Method for identifying target of active natural product based on protac protein targeted degradation technology and protac compound

Abstract

The active natural product target identification method based on the PROTAC protein targeted degradation technology and the PROTAC compound belong to the technical field of medicines, and relate to the synthesis of target protein hydrolysis targeted chimeric molecules with different Linker lengths by taking the PROTAC technology as support and taking the active natural product as a POI ligand, the determination of the target point of the active natural product through proteomics, molecular biology, structural biology and other technologies, and the further determination that the anti-inflammatory target point of the qianjin diterpene alkane compound is the MAFF protein. The PROTAC protein targeted degradation technology involved in the application provides a new strategy method and a new technical supplement for the target identification of the active natural product. Meanwhile, the application provides a new mechanism basis and a treatment strategy for the treatment of inflammation, autoimmune diseases and immune damage mediated by the qianjin alkane diterpene compound.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to an active natural product target identification method based on a PROTAC protein targeted degradation technology and a PROTAC compound. BACKGROUND

[0002] Active natural products have become an important source of discovery of lead compounds and new therapeutic drugs due to their unique biocompatibility, novel structure and extensive pharmacological activity. However, the biological activity of natural products does not come from the regulation of a single protein or pathway, but is achieved through interaction with a large number of cellular components. The complexity of this mechanism of action brings great difficulty to the accurate target identification of natural products. Moreover, the binding affinity of natural products to target proteins is not very strong, which also brings challenges to the target identification work. The protein-targeting chimera (PROTAC) technology utilizes the function of proteasome to specifically degrade protein substrates, and relies on chemical synthesis to connect the ligand of the target protein and the ligand of ubiquitin ligase E3 through a linker to form a bifunctional molecular compound that can spontaneously mediate the degradation of the target protein, which has broad research and application value. At present, PROTAC is widely used to synthesize bifunctional small molecule inhibitors with better activity. Since PROTAC degrades target proteins without strong binding force, we speculate that this technology is suitable for identifying the exact target of traditional Chinese medicine and small molecule compounds of natural origin. (See: Lai A.C., et al. Nat. Rev. Drug Discov. 2017, 16, 101-104; Schneekloth J.S., et al. J. Am. Chem. Soc. 2004, 126, 3748-3754; Deshaies R.J., Nat. Chem. Biol. 2015, 11, 634-635; Zeng S., et al. Eur. J. Med. Chem. 2021, 210, 112981; Burslem G.M., et al. Cell. 2020, 181, 102-114.)

[0003] Chongjinditerpenoid compounds are the main active components of Chinese medicine Semen Vaccariae, which have a wide range of biological activities, including anti-inflammatory, anti-tumor, anti-tumor multidrug resistance, etc. In recent years, more and more studies have been conducted on the anti-inflammatory activity of chongjinditerpenoid compounds isolated from Semen Vaccariae. The applicant's research group found that the new compound 2S, 3S, 4S, 5R, 9S, 11R, 15R)-15-acetoxy-3-cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17), 12E-diene showed the strongest inhibitory activity on the production of nitric oxide (NO) in lipopolysaccharide (LPS) stimulated mouse macrophage RAW264.7 cells, with an IC 50 value of 3.0±1.1μM. However, the exact target and anti-inflammatory mechanism of chongjinditerpenoid compounds are still unclear. Given the good anti-inflammatory activity of chongjinditerpenoid compounds, the exact target of chongjinditerpenoid compounds needs to be clarified in order to better understand the molecular mechanism of anti-inflammatory therapy and lay a foundation for subsequent optimization to find better lead compounds with anti-inflammatory activity. (See: Zhang C.Y., et al. J. Nat. Prod. 2019, 82, 756-764; Wang J.X., et al. Chem. Pharm. Bull. (Tokyo), 2018, 66, 674-677)

[0004] Currently, there is no report in the art on using PROTAC technology as a target identification method for natural source small molecule compounds, and there is no research on the exact target of chongjinditerpenoid compounds and related applications based on the target. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide an active natural product target identification method based on PROTAC protein targeted degradation technology and a PROTAC compound. Specifically, the PROTAC molecule targets the intracellular target protein of the active natural product as the target protein (POI) ligand, to identify the target of the active natural product in the cell, providing a new method for target identification of traditional Chinese medicine and natural medicine derived small molecule compounds. At the same time, it provides a treatment strategy for chongjinditerpenoid compounds in treating inflammation-related diseases in vivo and in vitro. On this basis, the potential target of chongjinditerpenoid compounds is found, including MAFF, TRIR (C19orf43), CHTOP, CDV3, MTDH, etc. It is found that chongjinditerpenoid compounds can target MAFF to exert anti-inflammatory effect in vivo and in vitro, and be used for treating autoimmune diseases, and for preparing a drug for treating autoimmune diseases and immune damage diseases.

[0006] A method for identifying a target of an active natural product based on a PROTAC protein targeted degradation technology, comprising the following steps:

[0007] (1) A series of bifunctional PROTAC compounds based on the active natural product are synthesized by using the active natural product as a POI ligand and different E3 ligase ligands;

[0008] (2) The best active PROTAC compound is determined through activity screening, and differential proteomics analysis is performed on cell lysates treated with the active natural product and the PROTAC compound respectively, differential expression proteins in the protein lysate are identified, and KEGG analysis is performed to determine the potential target protein of the PROTAC compound and the active natural product;

[0009] (3) The potential target protein is verified by western blot to determine its degradation activity and degradation characteristics by the PROTAC compound, and the direct binding force between the active natural product and the target protein is verified by in vitro target verification methods such as MST, CETSA and DARTS, and the function of the active natural product on the target protein and the downstream proteins affected by the target protein are determined by cell biology techniques such as RNAi and in vivo target-related pathway verification, to further determine whether the active natural product acts on the target, thereby verifying the authenticity of the target protein, and finally confirming the target protein of the active natural product.

[0010] The method for identifying the target of the active natural product based on the PROTAC protein targeted degradation technology is based on the formation of a POI-PROTAC-E3 ligase ternary complex, does not require strong affinity between the active natural product and the POI, can effectively degrade target proteins with low affinity, and is suitable for target identification of traditional Chinese medicine and natural medicine active small molecule compounds.

[0011] In step (1), the active natural product is a compound or natural product with a lathyrol diterpene alkane nucleus, and the natural product includes but is not limited to lathyrol and ZCY020, and the structural formulas of lathyrol and ZCY020 are shown in general formula I-1 and general formula I-2 as follows:

[0012]

[0013]

[0014] In step (1), the PROTAC compound is a bifunctional (targeting the target protein and E3 ligase) compound based on different structures of the quinone diterpene alkyl compound, different E3 ligase ligands, and different linker arms, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and the structure of the PROTAC compound is as shown in the following general formula I-3:

[0015]

[0016] In the general formula I-3, R1 and R2 are H, Cinnamoyl, P-hydroxycinnamoyl, Acetyl, Butyryl, Benzoyl or Nicotinoyl.

[0017] R1 and R2 are H, Cinnamoyl, P-hydroxycinnamoyl, Acetyl, Butyryl, Benzoyl or Nicotinoyl.

[0018] B is a small molecule ligand of E3 ubiquitin ligase complex, which is selected from Cereblon protein ligand (Thalidomide, Lenalidomide, Pomalidomide and derivatives thereof); VHL ligand; CIAP ligand; MDM2 ligand; UBR7 ligand; RNF114 ligand; CBLB ligand; KEAP1 ligand, etc.

[0019] L is a linker arm, which is connected to the hydroxyl group and B through a covalent bond, and together constitutes a bifunctional molecular compound.

[0020] B is any one of the following structures:

[0021]

[0022] wherein: W is selected from CH2, C=O, SO2, NH, N-C1-C4 alkyl; X is selected from O, S; Z is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, halogen; G, G' are selected from H, C1-C4 alkyl, -OH, 5-10 membered heterocyclic group substituted with C1-C4 alkyl, said heterocyclic group containing 1-3 N, O or S heteroatoms; R 3 H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, deuterated C1-C4 alkyl.

[0023] L is connected to the hydroxyl group and B through a covalent bond, and L is preferably any one of the following structures:

[0024]

[0025] wherein: n is an integer selected from 1-10.

[0026] The present application preferably comprises a bifunctional molecule compound as shown in general formula II or a pharmaceutically acceptable salt, hydrate or prodrug thereof:

[0027]

[0028] L is any one of the following structures:

[0029]

[0030] n is an integer between 1-10.

[0031] Preferred compounds of the present application include, but are not limited to:

[0032]

[0033] In step (1), the pharmaceutically acceptable salt includes addition salts with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, pyruvic acid, succinic acid, benzoic acid, and the like. In addition, the present application also includes prodrugs of the derivatives of the present application. They can have weak activity or even no activity themselves, but are converted into the corresponding biologically active forms under physiological conditions (such as by metabolism, solvolysis or other means) after administration.

[0034] In step (2), the activity screening method includes but is not limited to anti-inflammatory activity screening in inflammation stimulation models, anti-tumor activity screening in tumor cells, anti-oxidative activity screening in oxidative stress models, etc., and the detection method varies depending on the specific model; the specific steps of the anti-inflammatory activity screening method in the inflammation stimulation model are as follows:

[0035] (1) A series of bifunctional PROTAC compounds based on active natural products synthesized above are added to cultured mouse macrophage RAW264.7 cells in the logarithmic growth phase for 3 hours, then the cells are stimulated with lipopolysaccharide LPS for 24 hours to establish a mouse macrophage inflammation model;

[0036] (2) The total nitric oxide (NO) content in the cell supernatant is detected with Griss reagent, and the anti-inflammatory activity of the synthesized PROTAC compound is analyzed and determined according to the inhibitory effect of the PROTAC small molecule on NO release, and the best anti-inflammatory activity PROTAC compound, ZCY-PROTAC, is screened.

[0037] The structure of ZCY-PROTAC is as follows:

[0038]

[0039] In step (2), the protein lysate for differential proteomics analysis can be a whole cell lysate or a lysate of organelle components of the cells.

[0040] In step (1), the method for preparing the PROTAC compound comprises the following steps:

[0041] (1) Lathyrol is dissolved in anhydrous DMF, sodium hydride and bromopropargyl are added, and after reaction at room temperature, dilution, washing and purification are performed to obtain intermediate 1;

[0042] (2) Vitamin C sodium (23 mg, 0.36 mmol, 3.0 eq) and anhydrous copper sulfate are added to a mixture of intermediate 1 and intermediate 2 in tetrahydrofuran and water;

[0043] (3) The reaction system is reacted at room temperature for 20-30 minutes, after the reaction is completed, the solid is removed by filtration, and the filtrate is evaporated under reduced pressure and separated by column chromatography to obtain the corresponding final product; wherein:

[0044] The intermediate 2 is one of intermediate 2-1, intermediate 2-2, intermediate 2-3, intermediate 2-4 or intermediate 2-5.

[0045] The preparation process of intermediate 2-1 is as follows:

[0046] The thalidomide derivative is taken, a solvent is added, and azido-PEG-amine and DIPEA are added under stirring, after heating reaction, extraction, drying, concentration, purification, elution, a yellow oil is obtained, which is intermediate 2-1;

[0047] The preparation process of intermediate 2-2 is as follows:

[0048] M7 is weighed and dissolved, then azido-PEG-amine, EDCI, HOBt and DIPEA are added, after reaction at room temperature, dilution, washing, drying and purification, intermediate 2-2 product is obtained;

[0049] The preparation process of intermediate 2-3 is as follows:

[0050] B5 is weighed and dissolved, then azido-PEG-amine, EDCI, HOBt and DIPEA are added, after reaction at room temperature, dilution, washing, drying and purification, intermediate 2-3 is prepared;

[0051] The preparation process of intermediate 2-4 is as follows:

[0052] The intermediate 2-4 is prepared by weighing the B4 hydrochloride, dissolving it, adding azido carboxylic acid, EDCI, HOBt and DIPEA, reacting at room temperature, diluting, washing, drying, and purifying.

[0053] The preparation process of the intermediate 2-5 is as follows:

[0054] The intermediate 2-5 is prepared by weighing the VHL hydrochloride, dissolving it, adding azido carboxylic acid, EDCI, HOBt and DIPEA, reacting at room temperature, diluting, washing, drying, and purifying.

[0055] In step (2), when the intermediate 2 is the intermediate 2-1, n = 2, 3 or 4, the final product obtained is the final product 1-3, and the reaction process is as shown in the following formula III; when the intermediate 2 is the intermediate 2-2, n = 2, 3 or 4, the reaction process is as shown in the following formula IV, and the final product obtained is the final product 4-6; when the intermediate 2 is the intermediate 2-3, n = 2, 3 or 4, the reaction process is as shown in the following formula V, and the final product obtained is the final product 7-9; when the intermediate 2 is the intermediate 2-4, n = 1, 3 or 5, the reaction process is as shown in the following formula VI, and the final product obtained is the final product 10-12; when the intermediate 2 is the intermediate 2-5, n = 1, 3 or 5, R 1 is H or CH3, the reaction process is as shown in the following formula VII, and the final product obtained is the final product 13-18.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] A pharmaceutical composition containing a therapeutically effective amount of any one of the above based on the PROTAC protein targeted degradation technology has a thousand gold diterpene alkane mother nucleus class of substances or its stereoisomer, tautomer, pharmaceutically acceptable salt, hydrate, prodrug and pharmaceutically acceptable carrier, diluent, adjuvant, vehicle or their combination.

[0062] The dosage form of the pharmaceutical composition is any one of injection, tablet and capsule.

[0063] The application of the thousand gold diterpene alkane mother nucleus class of substances in the preparation of drugs for treating or preventing inflammation-related diseases.

[0064] The application confirms that the compound with the qingjidi diterpene alkane mother nucleus is an effective MAFF-Nrf2 activator at the cell level and the animal level, and can be used for treating inflammation, autoimmune diseases and immune damage, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage and nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, endotoxemia caused by infection, sepsis and the like.

[0065] The compound with the qingjidi diterpene alkane mother nucleus includes a qingjidi diterpene alkane natural small molecule compound, a targeted degrader based on the qingjidi diterpene alkane mother nucleus and a pharmaceutically acceptable salt thereof or a combination thereof, and application of the compound in preparation of a drug for treating or preventing an inflammation-related disease. The inflammation action target is MAFF.

[0066] Advantages of the application:

[0067] The application is supported by the PROTAC technology, and uses traditional Chinese medicines and natural active small molecule compounds as raw materials to synthesize target protein hydrolysis targeted chimeric molecules with different linker lengths. The application establishes a target identification method of traditional Chinese medicines and natural medicine small molecule compounds based on the bifunctional PROTAC protein targeted degradation technology. The application synthesizes a series of bifunctional PROTAC molecules based on traditional Chinese medicines and natural medicine small molecule compounds, determines the best PROTAC with the best activity through cell activity screening, and performs differential proteomic analysis and KEGG pathway analysis on cell lysates treated by the small molecule compound and the PROTAC respectively, verifies the degradation mechanism of the candidate proteins to be degraded, and obtains the candidate target protein. The target protein of the traditional Chinese medicines and the natural medicine small molecule is further verified by in-vitro target point verification methods such as MST, CETSA and DARTS, cell biology technologies such as RNAi, and in-vivo target-related pathway verification.

[0068] The application further verifies the direct binding force between the MAFF protein and the POI ligand of ZCY-PROTAC, lathyrol and the representative compound ZCY020 of the qingjidi diterpene alkane at the molecular level and the cell level, and determines that MAFF is the target protein of the qingjidi diterpene alkane, thereby providing a new action mechanism for the treatment of inflammation and / or autoimmune diseases mediated by the qingjidi diterpene alkane compound.

[0069] The application further confirms that the Qingjini diterpene alkyl compounds are effective MAFF-Nrf2 activators at the cell level and the animal level, and can be used for treating inflammation, autoimmune diseases and immune damage, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, endotoxemia caused by infection, sepsis and the like. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 Figure 1 is a degradation property diagram of ZCY-PROTAC mediated MAFF protein of an embodiment of the application, wherein A is the structure of ZCY-PROTAC, B is the anti-NO activity IC of Lathyrol and ZCY-PROTAC, C is the volcano plot of differential proteins in the control group and ZCY-PROTAC administered cells, D is the KEGG pathway analysis of inflammation related proteins, E is the concentration and time dependence of ZCY-PROTAC degrading MAFF in RAW264.7 and HEK293T cells, and F is the activity of ZCY-PROTAC degrading MAFF after pretreatment of RAW264.7 and HEK293T cells with MG132; 50

[0071] Figure 2 Figure 2 is a target verification diagram of Lathyrol and ZCY020 of an embodiment of the application, wherein A is the structure of Lathyrol and ZCY020, B is the MST result diagram of Lathyrol and ZCY020 and MAFF protein, C is the CETSA result diagram of Lathyrol and ZCY020 and MAFF protein, and D is the DARTS result diagram of Lathyrol and ZCY020 and MAFF protein;

[0072] Figure 3 ​Figures A, B, C, D, E and F are COIP experimental results of ZCY020 promoting the generation of MAFF-Nrf2 heterodimer, wherein A is the COIP experimental results after LPS and different concentrations of ZCY020 treatment, B is the COIP experimental results after LPS and 40 μM of ZCY020 treatment, C is the COIP experimental results after RAW264.7 cells are treated with different concentrations of ZCY020, D is the COIP experimental results after HEK293T cells are transfected with MAFF-GFP-HA plasmid or MAFF-GFP-HA plasmid plus MAFG-HIS plasmid or MAFF-GFP-HA plasmid plus MAFK-GST plasmid, and E is the COIP experimental results after HEK293T cells are transfected with MAFF-GFP-HA plasmid and Nrf2-FLAG plasmid;

[0073] Figure 4 Figures A, B, C, D, E and F are RT-qPCR results of ZCY020 activating MAFF-Nrf2 / HO-1 signaling pathway, wherein A is the RT-qPCR results of LPS-stimulated RAW264.7 cells treated with different concentrations of ZCY020, B is the expression of Nrf2 pathway-related proteins, C is the ROS level detected by flow cytometry, D is the expression of Nrf2 and HO-1 after ML385 intervention, E is the ROS level detected by flow cytometry after ML385 intervention, F is the expression of MAFF after RAW264.7 cells are treated with shRNA for 48 h, G is the expression of HO-1 after shRNA treatment, and H is the ROS level detected by flow cytometry after shRNA treatment.

[0074] Figure 5 Figures A, B, C, D, E and F are RT-qPCR results of ZCY020 activating MAFF-Nrf2 / HO-1 signaling pathway, wherein A is the RT-qPCR results of LPS-stimulated RAW264.7 cells treated with different concentrations of ZCY020, B is the expression of Nrf2 pathway-related proteins, C is the ROS level detected by flow cytometry, D is the expression of Nrf2 and HO-1 after ML385 intervention, E is the ROS level detected by flow cytometry after ML385 intervention, F is the expression of MAFF after RAW264.7 cells are treated with shRNA for 48 h, G is the expression of HO-1 after shRNA treatment, and H is the ROS level detected by flow cytometry after shRNA treatment. 50 Figures A, B, C, D, E and F are RT-qPCR results of ZCY020 activating MAFF-Nrf2 / HO-1 signaling pathway, wherein A is the RT-qPCR results of LPS-stimulated RAW264.7 cells treated with different concentrations of ZCY020, B is the expression of Nrf2 pathway-related proteins, C is the ROS level detected by flow cytometry, D is the expression of Nrf2 and HO-1 after ML385 intervention, E is the ROS level detected by flow cytometry after ML385 intervention, F is the expression of MAFF after RAW264.7 cells are treated with shRNA for 48 h, G is the expression of HO-1 after shRNA treatment, and H is the ROS level detected by flow cytometry after shRNA treatment.

[0075] Figure 6A is the expression of p62, Keap1 and Nrf2, B is the expression of BNIP3 and LC3B, C is the flow detection of mitochondrial superoxide mitoSOX level, D is the effect of ZCY020 on the expression of p62, BNIP3 and LC3B after NAC treatment, E is the effect of ZCY020 on the expression of BNIP3 and LC3B after ML385 intervention, F is the effect of ZCY020 on the level of mitoSOX after ML385 intervention, G is the effect of ZCY020 on the expression of p62, BNIP3 and LC3B after shRNA treatment;

[0076] Figure 7 The treatment effect of ZCY020 of the embodiment of the application on the LPS-induced acute lung injury model of mice, wherein A is the survival rate of mice after high-dose LPS treatment, B is the histopathological changes of the lungs and liver of mice, C is the level of inflammatory factors in the serum of mice, D is the mRNA level of inflammatory factors detected by RT-qPCR in the lung tissue of mice, E is the mRNA level of Nrf2-regulated genes detected by RT-qPCR in the lung tissue of mice, and F is the expression of related pathway proteins in the lung tissue of mice;

[0077] Figure 8 The treatment effect of ZCY020 of the embodiment of the application on the IMQ-induced psoriasis model of mice, wherein A is the degree of skin lesion score during the experiment, B is the picture of the back skin of mice on the last day of the experiment, and C is the histopathological changes of the back skin of psoriasis mice;

[0078] Figure 9 The treatment effect of ZCY020 of the embodiment of the application on the CIA arthritis model of mice, wherein A is the degree of joint swelling score during the arthritis experiment of mice, B is the picture of the joints of mice on the last day of the experiment, C is the histopathological changes of the joints of arthritis mice, and D is the spleen coefficient of arthritis mice. DETAILED DESCRIPTION

[0079] The application will be further described in detail below with reference to the examples.

[0080] Synthesis of intermediate 1 in Example 1

[0081]

[0082] Lathyrol was dissolved in anhydrous DMF and stirred in an ice bath for 20 min. Then 1.0 eq of sodium hydride was added and the ice bath was continued for another 10 min. Then 1.2 eq of bromopropyne was added and the ice bath was removed. The reaction was stirred at room temperature for 5 h. After the reaction was completed, the reaction system was diluted with ethyl acetate and washed with saturated sodium chloride solution for 3 times. The organic phase was dried and rotary evaporated. The final product was obtained as an oily liquid after column chromatography purification with a yield of 45%.

[0083] 1 H NMR (400 MHz, CDC13) δ 7.61 (s, 1H), 7.49 (dd, J = 7.6, 2.0 Hz, 1H), 7.34 - 7.28 (m, 2H), 4.22 (s, 2H), 3.70 - 3.57 (m, 4H), 3.44 (d, J = 2.4 Hz, 2H), 2.22 (t, J = 2.4 Hz, 1H), 1.69 - 1.62 (m, 4H), 1.22 (s, 3H).

[0084] Synthesis of Example 2 intermediate 2-1 (n = 2)

[0085]

[0086] 58 mg of thalidomide derivative (commercially available) was added to a flask with 3 mL of DMF. 50 mg of azido-PEG2-amine (1.2 eq, commercially available) and 47 μL of DIPEA (2 eq) were added sequentially while stirring. The reaction was carried out at 90°C for 3-4 hours. 30 mL of water and 30 mL of ethyl acetate were added for extraction. The organic layer was dried with anhydrous sodium sulfate. The crude product was obtained after concentration and purified by silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate 1:2 to 1:4. A yellow oily substance was obtained with a yield of 40.9 mg, 41%.

[0087] 1 H NMR (400 MHz, CDC13) δ 9.06 (br s, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.09 (dd, J = 7.2, 2.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.50 (t, J = 5.6 Hz, 1H), 4.96 - 4.92 (m, 1H), 3.74 (t, J = 5.2 Hz, 2H), 3.70 - 3.67 (m, 6H), 3.48 (q, J = 5.6 Hz, 2H), 3.38 (t, J = 4.4 Hz, 2H), 2.80 - 2.73 (m, 3H), 2.13 - 2.10 (m, 1H).

[0088] Synthesis of Example 3 intermediate 2-1 (n = 3)

[0089]

[0090] The procedure and ratio were referred to the preparation of intermediate 2-1 (n=2). The product was yellow oil in 40% yield. 1 HNMR (400 MHz, CDC13) δ 8.81 (br s, 1H), 7.48 (dd, J = 8.0, 7.2 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.49 (t, J = 5.6 Hz, 1H), 4.95 - 4.90 (m, 1H), 3.72 (t, J = 5.2 Hz, 2H), 3.68 - 3.66 (m, 14H), 3.48 (q, J = 5.6 Hz, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.88 - 2.72 (m, 3H), 2.13 - 2.09 (m, 1H).

[0091] Synthesis of intermediate 2-1 (n=4) in Example 4

[0092]

[0093] The procedure and ratio were referred to the preparation of intermediate 2-1 (n=2).

[0094] The product was yellow oil in 40% yield. 1 HNMR (400 MHz, CDC13) δ 8.81 (br s, 1H), 7.48 (dd, J = 8.0, 7.2 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.49 (t, J = 5.6 Hz, 1H), 4.95 - 4.90 (m, 1H), 3.72 (t, J = 5.2 Hz, 2H), 3.68 - 3.66 (m, 14H), 3.48 (q, J = 5.6 Hz, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.88 - 2.72 (m, 3H), 2.13 - 2.09 (m, 1H).

[0095] Synthesis of intermediate 2-2 (n=2) in Example 5

[0096]

[0097] M7 was weighed out, dissolved in dichloromethane (1 ml per 100 mg), 1.05 eq of linker, 1.1 eq of EDCI and 1.1 eq of HOBt were added and the reaction was left to stir at room temperature for 3 hours. After the reaction was complete, the reaction was diluted with ethyl acetate and the organic phase was washed 3 times with saturated ammonium chloride solution. The organic phase was dried over anhydrous sodium sulfate, evaporated and purified by column chromatography on silica gel (dichloromethane:methanol 40:1-30:1) to obtain the product. The product was a white solid with a yield of 85%.

[0098] 1 H NMR (400 MHz, CDC13) δ 7.54 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 7.6 Hz, 4H), 6.86 (dd, J = 11.8, 8.4 Hz, 4H), 6.51 (dd, J = 8.5, 2.2 Hz, 1H), 6.45 (d, J = 2.1 Hz, 1H), 6.27 (t, J = 5.4 Hz, 1H), 5.59 (d, J = 9.9 Hz, 1H), 5.45 (d, J = 9.9 Hz, 1H), 4.59 (dt, J = 12.0, 6.0 Hz, 1H), 3.94 - 3.78 (m, 5H), 3.75 - 3.68 (m, 2H), 3.68 - 3.59 (m, 2H), 3.57 - 3.39 (m, 6H), 3.38 - 3.32 (m, 2H), 3.31 - 3.22 (m, 1H), 3.10 (t, J = 5.2 Hz, 2H), 1.37 (dd, J = 16.5, 6.0 Hz, 6H). Yield: 85%

[0099] Synthesis of intermediate 2-2 (n = 3) of example 6

[0100]

[0101] The procedure and proportions were as for the preparation of intermediate 2-2 (n = 2). The product was a white solid with a yield of 90%.

[0102] 1H NMR (400 MHz, CDC13) δ 7.59 (d, J = 8.5 Hz, 1H), 7.06 (dd, J = 20.9, 8.5 Hz, 4H), 6.91 (dd, J = 26.3, 8.4 Hz, 4H), 6.55 (dd, J = 8.5, 2.2 Hz, 1H), 6.51 - 6.40 (m, 2H), 5.57 (d, J = 9.7 Hz, 1H), 5.48 (d, J = 9.7 Hz, 1H), 4.62 (dt, J = 12.1, 6.0 Hz, 1H), 3.97 - 3.79 (m, 5H), 3.78 - 3.57 (m, 9H), 3.57 - 3.46 (m, 3H), 3.41 (dt, J = 10.1, 4.4 Hz, 4H), 3.32 - 3.19 (m, 1H), 3.11 (t, J = 5.2 Hz, 2H), 1.36 (dd, J = 14.8, 6.0 Hz, 6H). Yield: 90%

[0103] Synthesis of Example 7 intermediate 2-2 (n = 4)

[0104]

[0105] The procedure and ratio were referred to the preparation of intermediate 2-2 (n = 2). The product was a white solid with 86% yield.

[0106] 1 H NMR (400 MHz, CDC13) δ 7.62 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.56 (dd, J = 8.5, 2.2 Hz, 1H), 6.48 (d, J = 2.1 Hz, 1H), 5.61 (s, 2H), 4.69 - 4.52 (m, 1H), 3.94 (d, J = 15.7 Hz, 1H), 3.86 (s, 3H), 3.85 - 3.75 (m, 2H), 3.68 - 3.57 (m, 16H), 3.51 (dd, J = 12.7, 7.7 Hz, 5H), 3.43 - 3.34 (m, 4H), 3.29 (dd, J = 13.1, 6.2 Hz, 1H), 3.13 (s, 2H), 1.37 (dd, J = 16.9, 6.0 Hz, 6H). Yield: 86%

[0107] Synthesis of Example 8 intermediate 2-3 (n = 2)

[0108]

[0109] B5 was weighed out, dissolved in dichloromethane (1 ml per 100 mg), 1.05 eq of linker, 1.1 eq of EDCI and 1.1 eq of HOBt were added and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate and the organic phase was washed with saturated ammonium chloride solution three times. The organic phase was dried over anhydrous sodium sulfate, rotary evaporated and purified by silica gel column chromatography (dichloromethane:methanol 50:1-40:1) to obtain the product. The product was a white solid with a yield of 85%.

[0110] 1 H NMR (400 MHz, CDCl3) δ 7.33-7.27 (m, 2H), 7.23 (d, J = 7.5 Hz, 3H), 6.57 (s, 1H), 5.01 (d, J = 8.0 Hz, 1H), 4.45 (d, J = 4.7 Hz, 1H), 4.13 (s, 1H), 3.98 (s, 1H), 3.67-3.60 (m, 2H), 3.59-3.52 (m, 2H), 3.37 (dd, J = 21.2, 16.4 Hz, 3H), 3.18 (d, J = 13.9 Hz, 1H), 3.03 (s, 1H), 1.43-1.33 (m, 8H), 0.95-0.86 (m, 6H). Yield: 85%

[0111] Synthesis of intermediate 2-3 (n = 3) of Example 9

[0112]

[0113] The specific operation and ratio refer to the preparation of intermediate 2-3 (n = 2). The product was a white solid with a yield of 85%.

[0114] 1 H NMR (400 MHz, CDCl3) δ 7.32-7.27 (m, 2H), 7.25-7.19 (m, 3H), 6.67 (s, 1H), 5.58 (s, 1H), 5.04 (d, J = 8.2 Hz, 1H), 4.45 (dd, J = 11.6, 6.5 Hz, 1H), 4.09 (dd, J = 26.7, 24.2 Hz, 2H), 3.71 -3.58 (m, 7H), 3.55 (dd, J = 9.4, 5.0 Hz, 2H), 3.51-3.43 (m, 2H), 3.39 (dd, J = 11.6, 6.6 Hz, 3H), 3.07 (d, J = 40.9 Hz, 2H), 1.62 (dt, J = 15.9, 10.2 Hz, 3H), 1.38 (s, 9H), 0.91 (dd, J = 12.5, 6.1 Hz, 6H). Yield: 85%

[0115] Synthesis of intermediate 2-3 (n = 4) in example 10

[0116]

[0117] The specific operation and ratio refer to the preparation of intermediate 2-3 (n = 2). The product is a white solid with a yield of 90%.

[0118] 1 H NMR (400 MHz, CDC13) δ 7.31 - 7.26 (m, 2H), 7.22 (dd, J = 8.6, 4.5 Hz, 3H), 6.88 (s, 1H), 5.04 (s, 1H), 4.48 (s, 1H), 4.13 (s, 2H), 3.70 - 3.50 (m, 16H), 3.40 (dd, J = 18.6, 13.7 Hz, 4H), 3.02 (d, J = 16.1 Hz, 2H), 1.71 (dd, J = 11.1, 6.4 Hz, 2H), 1.64 - 1.51 (m, 2H), 1.38 (d, J = 11.6 Hz, 8H), 0.92 (dd, J = 11.3, 6.2 Hz, 6H). Yield: 90%

[0119] Synthesis of intermediate 2-4 (n = 1) in example 11

[0120]

[0121] B4 hydrochloride was weighed and dissolved in dichloromethane (1 ml per 100 mg), 1.05 eq of azide carboxylic acid, 1.1 eq of EDCI, 1.1 eq of HOBt and 2.1 eq of DIPEA were added, and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate, the organic phase was washed with saturated ammonium chloride solution 3 times, the organic phase was dried with anhydrous sodium sulfate, rotary evaporation was performed, and the product was obtained by silica gel column chromatography (dichloromethane:methanol 50:1-40:1). The product was a white solid with a yield of 85%.

[0122] 1H NMR (400 MHz, CDC13) δ 7.31 (dd, J = 9.3, 5.3 Hz, 2H), 7.23 (d, J = 7.5 Hz, 3H), 7.08 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 5.13 (s, 1H), 4.64 - 4.49 (m, 1H), 4.37 (dd, J = 6.9, 2.9 Hz, 1H), 4.22 - 4.15 (m, 1H), 3.91 (d, J = 16.6 Hz, 1H), 3.82 (d, J = 16.6 Hz, 1H), 3.72 (s, 3H), 3.10 (dd, J = 13.7, 6.9 Hz, 1H), 3.00 (dd, J = 13.6, 8.8 Hz, 1H), 1.26 (d, J = 1.6 Hz, 1H), 0.93 (dd, J = 8.2, 6.2 Hz, 6H). Yield: 85%

[0123] Synthesis of Example 12 intermediate 2-4 (n = 3)

[0124]

[0125] The procedure and ratios were followed as for the preparation of intermediate 2-4 (n = 3). The product was obtained as a white solid in 88% yield.

[0126] 1 H NMR (400 MHz, CDC13) δ 7.32 - 7.27 (m, 3H), 7.25 - 7.18 (m, 3H), 6.36 (d, J = 8.1 Hz, 1H), 5.78 (s, 1H), 4.57 (td, J = 8.9, 5.0 Hz, 1H), 4.29 - 4.21 (m, 1H), 4.22 - 4.13 (m, 1H), 3.73 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H), 3.10 (dd, J = 13.8, 6.0 Hz, 1H), 2.98 (dd, J = 13.8, 9.8 Hz, 1H), 2.21 - 2.06 (m, 2H), 1.75 (dt, J = 14.9, 7.4 Hz, 3H), 1.71 - 1.65 (m, 3H), 1.60 (ddd, J = 16.9, 10.2, 4.3 Hz, 3H), 0.93 (dd, J = 8.0, 6.2 Hz, 6H).

[0127] Synthesis of Example 13 intermediate 2-4 (n = 5)

[0128]

[0129] The procedure and ratios were followed as for the preparation of intermediate 2-4 (n = 3). The product was obtained as a white solid in 92% yield.

[0130] 1 H NMR (400 MHz, CDC13) δ 7.39 (d, J = 8.6 Hz, 1H), 7.27 (dd, J = 8.1, 6.1 Hz, 2H), 7.21 (t, J = 7.6 Hz, 3H), 6.56 (d, J = 8.1 Hz, 1H), 4.57 (td, J = 8.9, 5.0 Hz, 1H), 4.29 - 4.13 (m, 2H), 3.72 (s, 2H), 3.28 (t, J = 6.8 Hz, 1H), 3.22 (t, J = 6.9 Hz, 2H), 3.11 (dd, J = 13.8, 5.8 Hz, 1H), 2.96 (dd, J = 13.8, 9.9 Hz, 1H), 2.35 (t, J = 7.4 Hz, 1H), 2.12 - 2.01 (m, 2H), 1.71 - 1.39 (m, 9H), 1.31 - 1.21 (m, 3H), 0.97 - 0.87 (m, 6H). Yield: 92%

[0131] Synthesis of Example 14 intermediate 2-5 (n = 1, R 1 = H)

[0132]

[0133] VHL hydrochloride was weighed out, dissolved in dichloromethane (1 ml per 100 mg), 1.05 eq of azidoacetic acid, 1.1 eq of EDCI, 1.1 eq of HOBt and 2.1 eq of DIPEA were added, and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate, the organic phase was washed with saturated ammonium chloride solution three times, the organic phase was dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The product was purified by silica gel column chromatography (dichloromethane:methanol 30:1-20:1). The product was a white solid, and the yield was 60%.

[0134] 1 H NMR (400 MHz, CDC13) δ 8.69 (s, 1H), 7.36 (q, J = 8.3 Hz, 4H), 6.93 (d, J = 8.5 Hz, 1H), 4.72 (t, J = 7.9 Hz, 1H), 4.61 - 4.46 (m, 3H), 4.34 (dd, J = 14.9, 5.2 Hz, 1H), 4.03 (dd, J = 17.9, 9.7 Hz, 2H), 3.63 (dd, J = 11.3, 3.6 Hz, 1H), 2.63 - 2.54 (m, 1H), 2.52 (d, J = 6.7 Hz, 3H), 2.13 (dd, J = 13.3, 8.0 Hz, 1H), 0, 95 (s, 9H). Yield: 36%

[0135] Synthesis of Example 15 intermediate 2-5 (n = 3, R = H) 1 = H)

[0136]

[0137] The procedure and ratios were followed as for the preparation of intermediate 2-5 (n = 1, R = H). The product was obtained as a white solid in 68% yield.

[0138] 1 H NMR (400 MHz, CDC13) δ 8.69 (s, 1H), 7.35 (q, J = 8.3 Hz, 5H), 6.52 - 6.37 (m, 1H), 4.69 (t, J = 7.9 Hz, 1H), 4.53 (dd, J = 12.3, 7.7 Hz, 3H), 4.35 (dd, J = 15.0, 5.4 Hz, 1H), 4.03 (d, J = 11.2 Hz, 1H), 3.65 (dd, J = 11.2, 3.6 Hz, 1H), 3.31 (td, J = 6.4, 1.9 Hz, 2H), 2.51 (s, 3H), 2.48 - 2.40 (m, 1H), 2.29 (dd, J = 14.3, 7.2 Hz, 2H), 2.13 (s, 1H), 1.93 - 1.80 (m, 2H), 0.95 (s, 9H).

[0139] Synthesis of Example 16 intermediate 2-5 (n = 5, R 1 = H)

[0140]

[0141] The procedure and ratios were followed as for the preparation of intermediate 2-5 (n = 1, R = H). The product was obtained as a white solid in 64% yield.

[0142] 1 H NMR (400 MHz, CDC13) δ 8.69 (s, 1H), 7.35 (q, J = 8.3 Hz, 5H), 6.52 - 6.37 (m, 1H), 4.69 (t, J = 7.9 Hz, 1H), 4.53 (dd, J = 12.3, 7.7 Hz, 3H), 4.35 (dd, J = 15.0, 5.4 Hz, 1H), 4.03 (d, J = 11.2 Hz, 1H), 3.65 (dd, J = 11.2, 3.6 Hz, 1H), 3.31 (td, J = 6.4, 1.9 Hz, 2H), 2.51 (s, 3H), 2.48 - 2.40 (m, 1H), 2.29 (dd, J = 14.3, 7.2 Hz, 2H), 2.13 (s, 1H), 1.93 - 1.80 (m, 2H), 0.95 (s, 9H). Yield: 64%

[0143] Synthesis of Example 17 intermediate 2-5 (n = 1, R = CH3) 1 = CH3) following the procedure described for the preparation of intermediate 2-5 (n = 1, R = H). The product was obtained as a white solid in 74% yield.

[0144]

[0145] Following the procedure described for the preparation of intermediate 2-5 (n = 1, R = H). The product was obtained as a white solid in 70% yield.

[0146] 1 H NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 7.38 (q, J = 8.5 Hz, 4H), 7.02 (d, J = 8.6 Hz, 1H), 5.14 - 5.03 (m, 1H), 4.73 (t, J = 7.8 Hz, 1H), 4.57 (d, J = 8.7 Hz, 2H), 4.10 - 4.04 (m, 1H), 3.99 (d, J = 16.7 Hz, 1H), 3.63 (dd, J = 11.3, 3.7 Hz, 1H), 2.60 - 2.50 (m, 4H), 2.08 (dd, J = 19.9, 6.3 Hz, 1H), 1.48 (d, J = 6.9 Hz, 3H), 1.06 (s, 9H). Yield: 74%

[0147] Synthesis of Example 18 intermediate 2-5 (n = 3, R 1 = CH3) following the procedure described for the preparation of intermediate 2-5 (n = 1, R = H).

[0148]

[0149] Following the procedure described for the preparation of intermediate 2-5 (n = 1, R = H). The product was obtained as a white solid in 70% yield.

[0150] 1 H NMR (400 MHz, CDC13) δ 8.70 (s, 1H), 7.38 (q, J = 8.5 Hz, 4H), 7.02 (d, J = 8.6 Hz, 1H), 5.14 - 5.03 (m, 1H), 4.73 (t, J = 7.8 Hz, 1H), 4.57 (d, J = 8.7 Hz, 2H), 4.10 - 4.04 (m, 1H), 3.99 (d, J = 16.7 Hz, 1H), 3.63 (dd, J = 11.3, 3.7 Hz, 1H), 2.60 - 2.50 (m, 4H), 2.08 (dd, J = 19.9, 6.3 Hz, 1H), 1.48 (d, J = 6.9 Hz, 3H), 1.06 (s, 9H). Yield: 74%

[0151] Synthesis of intermediate 2-5 (n = 5, R 1 = CH3) of Example 19

[0152]

[0153] The specific operation and ratio refer to the preparation of intermediate 2-5 (n = 1, R = H). The product is a white solid with a yield of 60%.

[0154] 1 H NMR (400 MHz, CDC13) δ 8.68 (s, 1H), 7.45 - 7.33 (m, 4H), 6.19 (d, J = 8.8 Hz, 1H), 5.15 - 5.03 (m, 1H), 4.72 (t, J = 7.9 Hz, 1H), 4.60 - 4.48 (m, 2H), 4.17 - 4.05 (m, 1H), 3.60 (dd, J = 11.4, 3.6 Hz, 1H), 3.26 (t, J = 6.8 Hz, 2H), 2.58 - 2.49 (m, 3H), 2.22 (t, J = 7.5 Hz, 2H), 2.12 - 2.02 (m, 1H), 1.70 - 1.54 (m, 4H), 1.48 (d, J = 6.9 Hz, 3H), 1.44 - 1.33 (m, 2H), 1.05 (s, 8H). Yield: 60%

[0155] Synthesis of final product 1 of Example 20

[0156]

[0157] To a mixture of intermediate 1 (0.12 mmol, 1.0 eq) and intermediate 2-1 (n = 2) (0.14 mmol, 1.2 eq) in tetrahydrofuran (THF, 1 mL) and water (0.3 mL), vitamin C sodium (0.36 mmol, 3.0 eq) and anhydrous copper sulfate (19 mg, 0.12 mmol, 1.0 eq) were added. The reaction system was reacted at room temperature for 20-30 minutes. After the reaction was completed, the solid was removed by filtration, and the filtrate was evaporated under reduced pressure, and then column chromatography was used to separate the final product 1. The product is a light yellow solid with a yield of 60%.

[0158] 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.13 (dd, J = 8.6, 3.9 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.60 (s, 1H), 5.76 (s, 2H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 4.84 (s, 1H), 4.73 (d, J = 12.0 Hz, 1H), 4.68 - 4.62 (m, 1H), 4.54 - 4.43 (m, 3H), 4.35 (d, J = 9.5 Hz, 1H), 3.86 - 3.76 (m, 2H), 3.61 - 3.56 (m, 2H), 3.54 (s, 4H), 3.47 - 3.41 (m, 2H), 2.95 - 2.77 (m, 2H), 2.62 - 2.52 (m, 2H), 2.17 (dd, J = 9.5, 3.5 Hz, 1H), 2.08 - 1.78 (m, 5H), 1.56 (s, 2H), 1.49 - 1.37 (m, 2H), 1.21 - 1.14 (m, 1H), 1.10 (d, J = 22.9 Hz, 4H), 1.04 (s, 2H), 1.01 (d, J = 6.7 Hz, 2H), 0.94 (d, J = 6.7 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 202.16, 173.24, 170.53, 169.43, 167.74, 150.20, 146.86, 145.91, 145.33, 144.86, 136.69, 133.91, 132.55, 124.29, 117.89, 113.05, 111.16, 110.24, 109.72, 88.32, 70.11, 70.05, 69.31, 69.25, 65.67, 65.51, 61.30, 56.28, 55.37, 50.10, 49.85, 49.03, 42.14, 38.04, 35.93, 31.45, 28.99, 28.46, 25.35, 22.61, 22.12, 16.65, 15.10, 12.93.

[0159] Synthesis of final product 2, Example 21

[0160]

[0161] The procedure and ratio were referred to the preparation of final product 1. The product was light yellow solid with a yield of 64%.

[0162] 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.63 - 7.51 (m, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.60 (t, J = 5.4 Hz, 1H), 5.76 (s, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 4.74 (d, J = 11.9 Hz, 1H), 4.66 (d, J = 8.1 Hz, 1H), 4.53 - 4.43 (m, 3H), 4.35 (d, J = 9.5 Hz, 1H), 4.15 (s, 1H), 3.83 - 3.75 (m, 2H), 3.61 (dd, J = 8.7, 5.0 Hz, 2H), 3.56 - 3.53 (m, 2H), 3.52 - 3.43 (m, 9H), 2.86 (ddd, J = 38.7, 20.1, 8.6 Hz, 2H), 2.57 (dd, J = 27.0, 15.4 Hz, 2H), 2.17 (dd, J = 9.3, 2.8 Hz, 1H), 2.07 - 1.79 (m, 4H), 1.56 (s, 2H), 1.48 (dd, J = 28.2, 15.7 Hz, 2H), 1.43 - 1.37 (m, 1H), 1.20 - 1.14 (m, 1H), 1.13 (s, 3H), 1.10 (s, 1H), 1.05 (s, 2H), 1.01 (d, J = 6.7 Hz, 1H), 0.95 (d, J = 6.6 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 202.16, 173.26, 170.52, 169.40, 167.75, 150.20, 146.86, 145.94, 145.31, 144.85, 136.68, 133.91, 132.55, 124.31, 117.89, 113.05, 111.14, 110.24, 109.70, 88.32, 70.23, 70.18, 70.03, 69.34, 69.22, 65.67, 61.34, 56.28, 55.37, 50.10, 49.81, 49.02, 42.16, 38.04, 35.93, 31.45, 28.99, 28.47, 25.36, 22.62, 22.12, 16.66, 15.10, 15.07, 12.93.

[0163] Synthesis of Example 22 final product 3

[0164]

[0165] Procedure and ratio are referred to the preparation of final product 1. The product is a yellow solid with 55% yield.

[0166] 1 H NMR (600 MHz, DMSO) δ 11.11 (s, 1H), 8.05 (s, 1H), 7.98 (s, 1H), 7.58 (dd, J = 8.3, 7.3 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.60 (t, J = 5.7 Hz, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 4.82 (d, J = 8.0 Hz, 1H), 4.77 - 4.71 (m, 1H), 4.69 - 4.63 (m, 1H), 4.50 (dt, J = 15.3, 5.2 Hz, 2H), 4.46 (s, 1H), 4.35 (s, 1H), 4.16 (t, J = 3.3 Hz, 1H), 3.83 - 3.75 (m, 2H), 3.61 (t, J = 5.3 Hz, 2H), 3.57 - 3.54 (m, 2H), 3.54 - 3.40 (m, 12H), 2.96 - 2.75 (m, 2H), 2.63 - 2.52 (m, 2H), 2.18 (dd, J = 9.5, 3.5 Hz, 1H), 2.01 (dd, J = 25.0, 18.1 Hz, 4H), 1.56 (s, 2H), 1.53 - 1.37 (m, 3H), 1.17 (d, J = 4.0 Hz, 2H), 1.12 (d, J = 13.9 Hz, 4H), 1.05 (s, 2H), 1.01 (d, J = 6.7 Hz, 2H), 0.95 (d, J = 6.7 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 202.15, 173.26, 170.52, 169.39, 167.75, 150.20, 146.86, 145.94, 145.31, 144.85, 136.68, 133.89, 132.55, 124.33, 117.90, 113.04, 111.13, 110.23, 109.69, 88.30, 70.28, 70.23, 70.09, 69.99, 69.34, 69.23, 69.19, 65.66, 65.48, 61.33, 56.29, 50.11, 49.79, 49.01, 42.15, 38.04, 35.93, 35.47, 31.44, 28.99, 28.46, 25.36, 22.60, 22.12, 16.66, 15.10, 15.07, 12.93.

[0167] Synthesis of final product 4

[0168]

[0169] The procedure and ratio are referred to the preparation of final product 1. The product is a white solid with a yield of 50%.

[0170] 1 H NMR (600 MHz, DMSO) δ 8.01 (d, J = 36.9 Hz, 1H), 7.92 (dt, J = 11.0, 5.5 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 21.6, 8.2 Hz, 4H), 7.01 (dd, J = 38.9, 8.1 Hz, 4H), 6.61 (d, J = 7.2 Hz, 2H), 5.66 (d, J = 9.8 Hz, 1H), 5.58 (d, J = 9.8 Hz, 1H), 4.89 - 4.78 (m, 1H), 4.77 - 4.63 (m, 3H), 4.53 - 4.44 (m, 3H), 4.35 (t, J = 8.7 Hz, 1H), 4.16 (s, 1H), 3.83 (s, 4H), 3.77 (dd, J = 10.0, 5.1 Hz, 2H), 3.72 (d, J = 15.9 Hz, 2H), 3.59 (d, J = 17.1 Hz, 1H), 3.42 - 3.35 (m, 3H), 3.17 (dd, J = 6.8, 4.3 Hz, 4H), 2.99 (s, 2H), 2.81 (dd, J = 12.8, 9.1 Hz, 1H), 2.56 (dd, J = 12.7, 6.7 Hz, 1H), 2.24 (s, 1H), 2.18 (dd, J = 9.4, 3.3 Hz, 1H), 1.97 - 1.90 (m, 1H), 1.84 (s, 1H), 1.56 (s, 2H), 1.53 - 1.38 (m, 3H), 1.27 (d, J = 5.9 Hz, 3H), 1.22 (t, J = 6.4 Hz, 5H), 1.12 (d, J = 14.8 Hz, 5H), 1.05 (s, 2H), 1.01 (d, J = 6.5 Hz, 2H), 0.95 (d, J = 6.6 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 202.18, 167.81, 164.84, 162.83, 160.39, 156.97, 154.65, 150.20, 145.93, 145.38, 144.91, 137.82, 136.88, 133.90, 132.42, 131.71, 131.58, 130.15, 129.18, 127.93, 127.89, 124.32, 113.80, 113.06, 110.25, 105.46, 99.78, 88.30, 71.52, 70.32, 69.08, 68.92, 68.30, 65.64, 61.29, 56.27, 55.89, 50.10, 49.69, 49.44, 49.00, 46.93, 42.48, 38.77, 38.05, 35.92, 28.99, 28.46, 26.80, 25.35, 22.19, 22.12, 16.66, 15.09, 14.54, 14.41, 12.93.

[0171] Synthesis of end product 5, example 24

[0172]

[0173] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 56% yield.

[0174] 1H NMR (600 MHz, DMSO) δ 7.98 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.2 Hz, 2H), 6.62 (s, 2H), 5.65 (d, J = 9.7 Hz, 1H), 5.58 (d, J = 9.7 Hz, 1H), 4.77 - 4.70 (m, 2H), 4.68 - 4.64 (m, 1H), 4.50 (dt, J = 15.6, 5.3 Hz, 3H), 4.38 - 4.33 (m, 1H), 4.16 (t, J = 3.4 Hz, 1H), 3.83 (s, 3H), 3.80 (dt, J = 10.3, 5.3 Hz, 2H), 3.75 - 3.66 (m, 2H), 3.62 - 3.56 (m, 1H), 3.53 - 3.49 (m, 2H), 3.46 (d, J = 2.6 Hz, 6H), 3.37 (td, J = 5.9, 1.9 Hz, 3H), 3.25 - 3.14 (m, 3H), 2.99 (s, 2H), 2.81 (dd, J = 13.0, 8.9 Hz, 1H), 2.57 (dd, J = 13.1, 6.7 Hz, 1H), 2.24 (s, 1H), 2.18 (dd, J = 9.5, 3.6 Hz, 0H), 1.97 - 1.90 (m, 1H), 1.89 - 1.81 (m, 1H), 1.56 (s, 2H), 1.53 - 1.46 (m, 1H), 1.46 - 1.38 (m, 2H), 1.27 (d, J = 6.0 Hz, 3H), 1.22 (d, J = 6.0 Hz, 3H), 1.13 (s, 3H), 1.10 (d, J = 8.9 Hz, 1H), 1.05 (s, 1H), 1.01 (d, J = 6.8 Hz, 2H), 0.97 - 0.93 (m, 1H). 13CNMR (151 MHz, DMSO) δ 167.79, 164.82, 162.83, 160.41, 156.98, 154.68, 150.22, 145.97, 145.33, 144.87, 137.84, 136.90, 132.42, 131.71, 131.58, 130.16, 129.19, 127.94, 127.90, 124.32, 113.83, 113.04, 110.25, 105.48, 99.78, 88.30, 71.55, 70.32, 70.15, 70.10, 70.08, 70.02, 69.39, 69.24, 69.20, 68.31, 65.66, 61.36, 56.29, 55.90, 50.11, 49.81, 49.77, 49.47, 48.97, 46.91, 42.48, 40.52, 38.99, 38.04, 35.94, 29.00, 28.47, 26.81, 25.37, 22.19, 22.12, 16.67, 15.10, 15.07, 12.94.

[0175] Example 25 Synthesis of end product 6

[0176]

[0177] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 53% yield.

[0178] 1H NMR (600 MHz, DMSO) δ 7.96 (d, J = 4.7 Hz, 2H), 7.54 (d, J = 6.8 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 7.8 Hz, 2H), 6.62 (s, 2H), 5.67 (d, J = 9.4 Hz, 1H), 5.60 (s, 1H), 5.16 (s, 1H), 5.01 (s, 1H), 4.84 (d, J = 4.4 Hz, 1H), 4.79 (s, 1H), 4.76 - 4.68 (m, 2H), 4.49 (t, J = 5.3 Hz, 2H), 4.45 (d, J = 10.3 Hz, 1H), 4.39 (s, 1H), 4.32 (s, 1H), 4.18 (d, J = 10.8 Hz, 1H), 4.11 (s, 1H), 3.83 (s, 4H), 3.80 (t, J = 5.2 Hz, 3H), 3.71 (s, 2H), 3.59 (d, J = 17.2 Hz, 1H), 3.51 (dd, J = 5.6, 3.0 Hz, 2H), 3.49 - 3.44 (m, 12H), 3.37 (t, J = 5.9 Hz, 3H), 3.24 (d, J = 10.7 Hz, 1H), 3.21 - 3.15 (m, 3H), 2.99 (s, 2H), 2.19 (dd, J = 11.1, 6.4 Hz, 1H), 2.12 - 2.05 (m, 1H), 2.02 (d, J = 13.4 Hz, 1H), 1.80 (s, 3H), 1.56 (dd, J = 15.9, 11.0 Hz, 1H), 1.49 (dd, J = 15.4, 8.8 Hz, 1H), 1.34 - 1.28 (m, 1H), 1.27 (d, J = 5.9 Hz, 3H), 1.22 (d, J = 5.9 Hz, 4H), 1.04 (s, 4H), 0.91 (s, 2H), 0.90 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.6 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 167.78, 164.79, 162.87, 156.95, 154.64, 148.10, 137.75, 136.81, 135.99, 132.37, 131.87, 131.73, 131.61, 130.14, 129.18, 127.94, 124.43, 116.86, 113.70, 105.48, 99.78, 81.70, 79.89, 72.45, 70.34, 70.21, 70.16, 70.07, 70.02, 69.98, 69.37, 69.16, 60.79, 55.90, 55.72, 49.75, 49.45, 48.95, 46.89, 42.46, 41.43, 38.98, 35.23, 33.63, 29.17, 26.59, 26.24, 24.65, 23.85, 23.76, 22.19, 22.11, 18.00, 16.16, 12.52.

[0179] Example 26 Synthesis of end product 7

[0180]

[0181] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 64% yield.

[0182] 1H NMR (600 MHz, DMSO) δ 8.04 (s, 1H), 7.98 (s, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.26 (t, J = 7.4 Hz, 2H), 7.19 (dd, J = 16.9, 7.2 Hz, 4H), 6.16 (dd, J = 9.3, 4.2 Hz, 1H), 5.97 (d, J = 5.7 Hz, 1H), 4.73 (s, 1H), 4.67 (d, J = 11.0 Hz, 1H), 4.52 - 4.43 (m, 3H), 4.34 (d, J = 8.5 Hz, 2H), 4.16 (s, 1H), 3.95 (d, J = 7.3 Hz, 1H), 3.81 (s, 1H), 3.77 (dt, J = 10.2, 5.1 Hz, 2H), 3.44 - 3.36 (m, 2H), 3.17 (d, J = 4.9 Hz, 2H), 2.77 (d, J = 7.3 Hz, 1H), 2.67 (s, 1H), 2.24 (s, 1H), 2.20 - 2.15 (m, 1H), 2.00 - 1.92 (m, 1H), 1.85 (s, 1H), 1.56 (s, 2H), 1.54 - 1.51 (m, 1H), 1.46 (dd, J = 19.5, 10.2 Hz, 3H), 1.40 (dd, J = 11.9, 8.2 Hz, 1H), 1.33 (s, 1H), 1.28 (s, 8H), 1.24 (d, J = 8.1 Hz, 3H), 1.15 (s, 2H), 1.13 (s, 3H), 1.10 (d, J = 9.3 Hz, 2H), 1.05 (s, 1H), 1.01 (d, J = 6.6 Hz, 2H), 0.95 (d, J = 6.7 Hz, 1H), 0.85 (d, J = 6.4 Hz, 3H), 0.81 (d, J = 6.3 Hz, 4H). 13 CNMR (151 MHz, DMSO) δ 202.17, 172.31, 171.98, 155.33, 150.19, 145.95, 145.39, 144.91, 139.18, 133.89, 129.85, 129.68, 128.59, 126.47, 124.29, 113.05, 110.25, 88.34, 78.04, 71.87, 69.08, 68.95, 65.65, 61.32, 56.29, 54.93, 50.75, 50.11, 49.70, 42.08, 38.80, 38.02, 35.93, 29.44, 29.00, 28.57, 28.46, 28.16, 25.36, 24.44, 23.59, 22.17, 21.52, 16.67, 15.10, 12.93.

[0183] Synthesis of Example 27 final product 8

[0184]

[0185] The procedure and ratios were as for the preparation of final product 1. The product was a white solid in 56% yield.

[0186] 1 H NMR (400 MHz, DMSO) δ 8.04 (s, 2H), 7.98 (s, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.26 (d, J = 7.3 Hz, 3H), 7.21 (d, J = 7.1 Hz, 4H), 6.16 (d, J = 9.4 Hz, 1H), 5.97 (d, J = 6.2 Hz, 1H), 4.86 (s, 1H), 4.81 (d, J = 8.0 Hz, 1H), 4.73 (d, J = 6.5 Hz, 1H), 4.70 - 4.63 (m, 1H), 4.49 (d, J = 5.0 Hz, 4H), 4.39 - 4.29 (m, 2H), 4.16 (d, J = 3.2 Hz, 1H), 3.95 (q, J = 7.4 Hz, 1H), 3.79 (d, J = 5.4 Hz, 4H), 3.56 - 3.42 (m, 5H), 3.17 (d, J = 5.2 Hz, 3H), 2.85 - 2.75 (m, 2H), 2.66 (dd, J = 13.3, 7.7 Hz, 1H), 2.29 - 2.14 (m, 1H), 2.04 - 1.79 (m, 4H), 1.48 (d, J = 66.2 Hz, 11H), 1.29 (s, 10H), 1.13 (t, J = 7.5 Hz, 9H), 1.05 (s, 2H), 1.01 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 2H), 0.84 (dd, J = 16.3, 6.4 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 202.14, 172.28, 171.95, 155.33, 150.21, 145.97, 145.34, 144.88, 139.19, 133.92, 129.68, 128.59, 126.47, 124.31, 113.03, 110.24, 88.37, 88.31, 78.05, 71.89, 69.93, 69.90, 69.87, 69.31, 69.24, 69.20, 65.67, 61.37, 56.29, 54.94, 50.77, 50.10, 49.77, 42.17, 39.01, 38.03, 35.94, 29.01, 28.92, 28.58, 28.47, 28.17, 26.81, 25.35, 24.44, 23.60, 22.20, 16.67, 15.10, 12.93.

[0187] Synthesis of end product 9, example 28

[0188]

[0189] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 50% yield.

[0190] 1H NMR (400 MHz, DMSO) δ 8.05 (s, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.26 (d, J = 7.3 Hz, 2H), 7.21 (d, J = 8.2 Hz, 4H), 6.15 (d, J = 9.4 Hz, 1H), 5.95 (t, J = 9.2 Hz, 1H), 4.77 (dd, J = 12.5, 10.0 Hz, 1H), 4.71 - 4.63 (m, 2H), 4.50 (d, J = 5.2 Hz, 4H), 4.39 - 4.30 (m, 2H), 4.18 - 4.12 (m, 1H), 3.95 (dt, J = 9.7, 5.0 Hz, 1H), 3.81 (d, J = 5.7 Hz, 3H), 3.54 - 3.50 (m, 2H), 3.47 (s, 11H), 3.38 (d, J = 5.8 Hz, 3H), 3.18 (dd, J = 10.2, 5.4 Hz, 3H), 2.84 - 2.74 (m, 2H), 2.66 (dd, J = 13.3, 7.7 Hz, 1H), 2.24 (d, J = 6.9 Hz, 1H), 2.18 (dd, J = 9.4, 3.5 Hz, 1H), 1.96 (dd, J = 15.5, 7.3 Hz, 2H), 1.87 (d, J = 18.8 Hz, 2H), 1.68 - 1.60 (m, 2H), 1.56 (s, 2H), 1.51 (dd, J = 13.1, 7.0 Hz, 2H), 1.48 (s, 3H), 1.40 (s, 3H), 1.29 (s, 9H), 1.24 (s, 2H), 1.15 (s, 2H), 1.13 (s, 3H), 1.11 (s, 2H), 1.05 (s, 2H), 1.01 (d, J = 6.6 Hz, 2H), 0.96 (d, J = 6.7 Hz, 2H), 0.86 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.4 Hz, 4H). 13C NMR (101 MHz, DMSO) δ 202.13, 172.28, 171.94, 155.34, 150.22, 146.00, 145.33, 144.87, 139.20, 133.93, 129.69, 129.13, 128.60, 126.48, 124.32, 113.02, 110.24, 88.39, 88.31, 78.05, 71.89, 70.23, 70.18, 70.12, 70.09, 70.04, 69.32, 69.25, 69.21, 65.68, 65.49, 61.39, 56.30, 54.94, 50.78, 50.10, 49.82, 42.21, 39.04, 38.03, 35.95, 29.01, 28.59, 28.48, 28.17, 26.81, 25.35, 24.45, 23.60, 22.23, 16.67, 15.09, 12.93.

[0191] Example 29 Synthesis of end product 10

[0192]

[0193] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 44% yield.

[0194] 1H NMR (400 MHz, DMSO) δ 8.22 - 8.14 (m, 1H), 7.87 - 7.82 (m, 1H), 7.78 (s, 1H), 7.26 (s, 2H), 7.23 (s, 3H), 6.30 (d, J = 5.8 Hz, 1H), 5.06 (dd, J = 16.1, 13.7 Hz, 1H), 4.93 (dd, J = 16.2, 8.8 Hz, 1H), 4.86 (s, 1H), 4.81 - 4.73 (m, 1H), 4.67 (d, J = 6.0 Hz, 1H), 4.37 (dt, J = 12.9, 8.6 Hz, 2H), 4.26 - 4.19 (m, 1H), 4.16 (s, 1H), 3.94 - 3.87 (m, 1H), 3.61 (s, 3H), 2.85 (dt, J = 19.2, 9.5 Hz, 2H), 2.71 - 2.62 (m, 1H), 2.25 (d, J = 8.6 Hz, 1H), 2.18 (dd, J = 9.5, 3.5 Hz, 1H), 1.97 (dd, J = 16.2, 6.8 Hz, 2H), 1.87 (d, J = 10.9 Hz, 2H), 1.71 - 1.58 (m, 3H), 1.56 (s, 2H), 1.55 - 1.38 (m, 5H), 1.27 - 1.15 (m, 4H), 1.13 (s, 5H), 1.05 (s, 1H), 1.02 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 202.11, 173.14, 172.15, 165.22, 150.26, 145.99, 145.18, 144.77, 138.90, 133.92, 129.64, 128.74, 126.69, 125.14, 113.02, 110.22, 88.42, 71.09, 65.74, 61.38, 61.08, 56.32, 54.15, 52.39, 51.91, 50.16, 38.04, 37.33, 35.97, 29.02, 28.50, 25.35, 24.64, 23.23, 21.69, 16.68, 15.10, 12.93.

[0195] Synthesis of end product 11, example 30

[0196]

[0197] The procedure and ratios are as for the preparation of end product 1. The product is a white solid in 43% yield.

[0198] 1 H NMR (600 MHz, DMSO) δ 8.03 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.26 (t, J = 7.5 Hz, 2H), 7.22 (d, J = 7.3 Hz, 2H), 7.17 (s, 1H), 6.19 (d, J = 6.1 Hz, 1H), 4.81 (d, J = 8.1 Hz, 1H), 4.73 (s, 1H), 4.72 (s, 1H), 4.66 (s, 1H), 4.45 (s, 1H), 4.34 (d, J = 8.7 Hz, 2H), 4.21 (dd, J = 11.7, 6.8 Hz, 3H), 4.15 (s, 1H), 3.87 (d, J = 2.4 Hz, 1H), 3.60 (s, 3H), 3.17 (d, J = 5.2 Hz, 1H), 2.80 (d, J = 7.4 Hz, 2H), 2.66 (dd, J = 13.2, 8.2 Hz, 1H), 2.57 (dd, J = 13.1, 6.9 Hz, 1H), 2.17 (dd, J = 9.5, 3.4 Hz, 1H), 2.11 - 2.04 (m, 1H), 1.99 (s, 2H), 1.93 (s, 2H), 1.91 (d, J = 7.2 Hz, 3H), 1.67 - 1.58 (m, 2H), 1.56 (s, 2H), 1.52 (s, 1H), 1.48 (d, J = 13.3 Hz, 2H), 1.45 (d, J = 3.8 Hz, 1H), 1.40 (dd, J = 11.8, 8.5 Hz, 1H), 1.23 (s, 2H), 1.17 (d, J = 7.4 Hz, 2H), 1.13 (s, 3H), 1.04 (s, 3H), 1.01 (d, J = 6.1 Hz, 1H), 0.95 (d, J = 6.7 Hz, 2H), 0.85 (d, J = 5.9 Hz, 1H), 0.82 (d, J = 6.5 Hz, 3H), 0.76 (d, J = 6.4 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 202.15, 173.18, 172.41, 171.08, 150.20, 145.41, 139.23, 133.86, 129.66, 128.61, 126.54, 123.83, 110.24, 88.36, 71.65, 65.71, 56.31, 53.62, 52.37, 50.13, 50.01, 49.26, 49.05, 38.05, 37.36, 35.93, 32.39, 28.99, 28.47, 26.53, 25.38, 24.47, 23.26, 22.13, 21.57, 16.68, 15.08, 12.93.

[0199] Synthesis of end product 12

[0200]

[0201] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 52% yield.

[0202] 1 H NMR (600 MHz, DMSO) δ 7.99 (s, 1H), 7.81 (t, J = 12.8 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.30 - 7.15 (m, 5H), 6.23 (s, 1H), 4.80 (dd, J = 29.2, 21.2 Hz, 1H), 4.68 (dd, J = 37.6, 9.7 Hz, 2H), 4.27 (s, 3H), 4.22 (s, 3H), 3.85 (s, 1H), 3.61 (s, 3H), 2.79 (dd, J = 12.8, 7.1 Hz, 1H), 2.70 - 2.60 (m, 1H), 2.19 (dd, J = 24.8, 8.4 Hz, 1H), 2.03 (dt, J = 15.1, 7.7 Hz, 1H), 1.95 (d, J = 7.4 Hz, 3H), 1.85 (s, 1H), 1.73 (s, 2H), 1.63 (d, J = 7.8 Hz, 2H), 1.56 (s, 3H), 1.44 (dd, J = 21.6, 8.7 Hz, 3H), 1.41 - 1.34 (m, 3H), 1.23 (s, 2H), 1.11 (d, J = 16.1 Hz, 6H), 1.05 - 0.99 (m, 3H), 0.92 (dd, J = 12.5, 7.0 Hz, 3H), 0.83 (d, J = 6.1 Hz, 3H), 0.78 (d, J = 6.2 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 173.19, 172.05, 167.42, 150.19, 145.95, 145.40, 144.90, 139.23, 133.90, 132.16, 131.98, 129.64, 128.61, 126.52, 123.75, 113.01, 110.24, 88.31, 71.60, 65.67, 65.48, 61.34, 61.04, 56.31, 53.62, 52.37, 50.94, 49.59, 38.00, 37.30, 35.37, 28.99, 28.43, 26.80, 25.86, 25.05, 24.48, 23.29, 21.64, 16.66, 15.10, 12.92.

[0203] Synthesis of example 32 final product 13

[0204]

[0205] The procedure and ratio were referred to the preparation of final product 1. The product was a white solid with a yield of 30%.

[0206] 1 H NMR (600 MHz, DMSO) δ 8.99 (s, 1H), 8.63 (s, 1H), 8.58 - 8.52 (m, 1H), 7.41 (t, J = 11.9 Hz, 5H), 5.26 - 5.19 (m, 1H), 5.14 (d, J = 3.5 Hz, 1H), 4.89 - 4.71 (m, 2H), 4.67 (dd, J = 14.8, 8.8 Hz, 1H), 4.54 (dd, J = 17.6, 8.3 Hz, 2H), 4.46 (s, 3H), 4.39 - 4.30 (m, 2H), 4.29 - 4.14 (m, 3H), 3.72 - 3.54 (m, 2H), 2.82 (dd, J = 12.8, 8.9 Hz, 1H), 2.56 (s, 1H), 2.47 - 2.42 (m, 4H), 2.30 (d, J = 53.5 Hz, 1H), 2.18 (dd, J = 9.5, 3.5 Hz, 1H), 2.03 (dd, J = 22.4, 14.7 Hz, 1H), 1.93 (s, 4H), 1.56 (s, 3H), 1.51 - 1.37 (m, 2H), 1.22 (d, J = 20.2 Hz, 1H), 1.13 (s, 4H), 1.06 (d, J = 2.4 Hz, 2H), 1.02 - 0.90 (m, 14H). 13 C NMR (151 MHz, DMSO) δ 172.34, 169.48, 165.81, 151.94, 150.24, 148.18, 145.16, 139.96, 131.63, 130.11, 129.10, 127.86, 125.55, 113.05, 110.23, 100.00, 88.33, 69.33, 65.75, 59.23, 57.31, 57.01, 56.31, 51.74, 50.10, 42.11, 38.38, 38.09, 36.04, 29.01, 28.47, 26.73, 25.37, 16.72, 16.42, 15.09, 12.93.

[0207] Synthesis of example 33 final product 14

[0208]

[0209] The procedure and ratio were referred to the preparation of the final product 1. The product was white solid with a yield of 45%.

[0210] 1 H NMR (600 MHz, DMSO) δ 8.99 (s, 1H), 8.54 - 8.40 (m, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 5.27 - 5.16 (m, 2H), 5.12 (d, J = 3.4 Hz, 1H), 4.93 (s, 1H), 4.83 (d, J = 8.0 Hz, 1H), 4.75 (s, 1H), 4.67 (s, 1H), 4.52 (s, 2H), 4.49 - 4.41 (m, 2H), 4.31 - 4.22 (m, 1H), 4.16 (t, J = 3.3 Hz, 1H), 3.62 (d, J = 7.4 Hz, 1H), 3.54 (dd, J = 19.6, 9.3 Hz, 1H), 2.82 (dd, J = 13.0, 9.0 Hz, 0H), 2.46 (s, 4H), 2.25 (s, 0H), 2.19 (dd, J = 9.4, 3.5 Hz, 1H), 2.07 - 2.00 (m, 1H), 1.95 (d, J = 2.4 Hz, 1H), 1.88 (d, J = 8.1 Hz, 1H), 1.78 (ddd, J = 24.2, 12.2, 7.8 Hz, 1H), 1.57 (s, 3H), 1.52 - 1.42 (m, 2H), 1.39 (d, J = 7.1 Hz, 4H), 1.23 (s, 1H), 1.15 - 1.09 (m, 4H), 1.06 (s, 2H), 1.04 - 0.92 (m, 14H). 13 C NMR (151 MHz, DMSO) δ 202.20, 171.01, 169.38, 165.77, 151.95, 150.24, 148.22, 145.95, 145.19, 145.10, 144.72, 133.88, 131.58, 130.17, 129.30, 126.85, 125.55, 113.05, 110.24, 88.36, 69.24, 67.43, 65.76, 65.49, 61.34, 60.22, 59.11, 57.38, 56.90, 56.29, 51.77, 50.10, 48.20, 38.17, 36.02, 36.01, 35.98, 35.95, 29.02, 28.49, 26.81, 25.37, 22.89, 22.14, 16.72, 16.45, 15.13, 15.09, 14.55, 12.94.

[0211] Synthesis of Example 34 end product 15

[0212]

[0213] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 50% yield.

[0214] 1 H NMR (600 MHz, DMSO) δ 8.99 (s, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.02 (s, 1H), 7.40 (dd, J = 22.7, 8.2 Hz, 5H), 5.16 - 5.11 (m, 1H), 4.81 (d, J = 8.0 Hz, 1H), 4.73 (s, 1H), 4.66 (s, 1H), 4.55 (dd, J = 9.3, 3.8 Hz, 1H), 4.43 (dd, J = 15.5, 6.8 Hz, 3H), 4.34 (dd, J = 15.2, 7.7 Hz, 4H), 4.21 (dd, J = 15.9, 5.4 Hz, 1H), 4.15 (t, J = 3.3 Hz, 1H), 3.66 (q, J = 10.6 Hz, 2H), 2.81 (dd, J = 12.9, 9.0 Hz, 1H), 2.45 (d, J = 2.9 Hz, 3H), 2.32 - 2.14 (m, 4H), 2.03 (d, J = 7.6 Hz, 3H), 1.97 - 1.82 (m, 4H), 1.56 (s, 2H), 1.46 (dd, J = 23.4, 10.8 Hz, 2H), 1.40 (dd, J = 11.8, 8.4 Hz, 1H), 1.24 (s, 2H), 1.13 (s, 3H), 1.10 (d, J = 7.1 Hz, 1H), 1.05 (s, 2H), 1.01 (d, J = 6.8 Hz, 2H), 0.97 - 0.91 (m, 12H). 13C NMR (101 MHz, DMSO) δ 202.14, 172.38, 171.55, 170.08, 151.89, 150.23, 148.19, 145.98, 145.47, 144.97, 139.97, 133.91, 131.63, 130.12, 129.10, 127.90, 123.84, 113.02, 110.23, 88.38, 88.30, 69.35, 65.73, 61.36, 59.17, 56.96, 56.83, 56.29, 55.36, 50.08, 49.42, 42.13, 38.41, 38.03, 35.94, 35.68, 32.11, 29.01, 28.47, 26.85, 26.66, 25.35, 22.13, 16.40, 15.09, 14.55, 12.93.

[0215] Example 35 Synthesis of end product 16

[0216]

[0217] The procedure and ratios were as for the preparation of end product 1. The product was a white solid in 54% yield.

[0218] 1H NMR (600 MHz, DMSO) δ 8.99 (s, 1H), 8.37 (s, 1H), 8.09 (s, 1H), 7.95 (dd, J = 9.1, 4.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 3H), 7.38 (d, J = 8.1 Hz, 2H), 5.11 (d, J = 2.3 Hz, 1H), 4.92 (dd, J = 14.3, 7.1 Hz, 1H), 4.80 (t, J = 11.6 Hz, 1H), 4.73 (s, 1H), 4.66 (s, 1H), 4.55 - 4.48 (m, 2H), 4.44 (d, J = 19.1 Hz, 2H), 4.37 - 4.31 (m, 3H), 4.28 (s, 1H), 4.16 (s, 1H), 3.61 (s, 2H), 2.81 (dd, J = 13.0, 9.1 Hz, 1H), 2.46 (s, 3H), 2.25 (ddd, J = 21.2, 14.2, 6.8 Hz, 2H), 2.18 (s, 2H), 2.06 - 1.98 (m, 4H), 1.98 - 1.90 (m, 2H), 1.88 (d, J = 7.3 Hz, 1H), 1.79 (ddd, J = 12.8, 8.4, 4.7 Hz, 1H), 1.56 (s, 2H), 1.48 (s, 2H), 1.37 (d, J = 7.0 Hz, 3H), 1.23 (s, 2H), 1.12 (d, J = 10.3 Hz, 5H), 1.05 (s, 2H), 1.02 (d, J = 6.8 Hz, 2H), 0.97 - 0.91 (m, 13H). 13 C NMR (101 MHz, DMSO) δ 202.15, 171.51, 171.07, 169.99, 151.92, 150.23, 148.23, 145.98, 145.48, 145.10, 144.98, 133.91, 131.58, 130.17, 129.28, 126.85, 123.84, 113.02, 110.24, 88.38, 69.24, 65.73, 65.51, 61.36, 59.02, 57.03, 56.72, 56.29, 50.08, 49.42, 48.16, 38.17, 38.04, 35.95, 35.64, 32.12, 29.01, 28.48, 26.91, 26.66, 25.35, 22.87, 22.12, 16.68, 16.44, 15.09, 12.93.

[0219] Synthesis of Example 36 final product 17

[0220]

[0221] The procedure and ratio were referred to the preparation of the final product 1. The product was a white solid with a yield of 50%.

[0222] 1 H NMR (600 MHz, DMSO) δ 8.98 (s, 1H), 8.56 (t, J = 6.0 Hz, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.86 (dd, J = 9.3, 3.7 Hz, 1H), 7.40 (dd, J = 22.6, 8.2 Hz, 6H), 5.13 (d, J = 2.8 Hz, 1H), 4.80 (t, J = 8.6 Hz, 1H), 4.76 - 4.63 (m, 3H), 4.53 (dt, J = 14.7, 7.3 Hz, 2H), 4.44 (d, J = 17.1 Hz, 4H), 4.33 (s, 5H), 4.21 (dd, J = 15.8, 5.6 Hz, 2H), 4.18 - 4.12 (m, 1H), 3.66 (dt, J = 17.4, 6.8 Hz, 2H), 2.81 (dd, J = 13.0, 8.9 Hz, 1H), 2.44 (s, 4H), 2.30 - 2.21 (m, 2H), 2.19 - 2.08 (m, 2H), 1.99 (s, 10H), 1.56 (s, 10H), 1.24 (s, 6H), 1.13 (s, 5H), 1.05 (s, 2H), 1.01 (d, J = 6.7 Hz, 2H), 0.96 - 0.89 (m, 13H). 13 C NMR (151 MHz, DMSO) δ 202.14, 172.41, 172.38, 170.16, 151.92, 150.21, 148.18, 145.97, 145.39, 144.89, 139.98, 131.63, 130.10, 129.10, 127.88, 123.81, 113.03, 110.24, 88.31, 69.33, 65.69, 61.37, 60.22, 59.15, 56.83, 56.75, 56.30, 50.11, 49.64, 42.11, 40.53, 38.42, 38.02, 35.94, 35.67, 35.09, 29.95, 29.00, 28.47, 26.84, 25.97, 25.37, 25.27, 22.13, 16.68, 16.41, 15.12, 15.09, 12.94.

[0223] Synthesis of final product 18

[0224]

[0225] Procedure and ratio are referred to the preparation of final product 1. The product is a white solid in 47% yield.

[0226] 1 H NMR (600 MHz, DMSO) δ 8.98 (s, 1H), 8.37 (s, 1H), 8.08 (s, 1H), 8.02 - 8.00 (m, 1H), 7.81 (dd, J = 9.2, 2.9 Hz, 1H), 7.43 (d, J = 8.2 Hz, 3H), 7.38 (d, J = 8.2 Hz, 3H), 5.11 (d, J = 3.2 Hz, 2H), 4.95 - 4.89 (m, 2H), 4.83 - 4.78 (m, 1H), 4.77 - 4.64 (m, 3H), 4.51 (t, J = 9.6 Hz, 2H), 4.42 (s, 3H), 4.33 (s, 5H), 4.16 (t, J = 3.3 Hz, 1H), 3.60 (s, 3H), 2.82 (dd, J = 13.0, 8.9 Hz, 1H), 2.46 (s, 4H), 2.28 - 2.20 (m, 2H), 2.18 (dd, J = 9.5, 3.5 Hz, 1H), 2.15 - 2.08 (m, 2H), 2.04 - 2.00 (m, 1H), 1.84 - 1.76 (m, 5H), 1.56 (s, 8H), 1.38 (d, J = 7.0 Hz, 5H), 1.23 (s, 4H), 1.15 - 1.08 (m, 5H), 1.06 (s, 2H), 1.01 (s, 2H), 0.94 (dd, J = 13.9, 4.5 Hz, 15H). 13 C NMR (151 MHz, DMSO) δ 202.15, 172.34, 171.09, 170.05, 151.94, 150.21, 148.22, 145.97, 145.40, 145.12, 144.89, 133.90, 131.58, 130.16, 129.29, 126.85, 123.81, 113.03, 110.25, 88.32, 69.23, 65.70, 61.38, 59.01, 56.82, 56.73, 50.11, 49.65, 48.16, 40.52, 38.19, 38.03, 35.94, 35.64, 35.10, 29.94, 29.00, 28.48, 26.90, 25.97, 25.37, 25.26, 22.89, 22.14, 16.45, 15.12, 15.10, 14.55, 12.93.

[0227] After the above-synthesized series of bifunctional PROTAC compounds based on active natural products were added to cultured mouse macrophage RAW264.7 cells in the logarithmic growth phase for 3 hours, the cells were stimulated with lipopolysaccharide LPS for 24 hours to establish a mouse macrophage inflammation model; the total nitric oxide (NO) content in the cell supernatant was detected with Griss reagent, and the anti-inflammatory activity of the synthesized PROTAC compounds was analyzed and determined according to the inhibitory effect of the PROTACs small molecules on NO release, so as to screen the PROTAC compound with the best anti-inflammatory activity, i.e. ZCY-PROTAC;

[0228] The cell lysates treated with active natural products and PROTAC compounds, respectively, were subjected to differential proteomics analysis to identify the differentially expressed proteins in the protein lysates, and KEGG analysis was performed to determine the potential target proteins of the PROTAC compounds and active natural products; the potential target proteins were subjected to western blot to verify the degradation activity and degradation characteristics of the PROTAC compounds, and the direct binding force between the active natural products and the target proteins was verified by in vitro target verification methods such as MST, CETSA and DARTS, and the function of the active natural products on the target protein and the downstream proteins affected by the target protein was determined by cell biology techniques such as RNAi and in vivo experiments, so as to further determine whether the active natural products act on the target, thereby verifying the authenticity of the target protein, and finally confirming the target protein of the active natural products, and the specific process is as follows:

[0229] Example 38: Detection of nitric oxide (NO) generation by Griess method

[0230] The logarithmic growth phase mouse macrophage RAW264.7 cells were inoculated in a 96-well plate at 2.5×10 4 cells per well, cultured for 24 h, the supernatant was discarded, and the culture medium containing a concentration gradient of compounds (Lathyrol and the synthesized PROTACs) was replaced. After 3 h of culture, LPS was added to each well to a final concentration of 1 μg / mL. After 24 h of continuous culture, the cell supernatant was taken, and according to the instructions of the nitric oxide kit, Griess reagent was added, and the OD value was detected at 540 nm. The NO inhibition rate of the compounds was calculated according to the following formula: NO inhibition rate = (OD 模型 -OD 化合物 ) / (OD 模型 -OD 对照 )×100%. The results showed that the thousand gold diterpene alkane parent compound Lathyrol had a good inhibitory effect on the generation of NO stimulated by LPS, and the IC 50 value was 11.10±1.14 μM. Among the synthesized PROTACs, the final product 2, i.e. ZCY-PROTAC,Figure 1 A) with the best anti-NO activity, IC 50 value of 8.34 ± 1.09 μM Figure 1 B).

[0231] Table 1. IC values of Lathyrol PROTAC end products for inhibiting NO production in RAW264.7 cells 50 Table

[0232]

[0233] Example 39. Mass spectrometry analysis of cell proteome

[0234] RAW264.7 cells were centrifuged after incubation with ZCY-PROTAC (8 μM), Lathyrol (11 μM) or DMSO for 48 hours, and 4 times the volume of lysis buffer (8 M urea, 1% protease inhibitor) was added. The supernatant was obtained by ultrasonic disruption, and protein quantification was performed using a BCA kit. Equal amounts of protein samples were taken from each sample for enzymolysis, dissolution, and peptide labeling using a 6-plex tandem mass tag (TMT) labeling kit. One microgram was subjected to mass spectrometry detection.

[0235] The results of quantitative proteomic mass spectrometry analysis showed that, compared with the DMSO group, the levels of 548 proteins were up-regulated after ZCY-PROTAC treatment, and the levels of 284 proteins were down-regulated. Among all the proteins, the potential degradation target proteins included MAFF, TRIR (C19orf43), CHTOP, CDV3, MTDH, etc., and the degradation fold of MAFF protein was the highest, with a protein level down-regulated by more than 7 times after ZCY-PROTAC treatment Figure 1 C). At the same time, we analyzed these inflammation-related proteins using KEGG pathway enrichment analysis. The KEGG analysis results showed that the proteins with differential expression were mainly related to lysosomes, ferroptosis, cholesterol metabolism and mitochondrial autophagy, and the MAFF protein was also closely related to inflammation Figure 1 D).

[0236] Example 40. ZCY-PROTAC-mediated degradation of MAFF protein in cells

[0237] Cell samples treated with different concentrations of ZCY-PROTAC for different times were collected, and Western blot analysis was performed after lysis of the proteins.

[0238] Results show that ZCY-PROTAC significantly reduces the protein level of MAFF in RAW264.7 cells and HEK293T cells in a concentration-dependent and time-dependent manner, and in addition, the proteasome inhibitor MG132 can completely block this process, indicating that ZCY-PROTAC has the effect of specifically inducing MAFF protein degradation through the proteasome pathway Figure 1 E-1F).

[0239] Example 41 Microscale thermophoresis experiment

[0240] MAFF protein was prepared into 10 μM with protein buffer (25 mM Hepes pH 7.5; 200 mM NaCl), 2.5 μL MST fluorescent dye was added, and it was incubated at room temperature for 30 min in the dark. The fluorescently labeled protein solution was separated by small molecules. The small molecule compounds (Lathyrol and ZCY020) were diluted with protein buffer to form 12 concentration gradients, and mixed with the same volume of fluorescently labeled protein. The samples were placed on the microscale thermophoresis instrument by standard capillary suction. Results show that the equilibrium dissociation constant K D of Lathyrol and ZCY020 with MAFF protein in vitro is 20.90 ± 2.34 μM and 19.50 ± 1.70 μM, respectively, both of which have strong in vitro binding ability with MAFF protein Figure 2 B).

[0241] Example 42 CETSA experiment

[0242] After RAW264.7 cells were treated with DMSO or 40 μM small molecule compounds (Lathyrol and ZCY020) for 4 h, the cells were centrifuged, resuspended with PBS, and aliquoted into 6 parts, heated at 64, 66, 68, 70 or 72°C for 10 min, then lysed by repeated freezing and thawing in liquid nitrogen for three times, centrifuged at 4°C at 12000g for 10 min, and the supernatant was added to SDS-PAGE loading buffer, heated at 95°C for 5 min, and then subjected to immunoblotting detection. Results show that Lathyrol and ZCY020 can directly bind to MAFF, and improve the thermal stability of MAFF protein Figure 2 C).

[0243] Example 43 DARTS experiment

[0244] The RAW264.7 cell lysate was divided into 5 equal parts, and divided into control group, enzyme hydrolysis group, drug administration group (10, 20 and 40 μM of Lathyrol or ZCY020), first incubate the compound with the lysate at room temperature for 1 h, then add 1:1000 diluted Pronase in the enzyme hydrolysis group and the drug administration group, and add the same volume of PBS in the control group, mix uniformly, and then incubate in a 37℃ water bath for 15 min. Immediately remove, add SDS-PAGE loading buffer, heat denaturation at 95℃ for 5 min, and then perform immunoblotting detection. The results show that after treatment with Lathyrol and ZCY020, the MAFF protein is more stable, and the degradation of MAFF by Pronase is significantly reduced Figure 2 D).

[0245] The above results prove that ZCY020 can directly bind to MAFF protein, so MAFF protein is the target of ZCY020.

[0246] Example 44 ZCY020 promotes the formation of MAFF-Nrf2 heterodimer

[0247] After cell centrifugation, non-denaturing lysis buffer containing 1% PMSF was used for lysis on ice, and the supernatant was quantified by BCA. 0.2-1 mg of each sample was added to 0.2-2 μg of protein primary antibody for immunoprecipitation, and slowly shaken at 4℃ overnight. Then add 20 μL of fully resuspended Protein A+G Agarose, slowly shake at 4℃ for 2 h. Instantly centrifuge at 13000 rpm, carefully aspirate the supernatant, and wash the precipitate with PBS for 5 times. Add 40 μL of 1×SDS-PAGE loading buffer to resuspend the precipitate, heat denaturation at 95℃ for 5 min, and then used for immunoblotting analysis.

[0248] The results show that under the induction of LPS, MAFF is overexpressed in macrophages, and ZCY020 down-regulates the overexpression of MAFF on this basis, while promoting the formation of MAFF-Nrf2 heterodimer Figure 3 A). At the same time, ZCY020 inhibits the binding of MAFF to SUMO-2 / 3 Figure 3 B), while inhibiting the formation of MAFF homodimer and promoting the formation of MAFF-Nrf2 heterodimer in RAW264.7 cells and HEK293T cells Figure 3 C-3E).

[0249] Example 45 ZCY020 activates the MAFF-Nrf2 / HO-1 signaling pathway

[0250] 1. ZCY020 promotes the transcription of Nrf2 regulated genes

[0251] After centrifugation, RAW264.7 cells were lysed at room temperature for 10 min using TRIZOL reagent. RNA was extracted and reverse transcribed to obtain cDNA. PCR amplification was performed using the obtained cDNA as a template, with the β-Atin gene as a positive internal control. The Ct value for each sample reaction was obtained from software analysis, and ΔCt was calculated. ΔCt = Ct value of the target gene - Ct value of the internal control gene. Then, 2... -△△Ct Data statistics were performed to calculate the relative mRNA transcription levels of each gene in each group of samples.

[0252] The results showed that in LPS-induced RAW264.7 cells, exogenous endotoxin LPS slightly increased the transcriptional levels of HMOX1, NQO1, and GCLM genes. Furthermore, ZCY020 concentration-dependently enhanced the transcription of HMOX1, NQO1, and GCLM. In response to LPS stimulation, GCLC transcription was downregulated, while ZCY020 restored the transcription of damaged GCLC genes. Therefore, ZCY020 can activate the transcription of important regulatory genes in the MAFF-Nrf2 pathway. Figure 4 A).

[0253] 2. ZCY020 promotes the nuclear translocation of Nrf2 and the expression of its regulatory protein HO-1.

[0254] After RAW264.7 cells adhered to the culture vessel, different concentrations of ZCY020 were added. After 2 hours of treatment, LPS was added to a final concentration of 1 μg / mL. Cells were collected 24 hours later for Western blotting analysis. The results showed that in LPS-induced RAW264.7 cells, ZCY020 significantly upregulated Nrf2 protein levels and attenuated LPS-induced overexpression of MAFF protein. Simultaneously, consistent with the changes in Nrf2, HO-1 protein expression also increased in a dose-dependent manner. Figure 4 B).

[0255] 3. ZCY020 inhibits the generation of reactive oxygen species.

[0256] RAW264.7 cells in the logarithmic growth phase were fed a 2×10⁻⁶ dose. 5 Cell suspension of 1 mL / well was added to 6-well plates. After cell attachment, different concentrations of ZCY020 were added for 2 hours, followed by incubation with LPS for 12 hours. The supernatant was discarded, and the cells were washed twice with PBS. 1 mL of 10 μM DCFH-DA was added, and the cells were incubated at 37°C for 20 minutes. The cells were washed three times with PBS, resuspended, and ROS levels were detected by flow cytometry.

[0257] Results showed that ZCY020 treatment could inhibit LPS-stimulated ROS production in a dose-dependent manner. It can be concluded that ZCY020 can exert antioxidant activity by activating the Nrf2 / HO-1 pathway Figure 4 C).

[0258] 4. Nrf2 inhibitor ML385 and MAFF shRNA inhibit the activity of ZCY020

[0259] After RAW264.7 cells were adhered, different concentrations of ZCY020 and Nrf2 inhibitor ML385 (5 μM) were added, and after 2 hours of treatment, LPS was added. After 24 hours, the cells were collected for immunoblotting and reactive oxygen detection experiments. The results showed that Nrf2 inhibitor ML385 inhibited the enhanced expression of Nrf2 and HO-1 by ZCY020, and reversed the inhibitory effect of ZCY020 on ROS Figure 4 D-4E).

[0260] After RAW264.7 cells were adhered, MAFF knockdown plasmid shMAFF was transfected, and after 24 hours, different concentrations of ZCY020 were added for 2 hours. LPS was added, and after 24 hours, the cells were collected for immunoblotting and reactive oxygen detection experiments. The results showed that in shMAFF-treated RAW264.7 cells, the enhanced expression of HO-1 by ZCY020 and the inhibitory effect of ZCY020 on ROS production were also significantly weakened Figure 4 F-4H). The above results showed that ZCY020 exerts antioxidant effects by promoting the formation of MAFF-Nrf2 heterodimers and activating the Nrf2 / HO-1 pathway.

[0261] Example 46. ZCY020 inhibits the NF-κB signaling pathway

[0262] 1. ZCY020 inhibits the expression of LPS-induced NF-κB pathway proteins

[0263] After preparing RAW264.7 cell samples treated with different concentrations of ZCY020 for 2 hours and then treated with LPS for 24 hours, immunoblotting was performed. The results showed that compound ZCY020 could inhibit the phosphorylation of IκBα induced by LPS, and could reduce the expression levels of iNOS and COX-2 in a concentration-dependent manner, and could inhibit the release of inflammatory mediator NO Figure 5 A-5C). ELISA analysis results showed that in RAW264.7 cells, ZCY020 significantly down-regulated the secretion of IL-6, IL-1β, and TNF-α induced by LPS Figure 6D). Meanwhile, RT-qPCR analysis showed that the transcriptional levels of IL-1β, IL-6 and TNF-α were significantly up-regulated after LPS stimulation, while their transcriptional levels were significantly decreased after ZCY020 treatment Figure 6 E).

[0264] 2. MAFF shRNA inhibits the anti-NF-κB pathway activity of ZCY020

[0265] After knocking down the expression of MAFF with MAFF shRNA plasmid, the inhibitory activity of compound ZCY020 on LPS-induced NF-κB pathway proteins was detected. The results showed that after MAFF shRNA (shMAFF) treatment, the inhibitory effect of ZCY020 on LPS-induced phosphorylation of IκBα and NO production was significantly weakened Figure 5 F and 5G). The results proved that ZCY020 inhibited the activation of NF-κB pathway by targeting MAFF.

[0266] Example 47 ZCY020 promotes mitochondrial quality control

[0267] 1. ZCY020 inhibits the production of excessive mtROS

[0268] RAW264.7 cells in logarithmic growth phase were inoculated in 6-well plates at 5x10 5 Each well, cultured for 24 h, treated with compound for 3 h, then added with LPS 1 μg / mL and incubated for 12 h. The mitochondrial superoxide (mitoSOX) detection reagent was prepared into a 5 mM stock solution with DMSO. The stock solution was diluted 1:1000 with HBSS buffer to prepare a 5 μM working solution. Discard the supernatant, add 1 mL of working solution to the well, and incubate at 37°C for 10 min in the dark. Wash the cells with preheated HBSS buffer for 3 times. Resuspend the cells with appropriate amount of HBSS buffer, and detect the level of MitoSOX with flow cytometry.

[0269] The results showed that LPS significantly increased the production of mitochondrial superoxide, and ZCY020 pretreatment dose-dependently reversed this situation Figure 6 C).

[0270] 2. ZCY020 promotes mitochondrial autophagy

[0271] The results of immunoblotting showed that after ZCY020 treatment, the expression of P62 was significantly up-regulated, while the level of Nrf2 protein was up-regulated and the level of Keap1 was decreased, and the protein level of BNIP3 was increased in a concentration-dependent manner, and the enrichment of LC3B-II was promoted, thus it was known that ZCY020 promoted mitochondrial autophagy to maintain mitochondrial homeostasis Figure 6A-6B).

[0272] 3. Reactive oxygen scavenger NAC, ML385 and MAFF shRNA inhibit the activity of ZCY020

[0273] After RAW264.7 cells were adhered, ① different concentrations of ZCY020 and reactive oxygen scavenger NAC (5mM) or ML385 were added, and after 2 hours of treatment, LPS was added, and the cells were collected after 24 hours; ② after transfection of MAFF knockdown plasmid shMAFF, different concentrations of ZCY020 were added for 2 hours, and then LPS was added, and the cells were collected after 24 hours. Western blot analysis and flow cytometry analysis were performed on the cells.

[0274] The results showed that NAC and ML385 pretreatment significantly weakened the effect of ZCY020 on p62, BNIP3 and LC3B-II (Fig. 6D-6E); the inhibitory effect of ZCY020 on mitochondrial superoxide production was blocked by ML385 (Fig. 6F); after MAFF knockdown, the effect of ZCY020 on p62, BNIP3 and LC3B-II was blocked (Fig. 6G). Figure 6 Figure 6

[0275] Example 48 Therapeutic effect of ZCY020 on LPS-induced acute lung injury model in mice

[0276] Forty BALB / C mice, 7-8 weeks old, half male and half female, were randomly divided into groups. At the beginning of the experiment, the first day, gavage administration or normal saline, 2 hours later, intraperitoneal injection of LPS (15 mg / kg), and at 12h, 48h, 72h and 96h, gavage administration of drug or normal saline, and the survival rate of each group of mice was observed for one week. On the eighth day, the liver, lung and spleen were dissected and subjected to HE staining, Western blot and RT-qPCR analysis. The results showed that after oral administration of ZCY020, the mortality rate caused by LPS was significantly reduced, and showed a dose-dependent effect (Fig. 8A). Serum ELISA analysis showed that compound ZCY020 could significantly reduce the production of inflammatory cytokines caused by LPS endotoxin (Fig. 8B). Pathological tissue analysis showed that compound ZCY020 treatment significantly improved the pathological damage of the lungs and liver of mice: inflammatory cell infiltration, alveolar wall thickening and hepatocyte damage (Fig. 8C). Tissue Western blot and RT-qPCR analysis showed that ZCY020 activated the transcription and protein expression of Nrf2 pathway regulated genes and inhibited the activation of NF-κB pathway, promoting the protective autophagy of mitochondria (Fig. 8D-7F). Figure 7 Figure 7 C). Pathological tissue analysis showed that compound ZCY020 treatment significantly improved the pathological damage of the lungs and liver of mice: inflammatory cell infiltration, alveolar wall thickening and hepatocyte damage (Fig. 8C). Tissue Western blot and RT-qPCR analysis showed that ZCY020 activated the transcription and protein expression of Nrf2 pathway regulated genes and inhibited the activation of NF-κB pathway, promoting the protective autophagy of mitochondria (Fig. 8D-7F). Figure 7 Figure 7 D-7F).

[0277] ​​​​Example 49 Therapeutic effect of ZCY020 on IMQ-induced psoriasis model in mice

[0278] Forty male BALB / C mice, 7-8 weeks old, were depilated on the back with an area of 2.5 cm x 2.5 cm. The mice were randomly divided into 4 groups, and an appropriate amount of imiquimod (IMQ) cream was continuously applied to the depilated skin on the back of the mice for 6 days. The control group was given the same dose of petrolatum ointment. At the same time of IMQ application, intragastric administration or normal saline was performed. During the experiment, body weight was measured and clinical symptom score PASI (Psoriasis Area and Severity Index) was performed every day. The results showed that ZCY020 treatment significantly reduced the severity of erythema, scales and skin lesion thickening, and improved IMQ-induced psoriatic-like skin inflammation Figure 8 A-8C).

[0279] Example 50 Therapeutic effect of ZCY020 on CIA arthritis model in mice

[0280] Forty male C57BL / 6J mice, 20-25 g, were randomly divided into 4 groups. Type II collagen solution and the same volume of complete Freund's adjuvant (CFA, 5 mg / mL) were completely emulsified in an ice bath, and 0.1 mL of CII / CFA emulsifier was injected intradermally into the tail root of each mouse in the model group and the drug group for primary immunization. On day 21, secondary immunization was performed in the same way. At the same time, each group of mice was given intragastric administration or normal saline every day until the 37th day after primary immunization. During this period, arthritis index score was performed every day. The results showed that ZCY020 treatment dose-dependently reduced the severity of foot swelling in CIA mice, and significantly improved the degree of cartilage and bone destruction and inflammatory infiltration Figure 9 A-9C). At the same time, the increase in spleen index caused by arthritis was also significantly reduced Figure 9 D).

Claims

1. A method for target identification of active natural products based on PROTAC protein targeted degradation technology, characterized in that, Comprising the following steps: (1) A series of bifunctional PROTAC compounds based on active natural products are synthesized with active natural products as POI ligands and different E3 ligase ligands; the active natural products are selected from Lathyrol and ZCY020; the structural formulas of Lathyrol and ZCY020 are shown in general formula I-1 and general formula I-2 respectively: (2) The best active PROTAC compound is determined through activity screening, differential proteomic analysis is performed on cell lysates treated by active natural products and PROTAC compounds respectively, differential expression proteins in the protein lysate are identified, and KEGG analysis is performed to determine the potential target proteins of PROTAC compounds and active natural products; (3) The potential target proteins are verified by western blot to determine their degradation activity and degradation characteristics by PROTAC compounds, the direct binding force between the active natural products and the target proteins is verified by in vitro target verification methods MST, CETSA and DARTS, the function of the target protein and the effect of the downstream proteins affected by the target protein are determined by cell biology techniques RNAi and in vivo target related pathway verification, and whether the active natural products act on the target are further determined, so as to verify the authenticity of the target protein, and finally determine the target protein of the active natural products.

2. The method for target identification of active natural products based on PROTAC protein targeted degradation technology according to claim 1, characterized in that, The structural formula of the PROTAC compound is shown in general formula I-3: In general formula I-3: R1, R2 are independently H, cinnamoyl, p-hydroxycinnamoyl, acetyl, butyryl, benzoyl or nicotinoyl; L is a connecting arm, which is connected to the oxygen atom and B by a covalent bond, and together constitutes a bifunctional molecular compound; B is a small molecule ligand of E3 ubiquitin ligase complex, which is any one of the following structures: Wherein: W is selected from CH2, C=O, SO2, NH, N-C1-C4 alkyl; X is selected from O, S; Z is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, halogen; G, G' are selected from H, C1-C4 alkyl, -OH, 5-10 membered heterocyclic group substituted by C1-C4 alkyl, the heterocyclic group containing 1-3 N, O or S heteroatoms; R 3 selected from H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4alkyl, haloC1-C4alkyl, deuterated C1-C4alkyl.

3. The method for target identification of active natural products based on PROTAC protein targeted degradation technology according to claim 2, characterized in that, L is any one of the following structures: Wherein: n is an integer between 1-10. 4.The method for identifying a target of an active natural product based on PROTAC protein targeted degradation technology according to claim 1, characterized in that, In step (1), the PROTAC compound comprises: 。 5.The method for identifying the target of active natural products based on PROTAC protein targeted degradation technology according to claim 1, characterized in that, In step (1), the preparation method of the PROTAC compound comprises the following steps: (1) Dissolve Lathyrol in anhydrous DMF, add sodium hydride and bromopropargyl, and react at room temperature; after dilution, washing and purification, intermediate 1 is obtained; (2) Add vitamin C sodium and anhydrous copper sulfate to the mixture of intermediate 1 and intermediate 2 in tetrahydrofuran and water; (3) The reaction system is reacted at room temperature for 20-30 minutes, after the reaction is completed, the solid is removed by filtration, the filtrate is evaporated under reduced pressure, and the corresponding end product is separated by column chromatography; wherein: The intermediate 2 is one of intermediate 2-1, intermediate 2-2, intermediate 2-3, intermediate 2-4 or intermediate 2-5; The preparation process of the intermediate 2-1 is as follows: The thalidomide derivative is taken, a solvent is added, and azido-PEG-amine and DIPEA are sequentially added under stirring, and after heating reaction, extraction, drying, concentration, purification, elution, a yellow oil is obtained, that is, intermediate 2-1; The preparation process of the intermediate 2-2 is as follows: M7 is weighed, dissolved, and then azido-PEG-amine, EDCI, HOBt and DIPEA are added, and after reaction at room temperature, dilution, washing, drying and purification, the intermediate 2-2 product is obtained; The preparation process of the intermediate 2-3 is as follows: B5 is weighed, dissolved, and then azido-PEG-amine, EDCI, HOBt and DIPEA are added, and after reaction at room temperature, dilution, washing, drying and purification, the intermediate 2-3 is prepared; The preparation process of the intermediate 2-4 is as follows: B4 hydrochloride is weighed, dissolved, and then azido carboxylic acid, EDCI, HOBt and DIPEA are added, and after reaction at room temperature, dilution, washing, drying and purification, the intermediate 2-4 is prepared; The preparation process of the intermediate 2-5 is as follows: VHL hydrochloride is weighed, dissolved, and then azido carboxylic acid, EDCI, HOBt and DIPEA are added, and after reaction at room temperature, dilution, washing, drying and purification, the intermediate 2-5 is prepared; When the intermediate 2 is intermediate 2-1, n=2, 3 or 4, the reaction process is as shown in the following formula III, and the final product obtained is final product 1-3; When the intermediate 2 is intermediate 2-2, n=2, 3 or 4, the reaction process is as shown in the following formula IV, and the final product obtained is final product 4-6; When the intermediate 2 is intermediate 2-3, n=2, 3 or 4, the reaction process is as shown in the following formula V, and the final product obtained is final product 7-9; When the intermediate 2 is intermediate 2-4, n=1, 3 or 5, the reaction process is as shown in the following formula VI, and the final product obtained is final product 10-12; When intermediate 2 is intermediate 2-5, n = 1, 3 or 5, R 1 is H or CH3, the reaction process is as shown in the following formula VII, and the final product obtained is final product 13-18; 。 6.The method for identifying the target of active natural products based on PROTAC protein targeted degradation technology according to claim 1, characterized in that, In the step (2), the activity screening method includes anti-inflammatory activity screening in an inflammation stimulation model, anti-tumor activity screening in tumor cells, and anti-oxidation activity screening in an oxidation stress model, and the detection method varies depending on the specific model; the anti-inflammatory activity screening method in the inflammation stimulation model is specifically as follows: (1) A series of bifunctional PROTAC compounds based on active natural products synthesized are added to cultured mouse macrophage RAW264.7 cells in the logarithmic growth phase for 3 hours, then the cells are stimulated with lipopolysaccharide for 24 hours to establish a mouse macrophage inflammation model; (2) The total nitric oxide content in the cell supernatant is detected by Griss reagent, and according to the inhibition of NO release after PROTAC small molecule treatment, the anti-inflammatory activity of the synthesized PROTAC compound is analyzed and determined, and the PROTAC compound with the best anti-inflammatory activity, that is, ZCY-PROTAC, is screened, and the structure of the ZCY-PROTAC is: 。 7. The method for target identification of active natural products based on PROTAC protein targeted degradation technology according to claim 2, characterized in that, The PROTAC compound is used for preparing a drug for treating or preventing an inflammation-related disease selected from acute lung injury, psoriasis, arthritis.

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