. S3. Dissolve compound 4, compound 5A or compound 5B, and potassium carbonate in anhydrous DMF, stir and heat to 90°C, react for 8 h, and after the reaction is complete, obtain compound 6a-6h. S4. Compound 6a-6h was dissolved in anhydrous dichloromethane and reacted with trifluoroacetic acid at room temperature to obtain a reaction solution. Petroleum ether was added to the reaction solution and stirred until homogeneous. The solvent was removed under vacuum to obtain a concentrated residue. Compound 7-1, EDCI, and HOBt were dissolved in DMF and added to the concentrated residue. The mixture was cooled to 5 °C, and DIPEA was added. The temperature was restored to room temperature, and the reaction was continued for 18 h to obtain an intermediate compound. S5. Cool the NH2OH solution to 5 °C, add an alkaline reagent, and stir to dissolve to obtain a hydroxylamine solution; dissolve the intermediate compound in MeOH / THF solution, then add it to the hydroxylamine solution. After the reaction is complete, add an acid reagent to neutralize, to obtain the corresponding compounds 9a, 9b, 9c, 9d, 9e, 9f, 9g, and 9h. The synthetic route for steps S1-S2 is as follows: ； The synthetic route for step S3 is as follows: ； Where m is an integer from 1 to 4, and n is an integer from 1 to 4; When m=1 for compound 5A, the synthesized compound is 9a; When m=2 for compound 5A, the synthesized compound is 9b; When m=3 for compound 5A, the synthesized compound is 9c; When m=4 for compound 5A, the synthesized compound is 9d; When n=1 for compound 5B, the synthesized compound is 9e; When n=2 for compound 5B, the synthesized compound is 9f; When n=3 for compound 5B, the synthesized compound weighs 9g. When n=4 for compound 5B, the synthesized compound is 9h; 。 3. The preparation method according to claim 2, characterized in that, Steps S1-S5 also include steps for concentrating, separating, and purifying the products obtained in each step.

4. The preparation method according to claim 2, characterized in that, In step S4, the concentrated residue does not require purification.

5. The preparation method according to claim 2, characterized in that, In step S5, the alkaline reagent is sodium hydroxide; the acidic reagent is acetic acid.

6. The preparation method according to claim 2, characterized in that, In step S5, the volume ratio of MeOH to THF in the MeOH / THF solution is 1:

1.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the target histone deacetylase 8 protein hydrolysis chimera as described in claim 1, or comprises the target histone deacetylase 8 protein hydrolysis chimera prepared by the preparation method described in any one of claims 2-6.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

10. The use of the target histone deacetylase 8 protein hydrolysis chimera as described in claim 1, and the pharmaceutical composition as described in any one of claims 7-9, in the preparation of a medicament for the prevention or treatment of diseases related to abnormal HDAC8 activity or expression.

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